Biomedical Research Journal - April 2015 Issue - 25.04

SCHOOL OF SCIENCE
BiomedicalResearchJournal
APRIL 2015 | VOLUME 2 | ISSUE 1
pISSN: 2349-3666; eISSN: 2349-3674
SCHOOL OF SCIENCE
BiomedicalResearchJournal
APRIL 2015 | VOLUME 2 | ISSUE 1
EDITORS-IN-CHIEF
EDITORIAL BOARD
Dhananjaya Saranath (Mumbai, India)
Ali Syed Arbab (Detroit, USA)
Alpana Ray (Missouri, USA)
Aparna Khanna (Mumbai, India)
Amit Agarwal (Bangalore, India)
Anandwardhan Hardikar (Sydney, Australia)
Anjali A. Karande (Bangalore, India)
Ashok B. Vaidya (Mumbai, India)
Basuthkar J. Rao (Mumbai, India)
Dhirendra Bahadur (Mumbai, India)
SECTION EDITORS
Hemant Malhotra (Jaipur, India)
Karuna Shanker (Lucknow, India)
Cancer Biology:
Kirti S. Laddha (Mumbai, India)
Mayur Yergeri (Mumbai, India)
Girish Maru (Navi Mumbai, India)
Mohan C. Vemuri (Frederick, USA)
Naganand Rayapuram (Evry, France)
Stem Cell Biology:
Nancy Pandita (Mumbai, India)
Partha Basu (Kolkata, India)
Vaijayanti P. Kale (Pune, India)
Paul J. Verma (Rosedale, Australia)
Prasad S. Adusumilli (New York, USA)
Nanotechnology:
Pritish Bhattacharya (New Jersey, USA)
Pulok Mukherjee (Kolkata, India)
Vilas G. Gaikar (Mumbai, India)
Purvish M. Parikh (Mumbai, India)
Ramesh Goyal (Ahmedabad, India)
Phytochemistry:
Sai Yendamuri (New York, USA)
Sukhinder Kaur Cheema (St. John's, Canada)
Lokesh Bhatt (Mumbai, India)
Sumitra Chanda (Rajkot, India)
Sunita Saxena (New Delhi, India)
Surinder K. Mehta (Chandigarh, India)
Tania Fernandez (San Francisco, USA)
Victoria M. Villaflor (Chicago, USA)
EDITORIAL OFFICE
EDITORIAL ASSISTANT
School of Science, NMIMS (Deemed-to-be University)
Bhaidas Sabhagriha Building,
Bhaktivedanta Swami Marg,
Vile Parle (W), Mumbai 400056, India.
Email: [email protected]
Brijesh S. (Mumbai, India)
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Contents
April 2015, Volume 2, Issue 1
Editorial: Precision/personalized medicine in cancer
Dhananjaya Saranath and Aparna Khanna ..........................................................................................1
Advances in omics technologies in GBM
Uday B. Maachani, Uma Shankavaram, Kevin Camphausen and Anita Tandle ..................................6
Dendrimers based electrochemical biosensors
Saumya Nigam, Sudeshna Chandra and Dhirendra Bahadur ...........................................................21
Developmental signalling in maintenance and regulation of cancer stem cells
Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare ..................................................37
Diverging role of Nrf2 in cancer progression and prevention
Lokesh Gambhir, Rahul Checker, Deepak Sharma and Santosh K. Sandur .....................................57
Physiology of embryo-endometrial cross talk
Deepak N. Modi and Pradeep Bhartiya …………………..…………….............................………….....83
Human EGFR-2, EGFR and HDAC triple-inhibitor CUDC-101 enhances
radiosensitivity of glioblastoma multiforme cells
Cody D. Schlaff, W. Tristram Arscott, Ira Gordon, Kevin A. Camphausen and Anita Tandle ............105
Phenotypic and functional characterization of a marrow-derived stromal cell
line, M210B4 and its comparison with primary marrow stromal cells
Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale ........120
Editorial
Precision/Personalized Medicine in Cancer
Dhananjaya Saranath and Aparna Khanna
Recent technological advances have provided
decisions.
unprecedented
develop
including 'Next Generation Sequencing'
'Precision/
(NGS) with both availability and affordability
Personalized Medicine', with confluence of
will enable understanding of cancer and other
medicine and technology making significant
diseases, a feasible proposition.
platforms
opportunities
for
to
implementing
Thus,
advanced
technology
advances in treatment. The Cancer Genome
Next Generation Sequencing is massively
Atlas (TCGA), a large scale initiative started in
parallel sequencing enabling rapid sequencing
2006, to generate a comprehensive landscape
of the entire genome or exome sequencing on
for identification of alterations in tumor types
whole genome or cDNA (RNA-Seq), builds
with a view to develop better therapies, is on a
on the concept of 'NGS taking us into
wind down. The next phase is use of the
expanded genomic testing for risk prediction,
information
'Precision/
diagnosis, prognosis, treatment response and
Personalized Medicine'. Implementation of
disease free survival or overall survival in real
'Precision/Personalized Medicine' requires
time'.The understanding of the mechanisms
understanding of the biology of each cancer
and processes in cancer with single aberrations
type with precise definition of the cancer
or cumulative alterations including mutations,
genome. The different genome alterations will
rearrangements, amplifications, deletions,
identify the 'Founder Mutations' involved in
insertions and other alterations in cancer will
the early phase, but may not be associated with
be discerned. It is important to remember that
a fully transformed phenotype; 'Driver
several countries have initiated studies in this
Mutations' required for fully transformed
direction. Prime Minister David Cameroon,
phenotype;
Mutations'
UK, endorsed the 'Genomes Project' for
considered as collateral damage. Treatment
collection of data for whole genome sequences
against key oncogenic driver mutations in
from 100,000 individuals, to be completed by
individual cases with targeted drugs will be the
2017, sanctioning USD 475 million for
benefit of the 'Precision Medicine' approach.
sequencing studies, with a view to better
Rational choices for treatment have to be
understand
preceded by full genomic data and expression
cancer. Barack Obama, President, USA,
data,
launched the 'Precision Medicine' initiative
generated
and
facilitating
for
'Passenger
combination
therapy
complex
diseases
including
Biomed Res J 2015;2(1):1-5
Editorial: Precision/personalized medicine in cancer
2
with a USD 215 million for genomic data on
examples are targeted drugs Dabrafenib and
one million volunteers to accelerate patient
Trametinib, mitogen activated protein kinase
powered research that promises to accelerate
1/2
biomedical discoveries and provide clinicians
BRAFV600E/K;
with new tools, knowledge and therapies for
Bevacizumab, against vascular endothelial
individual patients. A result of better
factor A is a targeted therapy in cancers of the
understanding of cancer, is the current
colon, lung, breast, kidney and brain; whereas
repertoire of targeted therapy drugs and
Ramicirumab, a monoclonal antibody against
personalized medicine on the global scene.
vascular endothelial growth factor receptor 2,
(MAPK1/2)
for
melanoma
angiogenesis
with
inhibitor,
In the past decade the advent of targeted
is used in gastric cancer and Non-small cell
therapy and new tailored drugs has led to a
lung cancer, and in combination with
revolution in treatment of lung cancer, with
Docetaxel improves outcomes in bladder
larger benefit, lower toxicity and better quality
cancer. HER2 gene antibody – Herceptin,
of life for the patient. The treatment is often
shows substantial survival benefits in all
based on molecular profiling of individual
newly diagnosed and recurrent breast cancer
patients with identified cancer, as also
patients
indicated in other cancers with a similar
expression of the gene. Development of
molecular profile. Thus, Tyrosine kinase
companion
inhibitors including Erlotinib, Gefitinib,
pathogenic molecular alterations and new
Afatinib targeting epidermal growth factor
targeted
receptors (EGFR), and ALK Inhibitors
guidelines for several molecular diagnostic
including Crizotinib, Ceritinibare beneficially
tests
used in patients with aberration of the genes in
Personalized Medicine' will enable the current
Non-small cell lung cancer, and indicated for
oncologists to 'Win the War against Cancer'.
with
diagnostics
drugs
are
amplification
go
and
indicating
hand-in-hand,
available.
Thus,
overthe
and
'Precision/
additional cancers with the appropriate
The current issue includes a review paper
molecular profile. Involvement of Kras,
and an original article on glioblastoma
EGFR, ALK, HER2, Braf, MET, AKTI,
multiforme
MAP2KI, PI3KC genes have been identified
aggressive brain tumor, a cancer with bad
in lung cancers, opening possibilities of
prognosis and median survival of 15 months.
targeted therapy with consequential benefits.
The conventional treatment of GBM using the
Thus, an additional aspect which has emerged
strategy
is combination therapy using two or more
chemotherapy,
targeted drugs, or targeted drugs plus the
incrementally in the last 30 years. With the
conventional chemotherapeutic drugs. A few
advent of molecular biology and consequent
Biomed Res J 2015;2(1):1-5
(GBM),
of
one
surgery,
has
of
the
radiation
advanced
most
and
only
Saranath and Khanna
3
improved understanding of basic tumor
for better contemporary treatment.
biology, targeted therapies have become
Nanomaterials
and
nanoparticles
cornerstones for cancer treatment. Several
including dendrimers, polymers, nanotubes,
signaling pathways including RTKs/PI3K/
oxides, and enzymes and their hybrids as
AKT/mTOR/VEGF/VEGFR are deregulated
catalysts for various sensors such as glucose
in
in
sensors, DNA sensors, neurotransmitters
and
sensors, are another facet of technological
progression of GBM. Dr. Anita Tandle and
advances with tremendous applications in
colleagues from National Cancer Institute,
health sciences. Dendrimers are synthetic
Bethesda, Maryland, USA, discuss the Omics
nanoscale compounds with unique properties,
of GBM and applications in novel therapies, in
resulting
the article, ‘Advances in Omics Technologies
applications. Dendrimers have a number of
in GBM’. The authors survey the technologies
features that make them ideally suited for
of genomics, transcriptomics, epigenomics,
sensor applications, such as, its high surface
proteomics,
post
area, high reactivity, easy dispersability and
transcriptional modifications of microRNAs
rapid fabrication. Dr. Saumya Nigam and Dr.
in GBM. A comprehensive information in
Dhirendra Bahadur, Indian Institute of
GBM will lead to better understanding of the
Technology Bombay, Mumbai, along with Dr.
cancer,
signal
Sudeshna Chandra, NMIMs (Deemed-to-be)
transduction pathways, and identify key
University, present a review on ‘Dendrimers
molecules associated with the pathogenesis,
based electrochemical biosensors’. The
culminating in development of new drugs and
review highlights the advanced development
'Personalized treatment'. The original article
of
by the group, ‘EGFR 2, EGFR and HDAC
electrochemical
triple
dendrimers. A must read review for all to
GBM,
playing
tumorigenesis,
a
treatment
major
resistance
metabolomics
highlight
inhibitor
the
role
and
various
CUDC-101
enhances
radiosensitivity of GBM cells’, convincingly
in
effective,
biomedical
rapid
and
and
biosensors
industrial
versatile
based
on
understand the technology.
shows enhancement of in vitro radiosensitivity
The concept of cancer stem cells (CSC)
of GBM and breast cancer cell lines
proposed earlier in the year 2000, are now well
selectively, with no effect on normal human
accepted to play a critical role in cancers. The
lung fibroblast cell line. The radiosensitization
CSCs are more of an enigma and relatively
of the cancer cell lines was attributed to
more difficult to decode the biology of CSCs.
inhibition of DNA double stranded break
The conserved Wnt/β-Catenin, Notch and
repair and modulation of cell cycle. A better
Sonic Hedgehog pathways regulate stem cell
understanding of the cancer will open avenues
pluripotency and cell fate decisions during
Biomed Res J 2015;2(1):1-5
Editorial: Precision/personalized medicine in cancer
4
normal embryonic development and adult
cells’ by Dr. Vaijayanti Kale and colleagues
tissue homeostasis, and aberrant activity
from the National Centre for Cell Science,
within these pathwaysis displayed in several
Pune, emphasizes importance of alternative
cancers. Human cancers contain a relatively
systems for investigating regulation of
dormant
hematopoietic stem cells (HSCs). The authors
cell
population,
CSCs,
with
characteristics similar to normal stem cells.
showed
Convincing evidence indicates that CSCs are
unequivocally
responsible
adipogenic lineage, and exhibited a higher
therapy
for
chemotherapy/radiation
resistance,
maintenance
that
the
cell
line
M210B4
differentiated
towards
and
HSC-supportive ability and conclude that the
consequent recurrence of the cancer. The roles
cell line M210B4 is an appropriate substitute
of Wnt, Notch and Hedgehog pathways in
to study HSC regulation in vitro.
cancers and their deregulation are of critical
The transcription factor Nrf2 containing
significance, directly linked to CSCs. In order
the conserved basic leucine zipper structure
to target the CSCs therapeutically, it is
belongs to the Cap 'N' Collar family, and plays
imperative to understand the molecular
a critical role in cell defense and survival
mechanisms regulating CSCs responsible for
pathways. Nrf2 often protects cells and tissues
maintenance and recurrence of cancer, and
from
develop combination therapies to target CSCs
transcription of cytoprotective genes, and
inhibiting the cumulative action of the
hence
deregulated genes. Dr. Sanjeev Waghmare and
protecting against redox-mediated injury and
his colleagues from Advanced Centre for
carcinogenesis. Paradoxically, the flip side of
Treatment, Research and Education in Cancer,
Nrf2 is protection of cancer cells from
Navi
the
chemotherapeutic agents and/or radiotherapy
intricately complex signalling cascades of
resulting in resistance to the therapy and
Wnt, Notch and Hedgehog genes, regulation
cancer
and maintenance of normal developmental
upregulated in several cancer types, and
processes, and their association in cancer, in
associated with poor prognosis in cancer
the article, ‘Developmental signalling in
patients. The dilemma of the dual action of
maintenance and regulation of cancer stem
Nrf2 has been well reviewed in the article,
cells’. Whereas, the article, ‘Phenotypic and
‘Divergent
functional
the
progression and prevention’ by Dr. Santosh
maintenance and regulation of a marrow-
Sandur and colleagues from Bhabha Atomic
derived stromal cell line, M210B4 and its
Research Centre, Mumbai. The review
comparison with primary marrow stromal
indicates a wider approach with better
Mumbai,
succinctly
characterization
Biomed Res J 2015;2(1):1-5
review
in
toxicants
and
considered
progression.
role
of
carcinogens
via
chemopreventive,
Nrf2
Nrf2
is
aberrantly
in
cancer
Saranath and Khanna
5
comprehension of the mechanisms of action of
infertility and intra-uterine growth retardation
Nrf2 and consequent design and development
are minimized or avoided in normal fetal
of drugs to handle the upregulation or
growth. The highly orchestrated embryo-
downregulation of Nrf2 in the preventive,
endometrial cross talk involves a plethora of
protective or destructive niche of normalcy
molecules including hormones, cytokines,
and diseases. Thus, 'One fit for all' is not a
growth factors, specific immune modulating
feasible solution in all conditions indicating
factors, to create the appropriate micromileu
importance
for establishing pregnancy. Dr. Deepak Modi
of
'Precision/Personalized
Medicine'.
and Mr. Pradeep Bhartiya from the National
The mechanisms of embryo implantation
Institute for Research in Reproductive Health,
and development resulting in pregnancy are
Mumbai, take us through the ‘Physiology of
comparable to cancer with respect to the
embryo-endometrial cross talk’ lucidly
growth
of
highlighting the various processes and
A receptive endometrium,
interactions. The networking interactions and
normal blastocyst, cross talk between fetal and
intricate physiology in a pregnancy is very
maternal compartments remodeling uterine
well explained. The applications in a clinical
vasculature, and selector activity comprise
scenario
innate requirements for successful pregnancy.
infertility treatment and development of
Adverse
contraceptive drugs are discussed.
processes
development.
events
and
such
mechanisms
as
preeclampsia,
for
successful
implantation,
Biomed Res J 2015;2(1):1-5
Review
Advances in OmicsTechnologies in GBM
Uday B. Maachani, Uma Shankavaram, Kevin Camphausen, Anita Tandle*
Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda MD,USA
Glioblastoma multiforme (GBM) is one of the most lethal human cancers and poses a great challenge in the
therapeutic interventions of GBM patients worldwide. Despite prominent recent advances in oncology, on
an average GBM patients survive 12–15 months with conventional standard of care treatment. To
understand the pathophysiology of this disease, recently the research focus has been on omics-based
approaches. Advances in high-throughput assay development and bioinformatic techniques have provided
new opportunities in the molecular analysis of cancer omics technologies including genomics,
transcriptomics, epigenomics, proteomics, and metabolomics. Further, the enormous addition and
accessibility of public databases with associated clinical demographic information including tumor
histology, patient response and outcome, have profoundly improved our knowledge of the molecular
mechanisms driving cancer. In GBM, omics have significantly aided in defining the molecular architecture of
tumorigenesis, uncovering relevant subsets of patients whose disease may require different treatments. In
this review, we focus on the unique advantages of multifaceted omics technologies and discuss the
implications on translational GBM research.
INTRODUCTION
Glioblastoma (GBM)
recurrence. As the name infers, it is
Brain tumors account for about 85–90% of all
multiforme microscopically showing regions
primary central nervous system (CNS) tumors.
of
Worldwide, approximately 343,175 new cases
multiforme genetically with various genetic
of brain and other CNS tumors were diagnosed
alterations leading to its aggressive nature.
pseudopalisading
and
hemorrhage,
in the year 2012 (http://www.cbtrus.org/).
The standard of care for treatment of GBM
Glioblastoma or Glioblastoma multiforme
includes surgical resection followed by
(GBM) is the most lethal and clinically
radiation and chemotherapy. The addition of a
challenging of brain tumors. Most patients die
chemotherapeutic agent, Temozolamide in
of their ailment in less than a year (Stupp et al.,
recent years changed the median survival of
2005). Some of the reasons for high fatality
for GBM patients to 14.6 months from 12.1
are the complex nature and diffuse character of
months with surgery and radiotherapy (Stupp
the tumor itself and the high rate of disease
et al., 2005). Also, currently there is no
Key words: Glioblastoma, Omics, Genomics, Transcriptomics, Epigenomics, Proteomics, Metabolomics.
*Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive
Magnuson Clinical Center Room B3-B100, Bethesda MD 20892, USA.
Email: [email protected]
Biomed Res J 2015;2(1):6-20
7
Maachani et al.
standard of care available for recurrent disease
instrumentation and bioinformatics data
and most of the patients die. Hence there is an
analysis have reshaped how we view the
urgent need to develop molecular targeted
cancer genome (Vucic et al., 2012). “Omics”
therapy for this devastating disease.
refers to the study of cancer as a whole entity
Some
responsible
of
the
for
molecular
molecules. It includes (but not limited to)
therapeutic resistance includes genetic and
DNA mutations, copy number changes,
epigenetic alterations, activation of stem cell
epigenetic changes like DNA methylation,
pathways,
tumor
transcriptome analysis and whole-genome
microenvironment and cellular metabolism.
DNA/RNA sequencing. The omics-based
However, the functional consequences of
recent
many of these alterations are largely unknown
transcriptomics, epigenomics, proteomics and
in GBM tumorigenesis (Frattini et al., 2013;
metabolomics have unveiled the molecular
Schonberg et al., 2013).
mechanisms behind various cancers and
changes
progression
focusing on the various micro- and macro-
and
and
GBM
alterations
in
the
approaches
including
genomics,
assisted in identification of next-generation
Omics
molecular markers for early diagnosis,
With the sequencing of the human genome, the
prognosis, predictive of response to treatments
study of biological systems underwent a major
and predisposition to gliomas (Cho, 2010;
genomic revolution. The major technological
Chin, 2013) (Fig. 1). The publically available
breakthroughs
multi-omics
in
high-throughput
assay
development, technological advancements in
databases
collected
by
International Cancer Genome Consortium
Figure 1: Omics in Glioblastoma.
Biomed Res J 2015;2(1):6-20
8
Advances in omics technologies in GBM
(ICGC) and The Cancer Genome Atlas
attempted to correlate mRNA signatures with
(http://cancergenome.nih.gov/)
network
the grades of gliomas and their clinical
group using a sample cohort of several
behavior to aid in overall prognosis and
hundred clinical specimens of GBM further
treatment response of patients (Mischel et al.,
elaborated the molecular processes funda-
2004; Kim et al., 2002). Transcriptomics is the
mental to GBM pathogenesis (Hudson et al.,
study of RNA transcripts that are produced by
2010; Verhaak et al., 2010).
the genome, under specific circumstances or
a
in a specific cell using high-throughput
Genomics/Transcriptomics
methods,
such
as
microarray
analysis,
Early work on gene expression analysis of
allowing the identification of genes that are
gliomas employed DNA microarrays and
differentially expressed in distinct cell
Table 1: Role of Omics in biomarkers identification and disease prognosis
GBM Biomarkers
Role in GBM prognosis
EGFR amplification
EGFR amplification is the most common event in primary GBM , with EGFRvIII being the most
EGFRvIII mutation
prominent mutated receptor tyrosine kinase receptor occurring in ~50% of GBM cases that
overexpress EGFR (Verhaak et al., 2010). A potential predictive biomarker for molecular
therapies.
PDGFRA
PDGFRA is mainly mutated and expressed in abnormally high amounts in proneural tumors
(Verhaak et al., 2010) and associated with poor prognosis in IDH1 mutant GBM (Brennan et al.,
2013).
TP53 mutation
TP53 gene although mutated, has no predictive or prognostic role. Can distinguish tumor grade
(Brennan et al., 2013).
1p/19q Co-deletion
1p/19q co-deletion is the most common genetic alteration in oligodendroglioma tumors and is
associated with favorable response to chemotherapy, radiation and survival (Alaminos et al.,
2005).
MGMT promoter
Promoter methylation of MGMT gene, inactivates DNA repair function (Esteller et al., 1999). It is
methylation
the first predictive epigenetic biomarker with a putative diagnostic role in detecting
pseudoprogression. MGMT methylation helps in molecular stratification of patients for
Temozolomide therapy (Malstrom et al., 2012).
VEGF
VEGF is considered to be the driving factor of tumor angiogenesis and has been identified in
64.1% GBMs. It is a strong predictor of survival , in patients with gliomas (Reynes et al., 2011)
PTEN
A gene level biomarker with poor survival outcomes for GBM (Baeza et al., 2003). PTEN is deleted
in 50–70% of primary and 54%–63% of secondary GBM. Also mutated in 14%–47% primary GBM.
Mutation is linked to resistance to targeted EGFR inhibitors in GBM (Deberardinis et al., 2008).
IDH1/2 mutation
IDH1 mutation is now recognized as an important driver in the etiology of low-grade and
secondary brain tumors (48). Has prognostic value in WHO grade III and IV GBM. Accumulation of
oncometabolite 2-hydroxygluatrate (2HG) considered as metabolomic imaging biomarker for
mutant IDH1 gliomas (Chen et al., 2014).
Biomed Res J 2015;2(1):6-20
9
Maachani et al.
Table 2: Molecular targeted therapies for glioblastoma
Other current strategies tested in GBMs
Pathways targeted
Agents
Molecular targets
Erlotinib (Roche)
Kinase inhibitors of
Epidermal Growth Factor Pathway
EGFR is amplified and frequently mutated in ~50% of GBMs and is
overexpressed in many malignant gliomas. Therefore could be used
Gefitinib (AstraZeneca)
EGFR
as a therapeutic targeted agent in GBM patients.
VEGF Pathway
Targeting vascular endothelial growth factor (VEGF) pathways to
Bevacizumab (Avastin;
Recombinant human
induce anti-angiogenic effects in the treatment of malignant gliomas
Genentech)
neutralizing monoclonal
has been in focus for past few years.
antibody to VEGF
Vatalanib (Novartis)
Kinase inhibitor of
VEGFR/PDGFR
Cediranib (AstraZeneca)
pan-VEGFR inhibitor
TGF-β is a multifunctional cytokine, which regulates gliomacell
Trabedersen (Antisense
Anti-sense TGF-β2
motility, invasion, and immune surveillance. Several small molecule
Pharma)
mRNA
Rapamycin (Sirolimus)
inhibitors of m-TOR
Transforming Growth Factor β(TGF-β) Pathway
inhibitors of TGF-β receptors have shown antitumor efficacy in
preclinical models of gliomas.
PI3K–AKT–mTOR Pathways
PI3K pathways regulate several malignant phenotypes including
antiapoptosis, cell growth, proliferation, and invasion. Activated PI3K
Temsirolimus (Sirolimus)
phosphorylates several downstream effectors, including AKT. mTOR
is a major player connecting multiple pathways downstream from
Everolimus (Novartis)
AKT.
PKC Pathways
Protein kinase C (PKC) is a serine/threonine kinase that regulates
enzastaurin (Eli-Lilly)
PKC-β inhibitor with
cell proliferation, invasion, and angiogenesis.
activity against glycogen
synthase kinase 3β
Note: Several of the above agents are being evaluated in clinical trials as monotherapies or in combination with other
treatment modalities such as chemotherapy or radiation in patients with malignant gliomas.
populations. Recent multi-omics (genomics,
(Verhaak et al., 2010). These subtypes were
transcriptomics
data
defined on the basis of distinct gene signatures
integration studies have utilized patient
and also characterized by different molecular
derived samples and cell lines to reveal
alterations and activated pathways (Verhaak et
heterogeneity among the primary GBM,
al., 2010; Brennan et al., 2013). The proneural
suggesting additional molecular subclasses:
subtype
neural, proneural, classical and mesenchymal
abnormalities in platelet-derived growth
and
proteomics)
was
mostly
characterized
by
Biomed Res J 2015;2(1):6-20
10
Advances in omics technologies in GBM
factor receptor-α (PDGFRA) or in isocitrate
methylation
dehydrogenase 1 (IDH1); whereas mutation of
promoter-associated
the epidermal growth factor receptor (EGFR)
specific loci accompanying tumor suppression
was found in the classical subgroup, and
in GBM (Sturm et al., 2014), such as
mutations in neurofibromin 1 (NF1) were
(CDKN2A), RB1, PTEN, TP53 (Costello et
common in mesenchymal tumors. The neural
al., 1996; Nakamura et al., 2001; Baeza et al.,
subtype seemed to be similar to the classical
2003; Amatya et al., 2005) and other
subtype but with a higher frequency of TP53
previously unrecognized regulatory genes
mutations (Brennan et al., 2013). Cytogenetic
EMP3, PDGFB (Alaminos et al., 2005; Bruna
and molecular studies have also identified a
et al., 2007). Most significantly, O-6-methyl-
number
chromosomal
guanine-DNA methyltransferase (MGMT)
abnormalities and genetic alterations in
promoter hypermethylation was identified
malignant
novel
occurring in ~45% of adult patients with GBM
candidates, particularly in GBMs. The
(Esteller et al., 1999; Brennan et al., 2013).
identification of molecular subtypes has
MGMT hypermethylation leads to gene
revealed a set of core signaling pathways
silencing, and reduced gene expression levels
commonly activated in GBM (Table1)
which compromises its ability to repair
(Furnari et al., 2007) and could be used in
damaged DNA by alkylating agents like
molecular targeted therapies (Table 2).
Temozolomide (Felsberg et al., 2011). Thus,
of
recurrent
gliomas,
as
well
as
have
identified
frequent
hypermethylation
of
gene methylation could be used as a biomarker
to predict sensitivity to chemo- radiotherapy
Epigenomics
enzymatic
(Wick et al., 2012; Malmström et al., 2012).
modifications of DNA and associated histone
Further, based on DNA methylation patterns,
proteins to regulate gene expression. In recent
proneural subtype is classified into CpG island
years these changes have been recognized as
methylator phenotype (CIMP) positive and
important causes of phenotypic changes in
CpG–CIMP-negative GBM subsets which
human
The
strongly correlates with IDH1 gene mutation
epigenetic changes are dynamic in nature and
status (Noushmehr et al., 2010; Turcan et al.,
play an important role in gene expression and
2012). Glioma CIMP (G-CIMP) is a powerful
DNA structure.
determinant
Epigenetic
changes
cancers
involve
(Esteller,
2007).
Epigenetic alterations,
of
tumor
aggressiveness
especially those related to changes in histone
(Riemenschneider et al., 2010; Brennan et al.,
acetylation, are a recent focus for therapeutic
2013). These epigenomic and other multi-
drug targeting in clinical trials. Genomic-array
omic
(microarray)
mutations,
techniques
Biomed Res J 2015;2(1):6-20
studying
DNA
analyses
have
altered
revealed
proteins,
several
miRNA
11
Maachani et al.
expressions and pathways associated with
efficient biomarker validation, treatment
GBM pathogenesis and prognosis.
monitoring and can be translated into clinical
applications in an affordable manner. Various
Proteomics
plasma/serum
Proteomic profiling represents the large-scale
identified earlier for GBM including YKL-40,
examination of protein expression, post-
GFAP
translational modification, and understanding
(Jayaram et al., 2014). Reynes et al. (2011)
how different proteins interact with each other.
reported inflammatory markers (C-reactive
Using various bioinformatics techniques, the
protein, IL-6 and TNF-) and angiogenesis
information can be unified into protein
markers such as VEGF and soluble VEGF
networks.
histopathology
receptor 1 to be significantly elevated in the
represents the gold standard for the typing and
plasma of GBM patients. Jung et al. (2007)
grading of gliomas and depends largely on
identified GFAP as a discriminatory serum
certain architectural similarities of tumor cells
biomarker
with normal glial cells (Tohma et al., 1998;
osteopontin (OPN), validated using IHC and
Riemenschneider et al., 2010). We feel that the
ELISA in GBM patients, was shown to
underlying disease pathology would result
correlate
into differential proteomic profiling of
(Sreekanthreddy et al., 2010). In an extended
diseased tissue and the surrounding disease-
effort, the TCGA group also generated protein
free normal tissue. Recent technological
expression data from 214 GBM patient
advances in proteomics has allowed analysis
samples using a high throughput antibody-
of glioma patient biopsies, proximal fluids,
based reverse phase protein arrays (RPPAs)
cerebrospinal fluid (CSF) and cyst fluid,
(Brennan et al., 2013) revealing several
plasma, glioma cell lines. This has allowed a
mutations, altered genes, proteins and their
comprehensive proteomic profiling of glioma
pathways underlying GBM pathophysiology
biology to aid the traditional histopathology in
(Dong et al., 2010).
Currently,
biomarkers
and
matrix
for
been
metalloproteinase-9
GBM.
with
have
Similarly,
poor
serum
prognosis
improving our understanding of glioma
Some of the challenges in using protein
processes and to better evaluation of drug
profiling more commonly in characterizing
responses to treatment (Somasundaram et al.,
and quantifying accepted protein biomarkers
2009). The techniques involve evaluation of
includes high costs, lengthy production times
protein arrays, including antibody and aptamer
and most importantly lack of high specificity
arrays. This allows simultaneous detection of
antibodies.
multiple
approach has the potential to identify novel
proteins/phosphoproteins.
These
high throughput techniques can be used for
diagnostic,
Moreover,
prognostic,
the
and
proteomic
therapeutic
Biomed Res J 2015;2(1):6-20
12
Advances in omics technologies in GBM
biomarkers
for
human
gliomas.
The
Spectroscopy (MRS), Nuclear Magnetic
application of proteomics in neuro-oncology
Resonance (NMR), have helped in profiling
is still in its developing stage. Please refer
global metabolomic signatures in cancers
recent reviews by Whittle et al. (2007) and
including glioma (Dunn et al., 2005; Serkova
Niclou et al. (2010) for more on the current
and Niemann, 2006). Several key differences
status of glioma proteomics and its clinical
in metabolite profiles have been identified in
applications.
GBM cancer cells when compared to normal
controls, providing a novel insight into GBM
Metabolomics
tumorigenesis (Spratlin et al., 2009). As
Nearly a century ago, Otto Warburg made a
metabolomics reflect underlying altered
seminal observation that even in the presence
genotype-phenotype, it can be used as a
of adequate oxygen cancer cells metabolize
predictive biomarker for measure of efficacy
glucose by aerobic glycolysis, termed as
and as a pharmacodynamic marker, for both
Warburg effect (Warburg et al., 1924; 1927).
traditional
Moreover,
disease-related
agents. Using the 1H-NMR spectra and neural
altered cellular metabolism has come into
networks, human glioma cell cultures can be
forefront of cancer research. Now, there is
separated into drug-resistant and drug-
increasing evidence that the underlying
sensitive groups before treatment with
genetic alterations contributing to glioma
nitrosourea treatment (El-Deredy et al., 1997).
pathogenesis is also responsible for altered
Frequent genetic alterations in glioma such as
cellular metabolism (Parsons et al., 2008).
MYC amplification, PTEN deletion or protein
Metabolomics refers to the global quantitative
loss and EFGR amplification are associated
assessment of endogenous enzyme kinetics,
with multiple downstream metabolic targets
cellular biochemical reactions, and synthesis
(Deberardinis et al., 2008). IDH1 and IDH2
of cellular metabolites within a biologic
metabolic genes are mutated in ~12% of
system, (Griffin and Shockcor, 2004; Boros et
primary gliomas, 86% of grade II and III
al., 2005). Although considerable progress has
gliomas and secondary glioblastoma through a
been made in understanding GBM biology
gain-of-function mutation that alters the
through genetic analysis, little is known about
enzymatic activity of the protein product,
the underlying metabolic alterations in glioma.
which results in the production of 2-
In recent years, several biochemical and
hydroxyglutarate (Dang et al., 2009). The
biophysical
very
Spectrometry
recently
techniques
(MS),
chromatography,
such
liquid-
Magnetic
Biomed Res J 2015;2(1):6-20
chemotherapy
and
hormonal
as
Mass
detection of 2-HG metabolic product has been
and
gel-
proposed to be a potential tool for in vivo
Resonance
distinction of secondary from primary
13
Maachani et al.
glioblastomas (Esmaeili et al., 2013). More
Srinivasan et al., 2011). More recently several
recently Chen et al. (2014) showed that
lines of evidence have implicated over-
IDH1-mutant glioma growth is facilitated by
expression of miR21 with chemo and
overexpression of glutamate dehydrogenase 2
radioresistance of GBM cells. Its expression
gene (GLUD2) and it could be targeted for
levels have been associated with glioma grade
growth
Hence,
and as a candidate independent marker for
metabolomics applications in a clinical
overall survival (Chao et al., 2013; Wu et al.,
perspective may have a favorable impact on
2013). Thus, integrative omics analysis has
glioma grade, metabolic state and treatment
revealed the importance and scope of
stratification of glioma patients.
translational
inhibitory
effects.
repression
in
microRNA-
mediated GBM pathogenesis. Please refer to
Other Omics: microRNAs
additional reviews (Sana et al., 2011; Karsy et
In recent years, microRNAs (miRNAs) have
al., 2012; Nikaki et al., 2012) for more
emerged in the forefront of cancer molecular
detailed coverage on miRNA expression and
biology.
function in GBM.
MicroRNAs
are
key
post-
transcriptional regulators that inhibit gene
expression by promoting mRNA decay or
Omics data integration methods
suppressing translation (Iorio and Croce,
The post-human genome project era has
2012). Experimental and clinical evidence
generated enormous heterogeneous and large
supports that miRNAs play pivotal role in
data sets. As vast gene profiling datasets and
cancer
proliferation,
technologies are being developed, they have
apoptosis and metastasis (Cho, 2011; Iorio and
created an unprecedented need to develop
Croce, 2012). The functional role of miRNAs
technologies to process the data in a
was first discovered in human gliomas (Li et
meaningful way. The efforts have yielded
al., 2013). Several miRNA expressions are
meaningful results in cancer biomarker
found to be dysregulated in GBM. TCGA
discovery, protein interactions and genotype
group identified alterations in 149 miRNAs
to phenotype correlations (Park et al., 2005).
(Dong et al., 2010) and an expression
However, current omics technologies cannot
signature comprising 10 miRNAs with
model
prognostic prediction (Srinivasan et al., 2011).
molecules by analyzing individual genes,
miR-128, miR-342 and miR-21 are known to
proteins or metabolites. This is often not very
play both oncogenic and tumor suppressive
effective
roles and are being explored as possible
heterogeneous nature of human cancers.
markers for GBM (Dong et al., 2010;
Cancer is a complex biological system and
gene
regulation,
interactions
due
to
between
the
multiple
complex
and
Biomed Res J 2015;2(1):6-20
14
Advances in omics technologies in GBM
requires a better understanding of the disease's
network and analyze experimental data in the
complexity at systems-level (Faratian et al.,
context of pathways using multiple source
2009; Hu et al., 2013). Pathway and network
omics data (Wang et al., 2012; Blazier and
based methods have taken more important role
Papin, 2012; Federici et al., 2013). Although
in analysis of high-throughput data, that can
currently there are tools available to process
provide a global and systematical way to
large datasets generated by one platform, it is
explore the relationships between biomarkers
expected that soon tools combining data
and their interacting partners (Wang et al.,
across multiple platforms will be available to
2015). Integration of data from multiple omic
researchers. This will help in integrating
studies can not only help unravel the
research results into a framework of whole
underlying
biological systems to support translation of
molecular
mechanism
of
carcinogenesis but also identify the signature
research into clinical applications.
of signaling pathway/networks characteristic
for specific cancer types that can be used for
Omics Advantages in GBM therapy
diagnosis, prognosis and designing tumor
So far clinical translation of an effective GBM
targeted therapy.
therapy has been hindered by multiple factors,
Most recently, attempts at integration of
including diffuse infiltration at the time of
multiple high-throughput omics data have
diagnosis, significant cellular heterogeneity
concentrated on comparing data acquired
(both
using
conditions/
difficulty in crossing the blood-brain barrier
platforms to explore functional and regulatory
by effective drugs, and the role of tumor
associations between genes and proteins
progenitor cells in reestablishment of resistant
(Faith et al., 2007; McDermott et al., 2009).
disease following chemo and radiotherapy.
This has culminated into combining functional
Current standard treatment of GBM consists
characterization and quantitative interactions
of attempted gross total surgical resection
extracted from various biomolecules such as
followed by concurrent temozolomide and
DNA, mRNA, proteins and metabolites (Chen
radiation therapy (RT) (Clarke et al., 2010).
et al., 2011; Coban and Barton, 2012; Mitchell
Although, RT provides good local control, it is
et al., 2013) (Fig. 1). Some analysis utilizes
not very beneficial in controlling the disease
pathways in the form of connected routes
recurrence. In case of GBM, majority of
through a graph-based representation of the
patients die from recurrent disease, as
metabolic network (Blum and Kohlbacher,
currently there is no effective therapy for
2008). Other approaches focus on the
recurrent GBM. Therefore, the addition of
functional module of protein interaction
systemic chemotherapy to RT can help in
various
experimental
Biomed Res J 2015;2(1):6-20
intratumoral
and
intertumoral),
15
Maachani et al.
controlling
recurrence
and
offering
an
methyl group to the middle guanine in a GGG
additional radiosensitization benefits in GBM,
sequence to convert it to O6-methylguanine.
benefiting both definitive and palliative
Temozolomideexerts
strategies for disease management (Stupp R et
activity by interfering with repair of damaged
al., 2006; Clarke et al., 2010). So far non-
DNA after radiation treatment (Mrugala and
omics studies have identified few GBM targets
Chamberlain, 2008). In a recent randomized
at the protein level, but fail to see an overall
trial,
role of molecules in signaling pathways,
Temozolomide chemotherapy with radiation,
protein-protein interactions, and role in
significantly
metabolic processes. Unfortunately so far only
survival from 12.1 months to 14.6 months, for
one drug has been identified (Temozolomide)
GBM patients (Stupp et al., 2005, Clarke et al.,
which can radiosensitize GBM patients. Thus,
2010). The consequent analysis of these
non-omics techniques will compliment whole
patients by Hegi et al. (2005) reported that
genomic/epigenomics/metabolomics approach
patients with methylated MGMT gene
of omics technologies. Without publically
promoter were benefited from this treatment
available databases, the surge of preclinical
compared to patients with unmethylated
and clinical information seen in the GBM field
MGMT promoter. The MGMT promoter
over last few years, would have not been
methylation silences the gene function
possible. As omics studies expand our
required
understanding of the molecular pathways
methylation and therefore cannot counteract
driving GBM tumorigenesis, more druggable
the action of Temozolomide. Thus, omics has
targets will be identified to treat GBM patients.
been helpful in predicting tumor response to
Also, understanding of ionizing radiation at
Temozolomide and to guide clinical decision
the level of molecular biology will lead to
making. The other most common types of
development and production of targeted
chemotherapies for GBM under investigation
radiosensitizers. Temozolomide is currently
include
the only radiosensitizing agent used for GBM
antiangiogenic therapies, immunotherapies,
with class I evidence of benefit (Mrugala and
gene
Chamberlain, 2008). It is a novel oral
therapies and drugs to overcome resistance
bioavailable
(Table 2).
second-generation
alkylating
its
concomitant
to
and
improved
reverse
targeted
therapies,
antineoplastic
adjuvant
progression
the
molecular
free
O6-guanine
therapies,
radiation-enhancement
agent. At physiologic pH it undergoes
hydrolysis to its active form methyltraizeno-
Challenges and Prospective
imidazoleoarboxamid
The
Oncogenic transformation is a complex,
mechanism of action of MTIC, is to transfer a
multistep process that differs widely between
(MTIC).
Biomed Res J 2015;2(1):6-20
16
Advances in omics technologies in GBM
and even within cancer types. Advances in the
therapeutic agents for GBM. The advances
large scale omics technologies have led to
with respect togene expression profiling,
identification of promising GBM disease
signaling pathway characterization, glioma
biomarkers. The major challenge is how to
stem cell identification, regulatory RNA
bring omics research into accurate and reliable
studies,
clinical use. In case of GBM, omics
immunomodulation approaches, have resulted
technologies have their limitations due to late
in several ongoing clinical studies evaluating
diagnosis of disease, intrinsic molecular
new therapeutic agents for GBM (Table 2). It is
complexity and genetic heterogeneity of
evident that omics based cancer research is
GBMs. To find consistencies that can be
going to play a pivotal role in diagnosis,
therapeutically targeted on the basis of
treatment and monitoring of GBM patients.
metabolomic
changes
and
molecular analysis, poses a major problem.
However, we are optimistic that the wealth of
CONFLICT OF INTEREST
information generated by omics techniques
The authors claim no conflict of interest.
has paved a roadmap for designing new
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Review
Dendrimers based Electrochemical Biosensors
Saumya Nigam1, Sudeshna Chandra2, Dhirendra Bahadur1*
1
Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai,
Mumbai, India
2
Department of Chemical Sciences, School of Science, NMIMS (Deemed-to-be) University, Vile Parle (W), Mumbai,
India
Electrochemical biosensors are portable devices that permit rapid detection and monitoring of biological,
chemical and toxic substances. In the electrochemical biosensors, the bioreceptor is incorporated into the
transducer surface; and when in contact with the analyte, generates measurable signals proportional to the
analyte concentration. Materials with high surface area, high reactivity, and easy dispersability, are most
suited for use in biosensors. Dendrimers are nanomaterial gaining importance for fabrication of
electrochemical biosensors. These are synthetic macromolecules with regularly branched tree-like and
globular structure. The potential applications of dendrimers as biosensors are explored due to their
geometric symmetrical structure, chemical stability, controlled shape and size, and varied surface
functionalities, with adequate functional groups for chemical fixation. The current review provides multifaceted use of dendrimers for developing effective, rapid, and versatile electrochemical sensors for
biomolecules. The redox centers in the dendrimers play an important role in the electron transfer process
during immobilization of biomolecules on the electrodes. This has led to an intensive use of dendrimer
based materials for fabrication of electrochemical sensors with improved analytical parameters. The review
emphasizes development of new methods and applications of electrochemical biosensors based on novel
nanomaterials.
INTRODUCTION
Dendrimers
are
globular
repetitive and radial branching units and the
macromolecules with well-defined, mono-
terminal functional groups. To achieve a high
disperse,
spatial
degree of precision and structural order,
conformations, and a wide spectrum of
dendrimers are synthesized in a stepwise
chemical and physical properties (Tomalia et
fashion. The number of repeat branching
al., 1985). These characteristics indicate
molecules used during the synthesis refers to
significant differences from the classical
the generation of dendrimers, which also
polymeric molecules. Structurally, these
governs the shape and size of the dendrimers.
macromolecules are divided into three
Generally, two different methods namely,
architectural
divergent and convergent, are adopted for the
three
hyperbranched
dimensional
regions:
the
central
core,
Key words: Dendrimers, Biosensors, Polyamidoamine, Polypropylene imine, Bioreceptors, DNA sensor.
*Corresponding Author: Dhirendra Bahadur, Department of Metallurgical Engineering and Material Science,
Indian Institute of Technology Bombay, Powai, Mumbai, India.
Email: [email protected]
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
22
synthesis of dendrimers, and classified into
geometric progression with every generation
different “generations”. It is the hyper-
followed by increase in molecular weight.
branching of the molecule from the centre of
This compromises the reaction kinetics,
the dendrimer towards the periphery that
making it slower and synthesis of high
results in homostructural layers between the
generation dendrimers becomes difficult,
focal points (branching points). The number of
further lowering the yield of desired product.
focal points from the core towards the outer
Addition of each branching unit requires care
surface is the generation number. Thus,
and precision to prevent structural defects and
generation refers to the number of repeated
asymmetry in the dendrimer structure.
branching cycles performed during the
Secondly, the separation of desired dendrimer
synthesis. The core part of the dendrimer is
from the by-products is hindered due to
denoted generation “zero” (G0). For example
molecular similarity exhibited by the by-
if a dendrimer is made by convergent
product as well as the desired dendrimer. On
synthesis, and the branching reactions are
the other hand, convergent method employs
performed onto the core molecule three times,
synthesis of small dendrites from the exterior
the resulting dendrimer is considered a third
and the reaction proceeds inwards to the
generation
central core. The convergent procedure results
dendrimer.
Each
successive
generation results in a dendrimer roughly
in
lesser
twice the molecular weight of the previous
purification of dendrimers resulting in high
generation.
degree
of
structural
defects
monodispersity.
and
easy
Despite
the
The two synthetic methods have inherent
possibility of purer and flawless dendrimers,
advantages and disadvantages. Using the
the convergent method falls short in synthesis
divergent synthesis method, the dendritic
of higher generation dendrimers. This choice
molecule is formed from a central core which
is limited due to the steric forces crowding the
then extends radially outwards through
dendrites around the central core molecule.
addition of branching molecules. The main
Despite the difficulties, these macro-
advantage of the divergent method is that high
molecules have gained interest over classical
molecular
is
polymers due to the varied options presented
attained with desired repetitive branching
by dendritic macromolecules. The freedom of
monomers. Thus, the dendrimer can be tailor
choice of central core, branching monomeric
made to achieve maximum functionalities and
units and surface functional groups from the
properties. However, two major challenges are
vast pool of molecules gives rise to a
encountered in divergent synthesis. First, the
multivalent system. Ethylenediamine, 1,4-
number of reaction points increase in
diaminobutane,
nanoscaffold
Biomed Res J 2015;2(1):21-36
architecture
1,12-diaminododecane,
23
Nigam et al.
cystamine, 1,6-diaminohexane and ammonia
sensor response to be used in biosensing
are the most common core molecules. The
applications.
Various
varied core and branching monomers affect
ferrocene,
polystyrene,
the internal chemical environment, three
carbohydrates, etc. have been explored for
dimensional structures and size of internal
surface modification (Ashton et al., 1997;
cavities in the dendrimer. Due to the different
Chen et al., 2014; Hung et al., 2013; Yoon et
structural and chemical properties, these user-
al., 2000). The conductivity of the moieties
customized dendrimers find applications in
plays an important role in enhancing response
the fields of drug delivery, gene delivery,
of the dendritic scaffold in sensing various
antimicrobials, magnetic resonance imaging,
biomolecules. The most common modifying
immunosensing and biosensing.
molecule is ferrocene which exhibits multi-
molecules
like
polyaniline,
Methyl acrylate alternating with ethylene
electron transfer in various redox interactions.
diamine forms the most widely synthesized,
Ferrocene has been exploited as central core,
studied and used class of polyamidoamine
branching monomer as well as for surface
(PAMAM) dendrimers (Esfand et al., 2002),
groups in various dendritic systems (Mehmet
with the internal amide groups providing an
et al., 2013; Villalonga-Barber et al., 2013).
abundance of lone pairs of electrons. Another
They
popular class of amine terminated dendrimers
moieties undergoing redox processes without
is
(PPI)
decomposition while maintaining the desired
synthesized by Michael's addition of primary
electrochemical reversibility (Sun et al.,
amines
2014).
the
poly
to
(propylene
acrylonitrile
imine)
followed
by
subsequent hydrogenation by Raney cobalt or
behave
The
as
non-interacting
molecular
redox
recognition
of
Raney nickel catalyst (de Brabander-van den
biomolecules by dendrimers is primarily
Berg et al., 2003). The interiors of PPI
governed
dendrimers are the tertiary nitrogen atoms with
conformation in higher generations. The
lone pairs of electron contributing to their
branches of lower generation dendrimers tend
reactive cavities. Both the classes of
to radiate out towards the periphery and exist
dendrimers have primary amine groups on the
in open conformation. On the other hand, as
surfaces governing the surface properties,
the number of generation is increased, the
reactivity and surface charge. Thus, any kind
branches tend to retract and adopt globular
of detection response observed in these
conformations in a three dimensional space
dendrimers is attributed to the amine groups.
with
The surface of dendrimers is further modified
governing the structures. The generation
to enhance the reactivity/interaction and
dependent conformational changes confirmed
by
the
intramolecular
three
dimensional
hydrogen
bonding
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
24
by X-ray analysis, demonstrated that the
applicable
in
several
biomedical
and
higher generations are more spherical as
environmental analyses.
compared to lower linear generations (Percec
a) Peroxide sensor: Copolymers of pyrrole-
et al., 1998). The globular conformations
PAMAM
closely resemble morphology of globular
electrochemical sensing of hydrogen
proteins and are useful in several biosensing
peroxide.
applications associated with the biomimetic
pyrrole-PAMAM with branched amine
macromolecular architecture. A vast variety of
periphery and focal pyrrole functionality
biomolecular species have been detected using
are synthesized by divergent method. The
dendrimer scaffolds. In the following sections,
conjugate is covalently attached to the
details of the various sensors using different
electrode
types of dendrimers are discussed.
peroxidase (HRP) immobilized on it to
dendrimer
Different
surface
are
used
generations
and
for
of
horseradish
form conducting films for H2O2 sensing.
Dendrimers in electrochemical biosensing
The steady state amperometric response is
By definition, a biosensor is an analytical
measured
device that makes use of bioreceptor molecule
concentration at +0.35V vs. Ag/AgCl, and
immobilized onto a transducer (recognition)
demonstrated that the dendritic wedge
surface and produces measurable signals in the
played
presence of an analyte, due to the bio-
immobilization of the HRP enzyme
recognition
the
(Mehmet et al., 2012). Yang et al. (2014)
concentration of the analyte. Biosensors are
described a magnetic electrochemical
classified based on either the bioreceptor or
sensor comprising Fe3O4 nanoparticles
transduction method or both. Common
with graphene oxide (GO) and subsequent
bioreceptors include enzymes, antibodies and
modification by PAMAM dendrimers.
DNA, while transducers include electro-
The
chemical, piezoelectrical, optical techniques.
modification of the gold electrode acting
The transducer techniques using electro-
as the working electrode and used for the
chemical biosensors have an edge over other
detection of H2O2 in phosphate buffer
methods due to excellent selectivity and
solution by the method of amperometric
sensitivity, and precise detection of the desired
i-t curve. The cyclic voltammograms of
species. These are relatively cheaper, faster
Fe3O4/GO
and more user friendly as compared to other
showed an increase in current while
techniques. The exceptional features render
displaying steady redox peaks which
the electrochemical biosensors increasingly
confirmed occurrence of a catalytic
event
proportional
Biomed Res J 2015;2(1):21-36
to
as
an
a
function
important
platform
and
was
of
role
employed
H2O2
for
for
Fe3O4/GO–PAMAM
25
Nigam et al.
reaction on the electrode interface. H2O2
was detected in a linear calibration range
-5
(Chandra et al., 2009).
b) Glucose Sensor: A dendritic wedge based
-3
of 2.0 × 10 –1.0 × 10 M with a correlation
on pyrrole-PAMAM dendrimer was used
coefficient of 0.9950 and detection limit of
to immobilize glucose oxidase (GOx) for
-6
2.0 × 10 M. The sensor platform also
the construction of an amperometric
displayed excellent recovery ratios of
glucose sensor (Mehmet and Cevdet,
96.9–108.1% H2O2 added to milk and juice
2012). Nanobiocomposite based glucose
samples. Another amperometric electro-
biosensor was prepared by electro-
chemical
was
polymerization of pyrrole containing
developed by modifying gold bead
PAMAM encapsulated Pt nanoparticles
electrodes
PAMAM
(Pt-PAMAM), and GOx. The developed
dendrimers of different generations of 2, 3
sensor had a sensitivity of 164 µA mM-1
and 4, followed by absorption of Prussian
cm-1 and a detection limit of 10 nM within
blue
bonded
a wide working range from 0.2−600 µM.
dendrimer/PB modified electrodes offered
Pyrrole provided electrical conductivity,
enhanced sensitivity and lower detection
stability and homogeneity to the thin film,
limits (Bustos et al., 2006). Metallic
while PAMAM provided a favorable
(Rhodium) nanoparticles stabilized with
microenvironment to maintain bioactivity
N, N-bis-succinamide-based dendrimer
of GOx (Tang et al., 2007). Yoon and
were immobilized on glassy carbon
colleagues used varying degrees of redox-
electrode (GCE) and electrocatalytic
active ferrocenyl in combination with
activity
PAMAM
biosensor
(PB).
reduction
with
The
towards
for
H2O2
starburst
covalently
hydrogen
investigated
using
peroxide
dendrimers
(Fc-D)
as
cyclic
recognition unit for fabrication of a
voltammetry and chronoamperometry.
glucose sensor (Yoon et al., 2000). GOx
The dendrimer stabilized nanoparticles
was deposited layer-by-layer on Au-
showed excellent electrocatalytic activity
surface to form an enzymatically active
for H2O2 reduction reactions and a steady-
GOx/Fc-D multilayered assembly. The
state cathodic current response was
bio-electrocatalytic signals from the
observed at −0.3 V (vs SCE) in phosphate
multilayer were directly correlated to the
buffer (pH 7.0). The electrochemical
number of layers deposited, confirming
sensor displayed a linear response to H2O2
the tunable sensitivity of the electrode and
concentrations ranging from 8 to 30 μM
hence a potential microbiosensor. Cyclic
with a detection limit and sensitivity of 5
voltammetry
μM and 0.031 × 10−6 A μM−1, respectively
resonance (SPR) was used to investigate
and
surface
plasmon
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
26
the
redox-orientation
changes
of
ferrocene-tethered dendrimers and GOx.
SPR monitors change in the refractive
index of the medium next to the Au
sensing surface and are used to monitor
immobilization of GOx onto the Au
surface (Frasconi et al., 2009). Redoxactive
dendrimer
fabricated
using
different generations of poly (propylene
imine) core with peripheral octamethyl
ferrocenyl units (Fig. 1) and deposited on
Pt electrodes for immobilizing GOx has
been used for detection of glucose
(Armada et al., 2006). The amperometric
response of all the dendritic mediators
towards glucose was determined at several
Figure
1: Structures of varying generations of
octamethyl ferrocenyl dendrimers for use as electrode
material for determination of glucose (Armada et al.,
2006).
applied potentials. Glucose biosensor has
been developed based on bioactive
the surface of PGLD and distribution of
polyglycerol
PANINT's (Santos et al., 2010).
(PGLD)
and
chitosan
dendrimer (CHD). Both the dendrimers
Ferrocenyl dendrimer (PAMAM-Fc)
were conjugated with GOx to form
has
PGLD-GOx
amperometric glucose biosensor. Series of
entrapped
and
in
CHD-GOx
polyaniline
and
nanotubes
also
asymmetric
used
for
fabricating
PAMAM
an
dendrimers
(PANINT's) during template electro-
containing a single ferrocene unit located
chemical polymerization of aniline. The
in the focal point have been synthesized.
prepared
and
The transducer consisted of a gold
biosensors
electrode covalently modified with 3-
PGLD-GOx/PANINT's
CHD-GOx/PANINT's
exhibited strong amperometric response
mercaptopropionic
to glucose concentrations in ranges
dendrimers and GOx enzyme. The
observed
PAMAM-Fc/GOx
in
human
GOx/PANINT's
was
blood.
more
PGLD-
acid,
PAMAM-Fc
biosensor
showed
sensitive
excellent performance for recognizing
(10.41 nA.mM ) as compared to CHD-
glucose at +0.25V with a high sensitivity
-1
-1
GOx/PANINT's (7.04 nA.mM ), due to
(6.54μA/mM) and low response time
specific organization of the GOx layer at
(~3s) in the concentration range of 1–22
Biomed Res J 2015;2(1):21-36
27
Nigam et al.
mM (Mehmet et al., 2013).
c) DNA Sensor: Dendrimers were also
exploited for their possible use in
fabricating DNA sensors. An electrochemical
DNA
nanobiosensor
was
Figure 2: Proposed charge transfer scheme between
PBS, DNA and PPI-AuNP (Arotiba et al., 2008).
developed by immobilization of 20-mer
thiolated probe ssDNA on electro-
sequence, immobilized on a multinuclear
deposited
nickel
poly
(propyleneimine)
(II)
salicylaldimine
metallo-
dendrimer (PPI) of generation 4 (G4),
dendrimer on GCE has been reported
doped with gold nanoparticles (AuNP)
(Arotiba et al., 2007). The authors studied
(Arotiba et al., 2008). Cyclic voltammetry
electrochemical
showed that the designed platform
immobilization layer of the PPI derivative
(GCE/PPI-AuNP) exhibited reversible
by
electrochemical behavior in pH 7.2
methods. The metallo-dendrimer was
phosphate buffer saline (PBS) solution
electroactive with two reversible redox
due to PPI. The redox chemistry of PPI
centers and was a strong DNA adsorbant.
involves a two electron and one proton
The sensor responded to 10 µL of 5 nM
process and is pH-dependent. PPI-AuNP
target DNA with detection limit as low as
was able to amperometrically detect target
3.4 × 10-12 M. Gold electrode has been
DNA concentrations at 0.05 nM in PBS.
modified with 3-mercaptopropionic acid
Using
and
electrochemical
spectroscopy
(EIS),
the
impedance
biosensor
-12
characterization
impedimetric
reacted
and
with
on
amperometric
amino-terminated
PAMAM G-4 dendrimer to obtain a thin
-9
film (Li et al., 2009). Recognition layer of
M for target DNA. The probe immobiliza-
single-stranded 3´-biotin-avidin combina-
tion effectiveness is apparently attributed
tion was immobilized onto the thin film to
to the AuNP's ability to connect to the
detect the complimentary target. Cyclic
thiolatedssDNA on the GCE surface via
voltammetry (CV), differential pulse
Au-S linkages. Further, the electrostatic
voltammetry (DPV) and electrochemical
interaction between the cationic platform
impedance spectroscopy (EIS) has been
and the anionic DNA probe improved the
used
immobilization process. Proposed charge
hybridization of DNA. The dynamic
transfer scheme between the electrolyte,
detection range of the sequence-specific
DNA and PPI-AuNP is shown in Fig. 2.
DNA was 1.4 × 10-11–2.7×10-14 M with a
exhibited a dynamic linearity of 10 –10
A DNA biosensor with probe DNA
to
study
immobilization
and
detection limit of 1.4 × 10-14 M. Sahoo et
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
28
al. (2013), demonstrated a label free
generation
impedimetric DNA biosensor based on
dendrimer was covalently functionalized
third generation G3 PAMAM dendrimer
onto
functionalized GaN nanowires (NWs).
(MWNT)
The developed nanosystem provided large
transducer
docking sites to immobilize probe (p-)
confinement of probe DNA. Impedance
DNA covalently. The biosensor was
spectroscopy revealed occurrence of
ultrasensitive and showed detection limit
hybridization between surface confined
as low as attomolar (aM) concentration of
ssDNA probe with target DNA in solution
complementary
DNA.
to form double stranded DNA (dsDNA).
Impedance spectroscopy revealed an
The interfacial charge-transfer resistance
increase in the resistance polarization (Rp)
of the electrode towards the redox
indicating efficient charge transfer due to
electrolyte changed due to occurrence of
strong covalent binding on NWs surface.
hybridization. The large number of amino
Zhu et al. (2006) modified gold electrodes
groups of the dendrimer enhanced the
with sub-monolayers of mercaptoacetic
surface binding of the probe DNA which
acid (RSH) and reacted with G-4 PAMAM
in turn resulted in increase in the
dendrimers to obtain thin films of
sensitivity of the impedimetric biosensor
PAMAM/RSH. DNA probe was then
for the target DNA. The interfacial charge-
immobilized onto the thin films to afford
transfer resistance responded linearly to
stable recognition layers. DPV was used to
the logarithmic concentration of the target
monitor
with
DNA within a concentration range from
daunomycin (DNR) as indicator. The
0.5 to 500 pM with a detection limit of 0.1
PAMAM-modified Au electrodes without
pM (S/N = 3) (Zhu et al., 2010). Single-
ssDNA showed good electrochemical
use electrochemical DNA biosensor has
response in DNR solution, while on
been fabricated based on pencil graphite
attachment with ssDNA the modified
electrode modified with succinamic acid
electrode showed a decrease in the DPV
and
response of DNR. This is attributed to less
PS/GCE). Calf thymus double stranded
accessibility of DNR molecules to ssDNA
DNA (ctDNA) and DNA oligonucleotide
probe on the electrode surface. Besides
(DNA ODN) immobilized on surface of
high
low
G2-PS/GCE under optimum conditions,
generation dendrimers are also used to
showed a detection limit of 4.2 µg/mL
develop DNA biosensor. A second
(Congur et al., 2014). Besides dendrimers,
DNA
target
(t-)
hybridization
generation
Biomed Res J 2015;2(1):21-36
dendrimers,
PAMAM
multi-walled
G2
and
and
(G2-PAMAM)
carbon
used
as
tether
PAMAM
nanotube
electronic
for
dendrimer
surface
(G2-
29
Nigam et al.
Figure 3: SEM images of Den-Au electrodes by electrodeposition in 2.8 mM HAuCl4 and 0.1 M H2SO4 at different time
points (A) 20s, (B) 100s, (C) 300s and (D) 600s (Li et al. 2011).
dendritic nanostructures have been used as
based on amino-terminated PAMAM
electrode
biosensing
dendrimer. Thrombin aptamer probe was
applications. Li et al. (2011) described
immobilized onto activated dendrimer
dendrimer-gold (Den-Au) nanostructure
monolayer film and detection of thrombin
modified electrode by directly placing the
was investigated in the presence of the
electrode into 2.8 mM HAuCl4 and 0.1 M
reversible [Fe(CN)6]3−/4− redox couple
H2SO4 solution at -1.5 V. Scanning
using impedance technique. The results
electron microscopic images show growth
showed that the charge-transfer resistance
evolution of Den-Au at different time
(Rct) value had a linear relationship within
period (Fig. 3). The Den-Au modified
concentrations
electrode respond to 1 fM complimentary
thrombin, and detection limit (S/N = 3) of
target DNA within a wide detection range.
0.01
material
in
Aptamers, as single-stranded DNA or
nM
Impedimetric
range
(Zhang
of
et
1–50
al.,
aptasensor
nM
2009).
based
on
RNA sequences that bind to specific target
succinamic
molecules was determined by a label-free
dendrimer was developed for monitoring
highly sensitive impedimetric aptasensor
interaction between DNA aptamer (DNA-
acid-terminated
PAMAM
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
30
APT) and its cognate protein, human
and their films, PDAMS with shorter
activated protein C (APC), a key enzyme
branches form rougher films and exhibit
in the protein C pathway. The dendrimer
higher rate constants (Kobs) and sensitivity
modified aptasensor showed detection
and smaller Michaelis constants (K'M),
limits of 1.81 µg/mL in buffer solution and
than
0.02 µg/mL in diluted FBS (Erdem et al.,
electrocatalytic activity towards NADH
2014).
oxidation (Jiménez et al., 2014).
PMDUS
indicating
better
Hyperbranched
e) Other biomolecules: Tang et al. (2007)
carbosilane polymers, polydiallyl methyl
reported enzyme based amperometric
silane
biosensor for determination of glutamate.
d) Coenzyme
Sensor:
(PDAMS)
and
polymethyl
with
A self-assembly of glutamate dehydro-
for
genase (GLDH) and PAMAM dendrimer
stabilization of Pt nanoparticles and as
encapsulated Pt nanoparticles on carbon
electrode material for NADH oxidation.
nanotubes
The modified electrodes worked in wide
were used as electroactive material (Fig.
linear concentration ranges for NADH
4). The electrochemical activity was
with a detection limit of 4.78 µM for
reported to be attractive with large
PDAMS/PtNPs/Pt and 6.18 µM for
determination range of glutamate (2-250
PMDUS/PtNPs/Pt. With regard to the
µM), short response time (< 3 s), high
structure of the two carbosilane polymers
sensitivity (433 µA/mM-1 cm2) and
diundecenyl
ferrocene
silane
moieties
(PMDUS)
were
used
(GLDH/Pt-PAMAM)n/CNT)
Figure 4: Schematic showing the procedure of immobilizing Pt-PAMAM onto CNTs (a) layer-by-layer self-assembly of
GLDH and Pt-PAMAM onto CNTs (b) Pt-PAMAM/CNTs heterostructures were covalently attached via EDC (Tang et al.
2007).
Biomed Res J 2015;2(1):21-36
31
Nigam et al.
stability.
PDATT/Den
Pt-PAMAM
and
GLDH
were
(AuNPs)/laccase
probe
(Rahman et al., 2008). The modified
alternately deposited until suitable layers
electrode
were obtained. PAMAM G-4 dendrimers
transfer (DET) process of laccase and a
crosslinked with reduced graphene oxide
catechin biosensor was fabricated based
were tested for performance as electro-
on the electrocatalytic process of laccase.
chemical biosensors by immobilizing
The linear range and detection limit for
enzyme tyrosinase (Araque et al., 2013).
catechin sensing was 0.1–10 and 0.05 µM,
The
respectively.
bioelectrode
electrocatalytic
showed
An
direct
electron-
electrochemical
towards
biosensor based on PAMAM dendrimers
determination of catechol with a response
was developed for the detection of
time of about 6s, linear range of 10 nM to
fructose in food samples by immobilizing
22 µM, sensitivity of 424 mAM-1 and a low
fructose
detection limit of 6 nM (Fig. 5).
cysteamine and PAMAM dendrimers. The
PAMAM
behavior
excellent
displayed
dendrimer
dehydrogenase
(FDH)
on
encapsulated
concentration range of the enzymatic
AuNPs were first immobilized to a
biosensor was 0.25–5.0 mM fructose
conducting polymer with two amine
(Damar et al., 2011). PAMAM dendrimers
groups
(3',4'-diamine-2,2',5',2''-terthio-
were also used to enhance signal response
phene (PDATT) through covalent bonding
of a nanobiocomposite fabricated to
between –COOH group of PAMAM and
obtain an immunosensor for alpha-feto
–NH2 group of PDATT. Laccase was
protein (AFP) in human serum (Giannetto
subsequently covalently bonded to the
et al., 2011). The binding of the dendrimer
–COOH of PAMAM dendrimers to form
with biologically active molecules like
Figure 5: (A) Amperometric response obtained with Tyr/PAMAM-Sil-rGO/GCE for different catechol concentrations at
Eapp= -150 mV (B) FE-SEM image of Tyr/PAMAM-Sil-rGO (Araqueet al., 2013).
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
32
antibodies can improve the activity and
was in accordance with referenced
sensitivity of the system. Response range
enzyme-linked
and precision were evaluated using cyclic
(ELISA) method.
immunosorbent
assay
voltammetry (CV) and double step
A multi-analyte sensing device based
chronoamperometry (DSCA) with limit of
on PAMAM dendrimer for simultaneous
detection of 3 ng/mL and limit of
at-line monitoring of glucose, ethanol,
quantification of 15 ng/mL. The enhanced
pO2- and cell density was fabricated (Akin
immunosensor could be useful for
et al., 2011). The device consisted of a
monitoring prognosis of pregnancy and
dual biosensor, a modified microscope
occurrence
diseases.
and a fiber optical pO2-sensor integrated
Recently, a redox-active silver-PAMAM
into a flow analysis (FA) system. The
dendrimer nanostructure was synthesized
electrochemical transducer consisted of
in situ by using wet chemistry (Xiaomei et
self-assembly of cysteamine on gold
al., 2013), and functionalized with mono-
surface. Alcohol oxidase and pyranose
clonal mouse anti-human antibody for free
oxidase were immobilized onto the gold
prostate specific antigen (fPSA). Using,
surface by means of PAMAM (poly-
graphite as the working electrode, a layer
amidoamine)
of gold nanoparticles modified with
aldehyde cross-linking. The responses for
prostate-specific antibody (mAb2). In
glucose and ethanol were linear up to 0.5
presence of the fPSA, specific immuno-
mM. The biosensor was used for
complex was formed on the functionalized
simultaneous determination of ethanol
antibody modified electrode. The Ag-
and glucose in yeast fermentation process.
mediated PAMAM dendrimer directly
A highly stable and sensitive ampero-
catalyzed reduction of H2O2 in the
metric biosensor was developed by
detection solution. Thus, PSA was
immobilizing alcohol oxidase (AOX)
detected primarily due to the antigen-
through PAMAM dendrimers on a
antibody immunocoupling. Under optimal
cysteamine-modified
conditions, the developed immunoassay
surface for determination of ethanol (Akin
could determine target fPSA in the
et al., 2009). The optimized ethanol
dynamic range of 0.005–5.0 ng/mL with a
biosensor showed a linearity from
detection limit (LOD) of 1.0 pg/mL (S/N =
0.025–1.0 mM with 100 s response time
3). In addition, the accuracy of the
and detection limit (LOD) of 0.016 mM.
electrochemical immunoassay evaluated
The analytical characteristics of the
for detection of clinical serum specimens,
system were also evaluated for alcohol
of
neoplastic
Biomed Res J 2015;2(1):21-36
dendrimer
gold
via
glutar-
electrode
33
Nigam et al.
determination in flow injection analysis
films. The multilayer film assembled with
(FIA) mode for analysis of ethanol in
the dendrimer stabilized Au nanoparticles,
various alcoholic beverage as well as
provided a new approach to fabricate
offline monitoring of alcohol production
biosensors and bioreactors based on direct
through yeast cultivation (Yuksel et al.,
electrochemistry of proteins and enzymes.
2012).
PAMAM dendrimer (generation G4)
CONCLUSIONS
stabilized with 1-hexadecanethiol was
Contemporary studies indicate that the most
used for immobilization of acetylcholin
elementary chemical reaction of electron
esterase from electric eel, and choline
transfer is widely prevalent in several
oxidase from Alcaligenes sp. was used as
biological systems and more importantly in
electrode material for fabrication of an
nanosystems with redox dendrimers. This is
amperometric
pesticides
possible by tailoring the nature and topology
(Snejdarkova et al., 2004). On similar
of the dendrimers to precisely control location
lines, urea electrochemical biosensor was
of the redox sites within the macromolecule
developed based on an electro-co-
and study its electron-transfer processes. The
deposited
dendrimer
increase in efforts to combine dendrimers with
modified screen printed carbon electrode.
other molecules like pyrrole, ferrocene,
Urease enzyme was immobilized onto
enzymes, etc. is promising in biosensing
electrodes and an amperometric response
applications.
sensor
for
zirconia-PPI
in urea concentration from 0.01 mM to
2.99 mM was obtained with sensitivity of
-1
-2
3.89µA mM cm (Shukla et al., 2014).
ACKNOWLEDGEMENTS
The authors acknowledge Department of
PPI dendrimers have also been used to
Science and Technology, Government of
reduce HAuCl4 to form core-shell PPI-Au
India, New Delhi, for providing financial
nanoclusters with several PPI molecules
support. The authors also acknowledge the
attached on the surface of one gold
publishers
nanoparticles (Zhang et al., 2007). PPI-Au
permissions for the figures.
for
providing
copyright
nanoclusters and myoglobin (Mb) were
alternately adsorbed on the surface of
CONFLICT OF INTEREST
pyrolytic
The authors claim no conflict of interest.
graphite
(PG)
electrodes
forming {PPI-Au/Mb}n layer-by-layer
Biomed Res J 2015;2(1):21-36
Dendrimers based electrochemical biosensors
34
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Review
Developmental Signalling in Maintenance and Regulation
of Cancer Stem Cells
Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare*
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar,
Navi Mumbai, India
Tissue stem cells self-renew throughout the life of an organism thereby maintaining tissue homeostasis and
prevent cancer. The major signalling pathways such as Wnt, Notch and Sonic hedgehog control the stem
cell regulation and their deregulation leads to cancer. Recent evidences showed that there exists a subset of
cells within tumour termed as cancer stem cells (CSCs). These CSCs escape the conventional chemoradiotherapy and further lead to tumour relapse followed by metastasis. This review focuses on the
developmental signalling pathways that are involved in the regulation and maintenance of normal stem cells
and CSCs. Understanding the molecular mechanism may be useful to specifically target the CSCs while
sparing the normal stem cells to reduce tumorigenecity.
INTRODUCTION
Tumour is composed of a heterogeneous group
implantation in mice (Furth et al., 1937). This
of cells with different morphologies and
suggested that certain cells within a tumour
behaviour. Research in cancer biology
may have the ability to give rise to tumour
indicates that several cancers are supported by
growth (Furth et al., 1937). Later, in 1994,
a small subset of cells with stem cell like
John Dick's group identified human acute
properties and are termed as cancer stem cells
myeloid leukaemia-initiating cells using
(CSCs) or tumour initiating cells (TIC).
CD34+CD38- markers and showed that these
Evidences of CSCs involved in resistance to
cells initiated tumour (Lapidot et al., 1994). In
conventional therapies, leading to metastasis
1997, Bonnet and Dick showed for the first
and tumour recurrence is abundant (Beck and
time that the CD34 CD38 population of cells
Blanpain, 2013; Chandler and Lagasse, 2010;
had the self-renewal property. The authors
Prince and Ailles, 2008).
performed limiting dilution assay to show that
+
-
+
-
As early as 1937, Furth and colleagues
low numbers of CD34 CD38 cells were able
demonstrated that a single cell was able to
to form tumours in NOD/SCID mice, identical
produce a haematopoietic malignancy on
to donors; whereas considerably higher
Key words: Cancer stem cells (CSCs); EMT, Epithelial to mesenchymal transition; Lineage tracing; β-catenin; NICD,
Notch intracellular domain.
*Corresponding Author: Sanjeev K. Waghmare, Advanced Centre for Treatment, Research and Education in Cancer
(ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
Email: [email protected]
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
38
numbers of non-CSCs (CD34+CD38+) were
of CSCs has come from the lineage tracing
unable to form tumours (Bonnet and Dick,
experiments in mice model for various cancers
1997). These cells were coined as cancer stem
such as glioblastoma, skin and colon cancers.
cells. In 2003, Michael Clarke's group
The assay showed that the individual
reported the first isolation of CSCs from breast
fluorescent tagged cells have the capability to
tumour (Al Hajj et al., 2003). Subsequently,
give rise to a tumour (Chen et al., 2012;
the presence of CSCs in other solid tumours
Driessens et al., 2012; Schepers et al., 2012).
like melanomas, hepatocellular carcinoma,
Although many different markers for
glioblastoma, pancreatic cancer, colorectal
CSCs have been identified in tumours of
cancer and head and neck cancer have been
different tissues, cells isolated by using these
identified (Keshet et al., 2008; Li et al., 2007;
markers are not a pure CSC population. Hence,
Ma et al., 2007; Prince et al., 2007; Ricci-
one of the major challenges is the isolation of a
Vitiani et al., 2007; Singh et al., 2004). The
pure population of CSCs. Recent study on
CSC markers from various cancers are listed in
quantitative proliferation dynamics of hair
the Table 1. The characterisation of CSCs uses
follicle stem cells showed the isolation of stem
various assays that include: sphere-forming
cells based on their cell division. This suggests
assay, serial transplantation assay in NOD/
that it may be possible to isolate pure stem cell
SCID mice and in vivo lineage tracing. Serial
population (Waghmare and Tumbar, 2013;
transplantation assay, is considered as 'gold
Waghmare et al., 2008). Another challenge is
standard' assay, and measures self-renewal as
to understand how these CSC populations are
well as the tumorigenic property of CSCs in
regulated and maintained. Therefore, it is
vivo (Al Hajj et al., 2003; Beck and Blanpain,
important to study the various signalling
2013; Bonnet, 1997; Prince et al., 2007).
pathways that are crucial for survival of CSC
Recently the strongest evidence for existence
population.
Table 1: Cancer stem cell markers in various cancers
Cancer
Cancer stem cell markers
Leukaemia
CD34 CD38 (Bonnet, 1997)
+
-
+
Breast Cancer
CD44 CD24- (Hajj et al., 2003); ALDH1+ (Ginesteir et al., 2007); CD133+ (Wright et al., 2008)
Head and Neck
CD44 Lin (Prince et al., 2007); A1DH1+ (Clay et al., 2010; Krishnamurthy et al., 2010); CD133
+
-
+
+
Cancer
(Zhang et al., 2010); CD10+ (Fukusumi et al., 2014); CD98 (Matens de Kemp et al., 2013)
Pancreatic Cancer
CD44 +CD24-ESA+ (Li et al., 2007); c-Met (Li et al., 2011)
Liver Cancer
CD133+ (Ma et al., 2007); CD90+ (Yang et al., 2008); CD13+ (Haraguchi et al., 2010); OV6+ (Yang et al.,
2008)
Glioblastoma
CD133+ (Singh et al., 2004); SSEA1+ (Son et al., 2009), MET (De Bacco et al., 2009)
Melanoma
ABCB5+ (Keshet et al., 2008)
Colorectal Cancer
CD133+ (Ricci-Vitiani et al., 2007); CD166+ (Dalerba et al., 2007; Vermeulen et al., 2008); Lgr5+ (Barker
et al., 2007; Vermeulen et al., 2008), CD44+ (Haraguchi et al., 2008), CD44v6+ (Todaro et al., 2014)
Biomed Res J 2015;2(1):37-56
Dash et al.
39
Embryonic developmental process and
(Boumahdi et al., 2014).
Another important phenomena common
cancer stem cells
Development of an organism is regulated at
to both the CSCs as wells as the embryonic
the molecular level by various signalling
stem cells is the occurrence of epithelial to
pathways, and deregulation in these molecular
mesenchymal transition (EMT). During EMT,
mechanisms leads to cancer formation. Recent
the cells lose their polarity and acquire
studies have shown various similarities
migration capabilities that results in loss of
between cancer and development. During the
epithelial marker E-cadherin and simulta-
normal developmental process, undifferentia-
neous increase in mesenchymal marker N-
ted embryonic stem cells further differentiate
cadherin. During embryogenesis, EMT is
and give rise to the differentiated tissues of an
associated with gastrulation required for the
organism. Similarly in cancer, undifferentia-
formation of the three germ layers. In cancer,
ted CSCs are involved in tumour progression
EMT leads to invasion, metastasis and cancer
that leads to metastasis (Bellacosa, 2013).
stem cell-like phenotype (Kalluri, 2009;
The embryonic stem cells have a core
Singh, 2010). A recent study showed that
of
Twist1, an EMT promoter protein, is expressed
transcription factors like Oct4, Sox2 and
during early stages of tumorigenesis and is
Nanog that contribute to self-renewal and
required for the initiation of skin tumours
pluripotency (Boyer et al., 2005). Similarly,
(Beck et al., 2015).
transcriptional
network
comprising
lung CSCs showed elevated levels of Oct4 and
All these indicate that regulation of
Nanog transcription factors (Chiou et al.,
embryonic stem cells and CSCs share similar
2010). In head and neck cancer, CD44 variant
mechanisms. Therefore, it suggests that
CD44v3 was shown to interact with Oct4-
deregulation
Sox2-Nanog leading to CSC like properties
pathways are involved in cancer formation and
such as self-renewal and cisplatin resistance
CSC regulation and maintenance. Hence,
(Bourguignon et al., 2012). Recently, it was
studying
shown that the lineage ablation of Sox2-
pathways will shed light on the regulation of
expressing cells in both benign and malignant
CSCs.
the
of
various
developmental
developmental
signalling
skin squamous cell carcinomas resulted in
tumour regression indicating an important role
Developmental signalling pathways and
of Sox2 in tumour initiation and CSC
CSCs
functions. Moreover, chromatin immuno-
The various pathways which are deregulated
precipitation analysis identified Sox2 target
in cancer include Wnt, Notch, Hedgehog,
genes involved in controlling tumour stemness
EGFR, PI3K, NFκB, etc. Among these, three
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
40
Figure 1: Wnt Pathway. A) In the absence of the Wnt ligand, β-catenin is phosphorylated by destruction complex (APC,
CK1α, GSK3 and Axin) and is subjected to proteasomal degradation resulting in no transcription of the Wnt target
genes.B) In the presence of the Wnt ligand, the destruction complex is disrupted and thereby β-catenin enters the
nucleus and brings about the transcription of Wnt target genes. APC: Adenomatous Polyposis Coli; CK1α: Casein kinase
1α; GSK3: Glycogen synthase kinase 3; TCF: T cell factor; LEF: Lymphoid enhancing factor; Dsh: Dishevelled; LRP:
Low-density lipoprotein like receptors.
well-known pathways such as Wnt, Notch and
(Nusse et al., 1984; Rijswijk et al., 1987;
Hedgehog play an important role in the
Sharma, 2013).
development
and
normal
homeostasis.
There are 19 highly conserved Wnt
Conversely, deregulation of these pathways is
ligands discovered till date. These ligands are
shown in CSC regulation and maintenance
secreted hydrophobic glycoproteins found to
(Ailles, 2012; Purow, 2012).
be associated with cell membranes and extracellular matrix. In Wnt producing cells, the
Wnt Pathway
endoplasmic reticulum produces Wnt ligands,
Wnt pathway is evolutionarily conserved and
lipid modified by porcupine (Mikels, 2006;
is involved in various organisms. It was first
Willert et al., 2003). Wnt ligands can act
discovered in Drosophila, when a mutation in
through two general categories of pathways:
wingless (wg) gene led to a distinct phenotype
canonical and non-canonical. The canonical
including absence of wings and halters. Later,
pathway is β-catenin dependent, while the
Nusse's group showed that the insertion of
non-canonical pathways include Wnt/Ca2+ and
Mouse Mammary Tumour Virus (MMTV) in
Wnt/JNK pathways. In the canonical pathway
mice led to mammary tumour by proviral
shown in Fig. 1, Wnt ligands bind to the
activation of the int oncogene. The int
conserved cysteine rich domain (CRD)
oncogene was later demonstrated as the mouse
domain of the frizzled receptors (Fz) which in
homologue of the Drosophila wg gene. From
turn forms co-receptors complexes with low-
these two studies, a new nomenclature Wnt
density lipoprotein like receptors (Lrp5/6).
(combination of wg and int) was obtained
Further,
Biomed Res J 2015;2(1):37-56
this
interaction
recruits
the
Dash et al.
41
Dishevelled (Dsh) protein to the cytoplasmic
gastrulation defect and perturbations in the
tail of Fz receptor and brings about inhibition
establishment of apical ectodermal ridge
of destruction complex surrounding β-catenin.
during development (Liu et al., 1999). Further,
The components of the destruction complex
absence of Wnt4 ligand led to defects in female
comprise of scaffold protein Axin, Glycogen
development, while Wnt7a deletion led to
synthase kinase 3β (GSK3β), Casein kinase 1α
female infertility in mice (Jeays-Ward et al.,
(CK1α) and adenomatous polyposis coli
2004; Parr et al., 1998). Axin1 knockout in
(APC). In the absence of the Wnt ligands, the
mice led to neuro-ectodermal and cardiac
destruction complex hyper-phosphorylates β-
abnormalities (Zeng et al., 1997). Wnt
catenin and targets it for proteasomal
signalling was shown to be crucial in hair
degradation by ubiquitination. The binding of
follicle development as targeted deletion of β-
Wnt ligand to Lrp5/6 causes phosphorylation
catenin in the epidermis led to failure in
of the cytoplasmic tail of Lrp6, which in turn
placode morphogenesis (Huelsken et al.,
recruits Axin to the receptor complex that
2000). Absence of Lef1 led to defects in the
disrupts the destruction complex and stabilises
pro-B-cell proliferation and abnormalities in
β-catenin. The stable β-catenin translocates to
several organs like teeth, mammary glands,
the nucleus and binds to the lymphoid
whiskers and hair (Reya et al., 2000;
enhancing factor/T-cell factor (LEF/TCF)
VanGenderen et al., 1994); while the knockout
thereby
the
of Tcf1 led to thymocyte proliferation and
different target genes involved in cell fate
differentiation defects (Schilham et al., 1998).
determination during embryonic development
Using the Wnt reporter, Axin2-LacZ, Wnt
and tissue homeostasis (Mikels, 2006; Willert
responsive cells were localised to the sub
et al., 2003).
ventricular zone (SVZ) of the developing brain
transcriptionally
activating
and basal layer of the mammary ducts, which
Wnt signalling in normal development and
are the stem cells niches. Furthermore, these
cancer
Wnt responsive cells showed high sphere
Wnt pathway is involved in different biological
forming ability and were able to differentiate.
processes such as embryonic development,
Hence, the Wnt pathway plays an important
self-renewal, proliferation, morphogenesis,
role in normal development and tissue
etc. Wnt3a and Wnt1 knock out in mice led to
homeostasis (Logan, 2004; VanAmerongen et
deficiencies in neural crest derivatives and
al., 2009).
neural tube formation during the development
There are strong evidences showing
(McMahon et al., 1990; Yoshikawa et al.,
involvement of Wnt pathway in regulation of
1997). Wnt3 knock out in mice led to early
various cancers. Frequent somatic mutations
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
42
in β-catenin were observed in both mice and
(HSC), overexpression of β-catenin increases
human hepatocarcinomas (Coste et al., 1998),
the stem cell pool size suggesting that Wnt
prostate cancers and colon cancers (Voeller et
pathway
al.,
adenoma
hematopoietic stem cell homeostasis (Reya et
initiation, the first step was APC inactivation
al., 2003). In mice hair follicle stem cells, live
followed by β-catenin stabilization, while
cell imaging showed that β-catenin activation
progression from adenoma to carcinoma
in hair follicle stem cells was involved in hair
required the synergistic action of k-ras
follicle tissue growth (Deschene et al., 2014).
activation and β-catenin nuclear localization
Further, Wnt target gene Lgr5, a G-protein
(Phelps et al., 2009). β-catenin was shown to
coupled receptor was identified as an intestinal
be essential for retaining tumorigenecity of
stem cell marker indicating an important role
MDA-MB-231 breast cancer cell lines both in
of Wnt pathway in the regulation of intestinal
vivo
β-catenin
stem cells (Ailles, 2012, Valkenburg, 2011).
knockdown cells implanted into mice showed
The deletion of Tcf4, a Wnt downstream gene
decrease in the tumour size. In addition, an in
showed loss of stem cell activity and reduced
vitro study in breast cancer cell lines showed
proliferation of the intestinal epithelium
reduction in aldehyde dehydrogenase 1
(Korinek et al., 1998). In addition, Lgr5 was
(ALDH1) positive cells (Xu et al., 2015).
identified as a marker of hair follicle stem cells
Wnt3a expression was associated with EMT
(Jaks et al., 2008) with multipotent properties.
and promoted colon cancer progression (Qi et
Moreover, Wnt inhibitor SFRP1 was shown to
al., 2014). Moreover, deletion of Axin1 was
play an important role in hematopoietic stem
reported in sporadic medulloblastomas and
cell maintenance through extrinsic regulation
hepatocellular carcinomas (Dahmen et al.,
(Renstrom et al., 2009). Over-expression of
2001). Increased expression of Dsh protein in
Sfrp1 led to enhanced mesenchymal stem cell
non-small cell lung carcinoma and meso-
function in angiogenesis (Dufourcq et al.,
thelioma have been reported (Uematsu et al.,
2008). Besides, Sfrp1 was over-expressed in
2003).
hair follicle stem cells as compared to the non-
1998).
and
During
in
intestinal
vitro.
Further,
is
critical
to
maintain
the
stem cells (Tumbar et al., 2004; Zhang et al.,
Wnt signalling in normal and cancer stem
2009). Recently, it was shown that Sfrp1 gene
cell regulation and maintenance
is critical for maintaining proper mammary
Wnt signalling is important in adult stem
gland development wherein loss of Sfrp1
regulation and has been shown to be involved
promotes mammosphere formation; however
in stem cell proliferation, self-renewal and
the role in mammary stem cells needs further
maintenance. In hemato-poietic stem cells
investigation (Gauger et al., 2012).
Biomed Res J 2015;2(1):37-56
Dash et al.
43
In cancer, various reports have shown that
Notch Pathway
deregulation of Wnt pathway is crucial for the
Notch gene was first discovered in Drosophila
CSC regulation. Human head and neck CSCs
by Morgan and Bridges where they showed
treated with Wnt antagonist, secreted frizzled-
that a mutation led to wings notching and
related protein 4 (sFRP4), the CSCs showed
hence the name “Notch” was coined (Morgan
reduction in the sphere-forming capacity and
and Bridges, 1916; Mohr, 1919; Poulson,
decrease in the stemness markers like CD44
1940). There are four Notch genes, three
and ALDH1 (Warrier et al., 2014). In another
Delta-like and two Jagged genes in mammals,
report, β-catenin was shown to be required for
that are translated into different Notch ligands,
maintenance of cutaneous CSCs since deletion
Delta and Jagged. Recently, it was shown that
of β-catenin led to reduction in the CSCs and
for
tumour regression (Malanchi et al., 2008).
development, complex of Notch receptor-
CSCs isolated from mammary tumours of
Delta-Jagged acts in concert (Fiuza, 2004;
radiation treated p53-null mice showed altered
Boaretoa et al., 2015).
cell
fate
determination
during
DNA repair in response to radiation as well as
Since the Notch ligands such as Delta and
β-catenin activation (Zhang et al., 2010). In
Jagged proteins, as well as Notch receptors are
prostate cancer, Wnt signalling induced
transmembrane proteins, cell-cell contact is
tumour initiation, EMT and metastasis.
important for the signalling cascade. The
Additionally, in prostate cancer cell lines and
Notch receptors contain an extracellular
primary cultures, Wnt3a treatment increased
subunit, having multiple EGF-like repeats,
the self-renewal capacity of putative prostate
and a transmembrane subunit (Wharton et al.,
CSCs, emphasizing that Wnt signalling plays
1985). When the Notch ligand binds to its
an important role in prostate cancer (Barker,
receptor, the extracellular domain of the Notch
2006; Valkenburg, 2011; Verras et al., 2004).
receptor is dissociated from the trans-
Moreover, the inactivation of APC in Lgr5-
membrane domain and the S2 cleavage site is
positive stem cells at the intestinal crypts led to
exposed (Fig. 2). This site is cleaved by
transformation within days; while inactivation
ADAM (a disintegrin and metalloprotease)
of APC in progenitors or differentiated cells
generating an intermediate that is further
did not lead to tumour formation even after 30
cleaved by γ-secretase to generate Notch
weeks (Barker et al., 2009). In addition, the
Intracellular Domain (NICD). NICD then
deletion of CD44, a CSC marker and a Wnt
translocates to the nucleus where it binds to
target gene in mice having heterogeneous APC
ubiquitous transcription factor CSL (CBF-1,
mutation (APC
Min/+
), attenuates intestinal
tumorigenesis (Zeilstra et al., 2008).
Suppressor of Hairless, Lag-1). This complex
displaces a co-repressor complex containing
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
44
Figure 2: Notch Pathway. A) In the absence of the Notch ligands (Delta and Jagged), the S2 cleavage site remains
hidden and inaccessible to ADAM. Hence, the NICD is not formed, with consequent no transcription of the Notch target
genes. B) In the presence of the Notch ligands, the conformational change in the intracellular subunit of the Notch
receptor takes place exposing the S2 cleavage site, thus leading to the formation of NICD. Further, NICD translocates the
nucleus and brings about transcription of Notch target genes. NICD: Notch intracellular domain; ADAM: A disintegrin and
metalloprotease; MAML: Mastermind-like protein; SKIP: Ski-interacting protein; SHARP: SMRT associated protein.
SKIP, SHARP and histone deacetylases.
establishment of the central and peripheral
Further, it recruits a co-activator complex
nervous systems, spermatogenesis, oogenesis,
containing
(Mastermind-like
myogenesis and imaginal disc development
protein), p300 and other chromatin modifying
(Artavanis-Tsakonas et al., 1999). In normal
enzymes, thereby bringing about transcription
mammary development, Notch pathway
of different Notch target genes (Ailles, 2012;
activation is required for regulation of cell
Andersson, 2011; Fiuza, 2004).
fate, proliferation and stem cell self-renewal.
a
MAML
The Notch pathway is also shown to be
Notch signalling in normal development
important for tip-cell formation during
and cancer
mammalian astrocyte differentiation and
Notch
pathway
is
also
evolutionarily
angiogenesis. In vertebrates, the Notch
conserved and is important in cell to cell
pathway leads to patterning during inner ear
communication that regulates cell fate
hair cell formation and insulin-secreting
determination
pancreatic β cell production (Ailles, 2012;
during
development,
cell
proliferation, differentiation and apoptosis.
Fortini, 2009).
The loss of Notch function in vertebrates is
Notch signalling has been shown to be
associated with disruption of neurogenesis,
involved in various cancers. For instance,
somite formation, angiogenesis, and lymphoid
Notch1 regulates breast cancer cells by
development. In Drosophila, Notch is shown
inducing Slug expression (Shao et al., 2015).
to control the fate of various cell types in the
Notch4 promotes growth of gastric cancer
eye. In vertebrates, Notch is involved in the
cells through activation of Wnt1, β-catenin
Biomed Res J 2015;2(1):37-56
Dash et al.
45
(Quian et al., 2015) and the downstream target
expressed in stem cells and disruption led to
such as c-myc and cyclin-D1. In mice, Notch1
defective stem cells proliferation (Kitamoto
activation combined with p53 loss showed
and Hanaoka, 2010).
synergistic effect in the formation of
Osteogenic sarcoma (Tao et al., 2014).
Notch signalling plays an important role in
a number of hematopoietic and solid tumours,
but the strongest evidences for its role in CSC
Notch signalling in normal and cancer stem
regulation has been shown in breast cancer,
cell regulation and maintenance
embryonal brain tumours and gliomas (Fan et
Notch has been shown to be involved in self-
al., 2006; Pannuti et al., 2010). In various
renewal, proliferation and differentiation of
human breast cancer cell lines and primary
adult stem cells in various tissues. In mice
patient tissues, a significant decrease in
mammary stem cells, the knockdown of Cbf-1,
mammosphere
a canonical Notch effector, showed increased
inhibition has been demonstrated (Abel et al.,
stem cell activity in vivo suggesting a role in
2014). Further, studies on the human
controlling mammary stem cells. (Bouras et
mammary mammospheres have shown a
al., 2008). Notch directly targets the crypt base
feedback loop between Her2/Neu and Notch,
columnar cells that maintain stem cell activity
as well as promotion of a hypoxia resistant
in mice (VanDussen et al., 2012). The Notch
phenotype (Pannuti et al., 2010). In brain
activation maintains the slow cycling property
tumours, blockade of Notch led to a 5-fold
of neural stem cells; however, blocking Notch
reduction in the CD133+ cell fraction and total
resulted in increased number of stem cell
depletion of the side population cells.
divisions followed by depletion of the stem
Additionally, differentiated cell growth was
cell
pool
(Chapouton
et
al.,
formation
after
Notch
2010).
observed after Notch inhibition, but lacked
Furthermore, constitutive activation of Notch
formation of tumour xenografts efficiently,
signalling promotes self-renewal in muscle
indicating that the CSCs required for tumour
stem cells through upregulation of Pax7 (Wen
propagation were absent (Fan et al., 2006). A
et al., 2012). Mice with satellite cell specific
recent study on primary human pancreatic
deletion of RBP-Jj (recombining binding
xenografts showed upregulation of the notch
protein-Jj), a nuclear factor required for Notch
pathway components in pancreatic CSCs.
signalling, showed depletion of the stem cell
Additionally, inhibition of notch pathway
pool and their muscles lacked ability to
reduced CSC percentage and tumour-sphere
regenerate in response to injury (Bjornson et
formation significantly (Abel et al., 2014).
al., 2012). Notch 3 has been shown to be
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
46
In
Sonic-hedgehog Pathway
Hedgehog
pathway
is
delineated
the
Sonic-hedgehog
pathway
in
elaborated in Fig. 3, absence of the hedgehog
Drosophila and determines the anterior-
ligand, Smoothened (Smo) is inhibited by
posterior orientation of developing structures
being bound to Patched (Ptc). When the
(Nusslein-Volhard and Wieschaus, 1980).
hedgehog ligand binds to Ptc, inhibition of
Similar to Wnt and Notch, the key components
Smo is released which acts on protein complex
of the Hedgehog pathway are evolutionarily
comprising of fused (Fu), suppressor of fused
conserved, although differences are observed
(Sufu) and cos-2-costa-2 (Wicking et al.,
in the mammalian and Drosophila Hedgehog
1999; Merchant, 2010). These proteins are
signalling. While Drosophila has only one
generally bound with Gli thereby inhibiting its
hedgehog gene, three homologues have been
action. Once the complex is disrupted, Gli
identified in vertebrates namely, Sonic (Shh),
translocates to the nucleus and brings about
Desert (Dhh), and Indian hedgehog (Ihh). The
transcription of different downstream targets
Sonic Hedgehog pathway is extensively
(Sasaki et al., 1999; Ruiz, 2007; Stecca, 2010).
investigated in the vertebrate system (Chen et
al., 2005; Varjosalo et al., 2006). The other
Sonic-hedgehog
components of the Hedgehog pathway include
development and cancer
patched
(Smo),
In vertebrates, the Sonic Hedgehog (Shh), is
transmembrane
expressed widely throughout the developing
glycoprotein and a 7-pass transmembrane
central nervous system (CNS), limb, gut, teeth
protein
and
(Ptc)
constituting
a
and
smoothened
12-pass
respectively
Wicking et al., 1999).
(Varjosalo,
2008;
hair-follicle.
signalling
Dhh
is
in
normal
involved
in
development of the germline, while the Ihh is
Figure 3: Hedgehog Pathway. A) In the absence of Hedgehog ligands (Indian, Sonic and Dessert), the Patched
receptor (ptc) exerts inhibitory action on the Smoothened receptor (smo). The Gli complex (Gli1 and Gli2) remains in the
cytoplasm followed by no transcription of Hedgehog target genes. B) In the presence of Hedgehog ligands, the inhibitory
action of Patched (ptc) on Smoothened (smo) is released, and hence Gli complex translocates to the nucleus and brings
about transcription of Hedgehog target genes.Fu: Fused; SuFu: Suppressor of Fused; Cos 2: cos-2costa-2.
Biomed Res J 2015;2(1):37-56
Dash et al.
47
involved in development of the skeletal system
Hedgehog almost completely blocks intestinal
(Bitgood et al., 1996; Wicking et al., 1999).
adenoma development (Buller et al., 2015).
Shh also plays a role in neural stem cells,
determining the neuronal cell fate (Merkle et
Sonic-hedgehog signalling in normal and
al., 2007). It was demonstrated that during
cancer
repair of acute airway injury, the Hedgehog
maintenance
pathway gets activated in the airway
Hedgehog signalling is involved in stem cell
epithelium (Watkins et al., 2003). Hedgehog
regulation of various tissues. Shh regulates
signalling components Ptc, Gli1, and Gli2
self-renewal of neural stem cells (Palma et al.,
were
mammary
2005). The components of Hedgehog pathway
progenitor cells grow as mammospheres (Liu
such as Ptc, Gli1 and Gli2 are expressed in the
et al., 2006). These reports indicate that the
mammary stem cells and down regulated
Hedgehog pathway plays a role in normal stem
during differentiation (Liu et al., 2006).
cell regulation (Ailles, 2012).
Hedgehog is involved in controlling neural
over-expressed
when
Hedgehog signalling is involved in
various cancers. For example, Ptc1 mutation
stem
cell
regulation
and
stem cells through the p53-independent
regulation of Nanog (Po et al., 2005).
was observed in patients with medullo-
In colon carcinoma, Hedgehog signalling
blastoma and rhabdomyosarcoma (Hahn et al.,
is activated in CSCs with higher expression of
1996; Johnson et al., 1996; Pietsch et al.,
Gli1 and Gli2. In non-small cell lung cancer,
1997). Sufu as well as Smo mutations were
the malignant phenotype of the tumours is
observed in medulloblastoma (Xie et al., 1998;
maintained by ligand-dependent Hedgehog
Taylor et al., 2002); Gli1 and Gli3 mutations
pathway activation (Watkins et al., 2003).
were seen in pancreatic adenocarcinoma; and
Furthermore, Bmi1, which is a downstream
Gli1
in
target of the Hedgehog pathway was activated
glioblastoma (Clement et al., 2007; Jones et
in breast CSCs and is also shown to regulate
al., 2008). Further, Kern et al showed that Gli
normal and leukemic stem cells (Liu et al.,
and
2006; Takebe, 2011).
gene
amplification
PI3K/AKT/mTOR
was
seen
signalling
act
synergistically to initiate and maintain chronic
lymphocytic leukemia (Kern et al., 2015).
CONCLUSION
Another report showed that Sonic hedgehog
Tumour maintenance and progression is
ligand over-expression led to increased
regulated by a subset of cells that are known as
number and size of intestinal adenomas in
cancer stem cells (CSCs). Recently, due to
APC (HET) mice, while loss of Indian
increase in evidences on the existence of
Biomed Res J 2015;2(1):37-56
Developmental signalling in cancer cells
48
CSCs, they have gained more attention but
targeted drugs approach can be fruitful.
how
Hence,
these
CSCs
escape
the
chemo-
further
detailed
research
on
radiotherapy is still unknown. Moreover, how
deregulation of the developmental pathways
the CSCs are maintained in the tumour micro-
in CSCs needs to be investigated. Eventually,
environment remains elusive. Several reports
elucidation of the signalling mechanisms will
showed signalling pathways such as Wnt,
enable to specifically target CSCs without
Notch and Sonic-hedgehog are deregulated in
affecting the normal cells.
cancer, and also involved in the CSC
regulation and maintenance. In addition,
ACKNOWLEDGEMENTS
evidence of cross-talk between the signalling
The authors acknowledge Mr. Rahul Sarate
pathways exists. Therefore, understanding
and
these signalling pathways at the molecular
suggestions.
Mr.
Gopal
Chovatiya
for
their
level will be of utmost importance. The study
will enable counteracting the issue of
CONFLICT OF INTEREST
signalling cross-talk, and perhaps, multi
The authors claim no conflict of interest.
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Review
Diverging Role of Nrf2 in Cancer Progression and
Prevention
Lokesh Gambhir, Rahul Checker, Deepak Sharma and Santosh K. Sandur*
Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai,
India
The role of transcription factor, nuclear factor [erythroid-derived 2]-like 2 (Nrf2), is detoxification of
xenobiotics, overcoming oxidative stress and offering resistance to ionizing radiation induced cell death.
However, the role of Nrf2 in cancer progression remains debatable. Activation of Nrf2 dependent proteins is
crucial in maintaining cellular redox homeostasis and combating toxicity of carcinogens. Thus, employing
natural or synthetic activators of Nrf2 pathway is a promising approach for development of chemopreventive
modalities. Intriguingly, recent reports have highlighted the dark side of Nrf2 suggesting that multiple cancer
cells demonstrate constitutive activation of Nrf2 caused by mutations in Nrf2 or Keap-1 proteins, offering
survival advantage. Additionally, Nrf2 pathway is also up-regulated in chemoresistant cells and may be a
major contributor in acquired chemoresistance. Thus, targeting Nrf2 pathway has emerged as a novel
strategy to improve efficacy of chemotherapeutic drugs. This review discusses the dark and bright sides of
this transcription factor in line with the recent literature.
INTRODUCTION
The transcription factor, nuclear factor
potentially
[erythroid-derived 2]-like 2 (Nrf2) was
transactivation domain (Moi et al., 1994).
identified as NF-E2-like basic leucine zipper
Further characterization demonstrated it as
transcriptional activator that binds to the
Cap`n`Collar (CNC) protein involved in the
tandem NF-E2/AP1 repeat of the β-globin
control
locus control regions (Moi et al., 1994). The
development by basic leucine zipper DNA
Nrf2 gene was cloned and characterized by
binding domain (bZip) homeotic gene. The
using the tandem repeats of nuclear factor like
CNC family comprises four members, namely
erythroid factor-2 (NF-E2)/activator protein-1
Nrf1, Nrf2, Nrf3 and p45NF-E2. Nrf1 and
(AP1) of the β-globulin locus as a recognition
Nrf2 are ubiquitously expressed and are
site probe. Nrf2 contains a basic leucine zipper
essential for normal development in mice. The
DNA binding domain at the C-terminus and an
expression of Nrf3 is restricted to placenta and
N-terminal acidic domain (rich in glutamic
liver, while p45NF-E2 expression is restricted
and aspartic acid residues), which could
to erythrocytes (Ikeda et al., 2004; Motohashi
of
function
Drosophila
as
an
head
acidic
segment
Key words: Cancer, Nrf-2 transcription factor, Keap-1 protein, β-TrCP protein.
*Corresponding Author: Santosh K Sandur, Free Radical Biology Section, Radiation Biology and Health Sciences
Division, Bio-Science Group, Modular Laboratories,Bhabha Atomic Research Centre, Trombay, Mumbai, India.
Email: [email protected]
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
58
et al., 2002). Expression of Nrf1 is essential for
seven lysine residues that direct ubiquitin
embryonic development and its deficiency
mediated proteasomal degradation of Nrf2
leads to hepatic abnormality. The Nrf2
(Fig. 1) (Itoh et al., 1999; Zhang et al., 2004).
knockout mice are viable and exhibit no
Neh3 is essential for interaction of Nrf2 with
phenotypic defects, but are sensitive to
CHD6 (a chromo-ATPase/helicase DNA
oxidative stress (Chan and Kwong, 2000;
binding protein) suggesting involvement in
Chan et al., 1998; Leung et al., 2003; Ohtsuji
interaction with co-transcription factors (Nioi
et al., 2008; Ramos-Gomez et al., 2001; Xu et
et
al., 2005). Human Nrf2 is homologus to
transactivation domains that interact with the
mouse and contains six highly conserved
CREB-binding protein (CBP) (Katoh et al.,
domains called Nrf2-ECH homology domains
2001). Neh6 domain interacts with β-
(Neh). Neh1 domain has a nuclear localisation
transducin repeat-containing protein (β-TrCP)
signal and CNC-type basic leucine zipper
(Jain and Jaiswal, 2007). Binding of Keap1 to
necessary for DNA binding and dimerization.
Nrf2 brings it close to E3 ligase complex
The Neh2 domain contains a Keap1 (Kelch-
through two major domains: BTB (Bric a
like ECH-associated protein 1a, negative
Brac, tramtrack, broad complex) domain
regulator of Nrf2) binding pocket and has
which interacts with Cul3; and kelch domain
al.,
2005).
Neh4
and
Neh5
are
Figure 1: Structures and functions of Nrf2 and its repressors Keap1 and β-TrCP1. The relative position of the Neh
domains is shown. The DLG and ETGE motifs present in Neh2 domain that bind to Keap1 are represented above with the
numbering of amino acids based on the human cap'n'collar (CNC)-basic-region leucine zipper (bZIP) protein.
Biomed Res J 2015;2(1):57-82
59
Gambhir et al.
which binds to Nrf2. Interaction of Neh2
reductase (TrxR), peroxiredoxin (Prx), heme
domain with Keap1 depends on low-affinity
oxygenase-1 (HO-1) and transporters like
binding via DLG motif and high-affinity
multidrug
binding of an ETGE motif which results in a
(MRP) (Banning et al., 2005; Ishii et al., 2000;
hinge and latch mechanism of binding. The N-
Ishii and Yanagawa, 2007; Kim et al., 2001;
terminal BTB/POZ (Pox virus Zinc finger)
Maher et al., 2005; Moinova and Mulcahy
domain
enabling
1999; Sakurai et al., 2005). Phase II enzymes
Keap1–Nrf2 interaction (Adams et al., 2000;
reduce the toxicity of xenobiotics by making
Kensler et al., 2007, Li et al., 2004; Lo et al.,
them water soluble, thereby facilitating their
2006; Padmanabhan et al., 2005).
elimination. Efflux of endogenous molecules
forms
homodimers
resistance-associated
protein
and xenobiotics is also governed by Nrf2
Activation of Nrf2 dependent genes
mediated
expression
Exposure of cells to low levels of oxidative
Constitutive expression of Nrf2 by tumor cells
stress, electrophiles or chemopreventive
may offer an advantage for ambient growth
compounds leads to activation of Nrf2. Upon
and
activation, Nrf2 dissociates from inhibitory
phenomena coined as “dark side of Nrf2” (Lau
protein Keap1 and translocates to the nucleus.
et al., 2008; Wang et al., 2008c). The present
In the nucleus it forms a heterodimer with co-
review emphasizes the putative dual role of
transcription factor Maf and binds to the anti-
Nrf2 pathway during cancer progression and
oxidant response element (ARE) sequence to
highlights its potential as a target for
induce transcription of several different genes
chemoprevention.
detoxification
of
of
transporters.
xenobiotics,
the
(Zhang, 2006). ARE sequence is the 'core'
sequence of 5´-RTGACnnnGCR-3´ identified
Mechanism of Nrf2 Activation
using murine GST-Ya ARE. The sequence was
Nrf2 is sequestered in the cytoplasm by Keap1
used to identify genes present in the promoter
which regulates Nrf2 stabilization and levels
region (Rushmore et al., 1991). The Nrf2
inside the cell. The interaction between the
downstream
II
two proteins is a dynamic process regulated in
detoxifying enzymes like glutathione S-
such a manner that enables Nrf2 to control
transferase
both the basal and inducible expression of
genes
(GST),
oxidoreductase-1
include
phase
NAD(P)H
(NQO1),
quinone
and
UDP-
dependent
genes.
Under
homeostasis
glucuronosyltransferase (UGT), intracellular
conditions, Nrf2 is maintained at low basal
cytoprotective
glutamate
levels for expression of cytoprotective genes
cysteine ligase (GCL), glutathione peroxidase
(Fig. 2) (Itoh et al., 1999). Nrf2 is at low levels
(GPx),
when bound to Keap1 homodimer through its
proteins
thioredoxin
like
(Trx),
thioredoxin
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
60
Figure 2: Schematic model of Nrf2 activation under normal and oxidative stress conditions.
and
in
vivo
kelch repeats domains at C terminal, leading to
alkylation
site-directed
Cullin3/Rbx1-mediated
polyubiquitination
mutagenesis, cys151 was identified as the
and subsequent proteasomal degradation.
major site directly alkylated by Nrf2 inducers
Keap1 protein contains numerous cysteine
along with critical residues cys273 and cys288
(cys) residues with potential to act as a redox
(Dinkova-Kostova et al., 2002; Eggler et al.,
sensor (Hong et al., 2005b).
2005; Hong et al., 2005a; Levonen et al.,
2004). Mutation at cys151 abolished induction
Role of cys residues in Nrf2 activation
of Nrf2 by activators like sulforaphane and
The significance of Keap1 as a central
tert-butylhydroquinone but had no impact on
regulator of Nrf2 activation was revealed
Keap1:Nrf2 binding. Keap1-cys151 restores
while addressing the negative regulation of
phenotypes like over-expression of Nrf2 and
antioxidant machinery by Keap1 dependent
post-natal lethality as observed in Keap1 null
proteasomal degradation of Nrf2 (McMahon
mice (Wakabayashi et al., 2004). However,
et al., 2003). The half life of Nrf2 increases
activation of Nrf2 by arsenite in cys151 Keap1
from 15 min to 30 min in cells expressing
mutant MDA-MB231 cells, indicated a
mutated ETGE motif containing Nrf2 and
possible redox independent mode of Nrf2
Keap1 (Du et al., 2008). Using in vitro
induction (Wand et al., 2008b). Further
Biomed Res J 2015;2(1):57-82
61
Gambhir et al.
cys273ser and cys288ser mutations showed
release of Nrf2 from the low affinity binding
abrogated repression of Nrf2 by Keap1
motif (Cullinan et al., 2004; Kobayashi et al.,
(Levonen et al., 2004; Wakabayashi et al.,
2006). The change confers stabilisation and
2004). These observations demonstrated that
accumulation of Nrf2 in the cytosol followed
cys151 is required for the activation of Nrf2,
by nuclear translocation. According to hinge
whereas cys273 and cys288 are needed for
and latch model, ETGE motif remains bound
Nrf2
significant
to the Keap1 following activation. This results
contribution of the critical cysteine residues
in saturation of Keap1 which is no longer able
during Nrf2 activation and regulation under
to
oxidative stress was indicated. Several cellular
translocation to the nucleus and binding to
redox modifiers modulate activation of Nrf2
ARE to induce expression of cytoprotective
via modification of the critical cysteine
machinery of the host cell (Jain and Jaiswal,
residues in Keap1. Further, the afore-
2006). An alternate model of induction is
mentioned three critical cysteine residues
attributed to the polyubiquitination of Keap1
undergo thiol modifications leading to
at lys63, leading to subverted Cullin3
conformational change in the Cul3–E3 ligase
interaction and dissociation of Nrf2 from
complex leading to loss of E3 ligase ubiquitin
Keap1 (Zhang et al., 2005). The ubiquitin-
activity. The cysteine residues act as redox
specific protease-15 deubiquitinase restored
sensors to further perturb the efficiency of
Keap1 activity (Villeneuve et al., 2013).
inhibition.
Besides,
a
compete
with
free
Nrf2
inducing
nuclear export signal on Keap1, and mutant
Apart from Keap1 and Cul3/Rbx1, other
form of Keap1 at leu308 and leu310 was
mediators also contribute in regulating the low
unable to locate in the cytoplasm (Kobayashi
basal levels of Nrf2. Phosphorylation status of
et al., 2009; Nguyen et al., 2005; Velichkova
tyr568 on Nrf2 is governed by Src subfamily
and Hasson, 2005). These studies suggested
kinases Fyn, Src and Fgr, which influence the
that under normal conditions, the signals from
nuclear export of Nrf2. Under oxidative stress
nuclear export sequence (NES) of Keap1
conditions, glycogen synthase kinase-3 beta
maintained the Keap1 dimer in association
(GSK-3β), a serine/threonine protein kinase,
with Nrf2 in the cytoplasm.
plays an important role in the nuclear export of
Exposure of cells to oxidative, xenobiotic
Nrf2 by phosphorylating Fyn. Another Src
or electrophilic stress abrogates Keap1
member Bach1 has been shown to govern
induced degradation of Nrf2. Perturbation in
export of Nrf2 from the nucleus, thereby
the
in
negatively regulating expression of its
modifications of critical cysteine residues in
dependent genes. Bach1 competes with Nrf2
Keap1. The conformational change renders
for binding to ARE sequence, resulting in
cellular
redox
status
results
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
62
suppression of ARE mediated expression of
rylated ser40 residue in the Neh2 domain
Nrf2 dependent genes (Jain and Jaiswal, 2006;
leading
Niture et al., 2011).
interaction in response to oxidative stress
to
disruption
of
Keap1/Nrf2
induced by tBHQ and β-naphthoflavone.
Keap1 independent activation of Nrf2
Mutation in the serine residue results in
Multiple studies have highlighted Keap1
abrogation of PKC induced activation of Nrf2
independent activation of Nrf2. Along with
(Huang
Keap1 dependent degradation of Nrf2, an
phosphorylation of ser40 was required for
alternate mechanism controls activation and
release of Nrf2 from Keap1, but does not play
stabilisation of Nrf2 mediated by β-transducin
a role in nuclear translocation (Bloom and
repeat-containing protein (β-TrCP) (Rada et
Jaiswal, 2003). Nuclear localisation sequence
al., 2012). Mouse Nrf2 contains two binding
(NLS) and nuclear export sequence in Nrf2
sites for β-TrCP which acts as an adapter for
regulates localization in the cell. The NLS
the
ligase
motifs are identified by adapter proteins like
complex. GSK-3β phosphorylates serine
importins that facilitate transfer inside to
residue in SCF/β-TrCP destruction motif
nucleus (Theodore et al., 2008). Another
“DSGIS” in Neh6 domain leading to Keap1
conserved protein kinase that influences Nrf2
independent degradation (Jain and Jaiswal,
activation is casein kinase II (CK2). CK2
2007). Post translational modification also
possesses an array of potential targets and
governs Nrf2 activation. Nrf2 contains
plays a role in complex cellular processes
multiple serine, threonine and tyrosine
including cytoprotection. Nrf2 contains 13
residues which serve as potential sites for
potential phosphorylation targets for CK2
phosphorylation. Different pathways for
abundant
activation of Nrf2 are identified including
domains. Phosphorylation dependent nuclear
protein kinase C (PKC), mitogen-activated
translocation of Nrf2 is sensitive to Ck2
protein kinases (MAPK), phosphatidylinositol
inhibitor (Apopa et al., 2008; Pi et al., 2007).
Skp1-Cul1-Rbx1
ubiquitin
et
in
al.,
2002).
Neh4/Neh5
Interestingly,
transcriptional
3-kinase (PI3K), and RNA-dependant protein
kinase-like endoplasmic reticulum kinase
Role of MAPK in activation of Nrf2
(PERK) (Cullinan and Diehl, 2004; Lee et al.,
PI3K
2001; Yu et al., 2000). PKC has multiple
protein kinase (ERK) are proposed to regulate
isoforms which play essential roles in growth,
Nrf2 pathway (Cullinan et al., 2003; Kang et
differentiation,
apoptosis,
al., 2001). tBHQ enhances NQO1 protein
survival and carcinogenesis and PKC can be
expression and activity in a PI3K dependent
activated by oxidative stimuli. PKC phospho-
manner in human neuroblastoma cells. tBHQ
cytoprotection,
Biomed Res J 2015;2(1):57-82
and
extracellular
signal-regulated
63
Gambhir et al.
elicited ARE mediated induction of GST in
activating
Nrf2.
The
authors
further
hepatoma cells in a PI3K dependent manner.
demonstrated Raf-1 mediated activation of
PI3K inhibitor (Ly294002) abrogated tBHQ
Nrf2 attributing it to up-regulation of the co-
mediated NQO1 induction, indicating a role of
activator CREB binding protein.
PI3K in Nrf2 activation (Lee et al., 2001).
PERK, a transmembrane kinase, phospho-
Pro-oncogenic Effects of Nrf2: The Dark
rylates Nrf2 in vitro leading to dissociation
Side
from Keap1. A pivotal role of PERK mediated
It is well documented that oxidative stress
activation of Nrf2 was proposed as a
plays a pivotal role in the initiation and
mechanism for maintenance of glutathione
progression of cancer, with magnitude of
levels that act as a cytoprotective buffer
oxidative stress a key determinant of the
against oxidative insult (Cullinan et al., 2003).
response of a cell towards the oncogenic
An important role of MAPK in the activation
stimuli. Chronic exposure of cells to oxidative
of Nrf2 via phosphorylation has been reported
insult causes cytotoxicity due to irreversible
by several investigators. Yu et al. (2000)
damage to vital macromolecules; whereas
studied MAPK mediated activation of phase II
transient increase leads to the activation of
detoxification
multiple
redox sensitive pro-survival transcription
inducers (Jeong et al., 2006). In hepatoma
factors. Therefore, in order to survive and
cells, sulforaphane and tBHQ induced
proliferate, tumor cells maintain a moderate
activation of ERK, MAPK kinase and Raf-1,
oxidative intracellular niche achieved by
to mediate induction of phase II detoxification
taking advantage of the antioxidant defense
enzymes via Nrf2/ARE pathway (Yuan et al.,
machinery of the cell like Nrf2 pathway.
2006). MAPK/ERK upon activation initiates
Constitutive activation of Nrf2 and expression
phosphorylation
modulates
of dependent cytoprotective genes, permits
activity of multiple downstream transcription
tumor cells to nurture and expand in an
factors (Shen et al., 2004; Zipper and
ambient redox niche. High levels of Nrf2
Mulcahy, 2000). Dithiolcarbamate was shown
expression is reported in multiple cancers
to activate ERK and p38 resulting in
including cancers of lung, breast, gall bladder,
transcriptional
Nrf2
pancreatic, colorectal and head and neck
synthetase
(Jaramillo and Zhang, 2013; Lau et al., 2008;
(Wild et al., 1999). Shen et al. (2004)
Shelton and Jaiswal, 2013; Sporn and Liby,
investigated the transactivation potential of
2012). Ikeda et al. (2004) demonstrated
different
observed
constitutive up-regulation of Nrf2 and GSTP1
differential effects of multiple MAPKs in
in hepatocellular carcinoma indicating role of
dependent
enzymes
cascade
using
that
up-regulation
γ-glutamylcysteine
Nrf2
domains
and
of
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
64
Nrf2 in cancer promotion. Nrf2 regulates
classified based on their functional impact into
expression of an exclusive neoplastic lesion
passenger mutations, null mutations and
marker GSTP1 in an ARE dependent
hypomorphic mutations. Passenger mutations
mechanism. Higher levels of Nrf2 have been
do not have any effect on Keap1/Nrf2
associated with poor clinical outcome and
interaction,
poor responsiveness in pancreatic, cervical
diminished the ability of Keap1 to repress
and lung cancer (Geismann et al., 2014; Sporn
Nrf2. Most of the mutations do not affect the
and Liby, 2012).
Nrf2 levels, but impact the activity as Keap1 is
whereas
null
mutations
unable to act as a negative regulator (Hast et
Dysregulation of Nrf2 pathway in cancer
al., 2014; Hayes and McMahon, 2009; Shibata
Persistent Nrf2 activation is responsible for the
et al., 2008). Japanese patients with lung
pro-tumorogenic effect due to genetic and
adenocarcinoma
epigenetic
Nrf2/Keap1
mutations (Ohta et al., 2008). Dysregulated
(frequencies of up to 30% in lung or ovarian
suppression of Nrf2 by Keap1 in breast cancer
cancer). Copy number loss in a member of E3
resulted due to mutation in cys23 residue (Nioi
ubiquitin ligase complex or oncogenic
and
pathways or persistent exposure to oxidative
reoxygenation
stress leads to persistent activation (Barbano et
upregulated and protected cancer cells from
al., 2013; Martinez et al., 2014; Zhang et al.,
deleterious effects of oxidative stress (Kim et
2010). A mutation in the Keap1 protein or loss
al., 2007).
alterations
in
Nguyen,
demonstrated
2007).
Under
conditions
Keap1
hypoxic/
Nrf2
was
of heterozygosity has been reported to result in
Mutations in the DLG motifs of the DC
persistent Nrf2 activation in multiple cancers
domain in Keap1 show highest frequency in
(Padmanabhan et al., 2006; Singh et al., 2006).
lung cancers (Ganan-Gomez et al., 2013).
DNA methylation of CpG sites in the promoter
Interestingly frame shift mutations in Keap1
region of Keap1 was observed in 51% of
are frequent in DGR domain (65%) essential
breast, 20% of colorectal, and 12% of lung
for interaction with Nrf2 (Taguchi et al.,
cancers, accounting for decreased levels of
2011). Other mutations in the intervening
Keap1
Nrf2
region and BTB domain of Keap1 occur in
activation (Bryan et al., 2013; Wang et al.,
prostate, lung and ovarian cancers. Mutation
2008a). Approximately 15% patients with
in these domains influence the critical cysteine
lung cancer posses somatic mutations in
residues that inhibit its interaction with
Keap1, resulting in impaired and inefficient
Cullin3, leading to inhibition of poly-
Nrf2 repression (Hayes and McMahon, 2009).
ubiquitination of Nrf2. Mutation in other
The
amino acids like ser104, gly186,423 and
and
consequent
prevailing
Keap1
Biomed Res J 2015;2(1):57-82
enhanced
mutations
were
65
Gambhir et al.
arg320 within the DC, BTB and IVR domains
Nrf2 from ubiquitination, whereas mutations
are cancer derived mutations that results in
in the DLG motif disrupt low affinity binding
impaired homo-dimerization of Keap1 needed
but Nrf2 remains bound to Keap1. Both these
for repression of Nrf2 (Hast et al., 2014). A
mutations result in Nrf2 stabilisation and
single nucleotide deletion in Keap1 gene was
accumulation in nucleus (Taguchi et al., 2011).
associated with marked drug resistance
Along with the somatic mutations in
against BRAF and cisplatin in melanoma cells
Keap1/Nrf2, an alternate mechanism for
(Miura et al., 2014). Although mutations in
activation of Nrf2 in tumorigenesis is
Keap1 play a central role in constitutive Nrf2
mediated by oncogenic signalling. Expression
activation, deficiency of Keap1 per se does not
of oncogenes like Kras, Braf and Myc activate
result
Nrf2,
in
cancer.
Interestingly,
Keap1
elevating
antioxidant
machinery
knockdown mice (floxed Keap1 allele) did not
resulting in depletion of the intracellular ROS
develop spontaneous cancer and survived for 2
levels, thus providing a conducive reduced
years. Keap1 knockdown mice showed
environment for tumor growth (DeNicola et
constitutive activation of Nrf2 in multiple
al., 2011).
tissues including lung and liver. These studies
indicated that impaired Nrf2/Keap1 pathway
Nrf2 in chemoresistance
may result in cancer cell proliferation or
A
resistance to anti-cancer modalities, but it does
activation
not set off cancer initiation (Taguchi et al.,
protecting cancer cells from anti-cancer drugs
2010). In addition, mutations in Nrf2 gene are
used in chemotherapy. Several studies have
focussed in Keap1 binding domain near ETGE
highlighted the pivotal role of Nrf2 in
and DLG motifs termed as hot spot regions.
chemoresistance such as cisplatin in ovarian
Mutations in Nrf2 were observed in lung, head
cancer, cervical cancer or endometrial serous
and neck, oesophagus and skin cancers, but are
carcinoma; gemcitabine in pancreatic cancer;
less abundant (Kim et al., 2010; Shibata et al.,
doxorubicin in liver cancer and 5-fluorouracil
2008). Nrf2 deficient mice were more
in gastric cancer (Chen et al., 2012; Duong et
susceptible to urethane induced lung cancer
al., 2014; Jiang et al., 2010; Ma et al., 2012).
compared to Nrf2 wild type (Bauer et al.,
Elevated Nrf2 induces autophagy in ovarian
2013). The Nrf2 mutations are clustered
carcinoma
within ETGE (57%) and DLG (43%) moti,
cisplatin and tamoxefin (Bao et al., 2014). Due
which were indispensable for Keap1 binding.
to
Mutations in ETGE motif disrupt the high
potential, several Nrf2 dependent genes are
affinity binding with Keap1 and thus prevent
implicated in conferring Nrf2 mediated
distinctive
the
of
property
of
Nrf2
chemoresistance,
is
imparting
cytoprotective
constitutive
resistance
and
against
detoxifying
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
66
chemoresistance, e.g., HO-1 is over-expressed
inhibition
in
TrxR
activity
abrogates
in multiple cancers. Due to the cytoprotective
resistance against cisplatin (Sasada et al.,
nature, over-expression is undesirable in
1999). NQO1 is another Nrf2 dependent gene
cancer cells. Over-expression of HO-1 was
over-expressed in adrenal gland, bladder,
associated with increased cell proliferation
breast, colon, liver, lung, ovary, and thyroid
and endothelial cell division leading to
cancers (Basu et al., 2004; Siegel and Ross,
angiogenesis (Was et al., 2006). Other Nrf2
2000). Suppression in NQO1 expression
dependent genes including NQO1, GPX, TrxR
sensitizes A549 cells to etoposide, cisplatin
and Prx1 were shown to be up-regulated in
and doxorubicin (Wang et al., 2008c).
multiple cancer cells. GPx, a selenoprotein
that detoxifies H2O2, is implicated in the
Chemopreventive Effects of Nrf2: The
control of malignant growth. Elevated GPx
Bright Side
levels were observed in advanced stages of
Several compounds derived from natural or
colorectal
synthetic
mucosa
cancer,
and
Barrett`s
origin
with
chemopreventive
cancers
activity act via Nrf2. Administration of
associated with cell proliferation, growth and
methylcholanthrene reduced cancer incidence
inhibition of apoptosis (Banning et al., 2005;
in rats caused by carcinogenic azo dyes, served
Chu et al., 2004; Was et al., 2006). Peroxi-
as a nucleation point for use of dietary
redoxins (Prx) are thiol specific antioxidants
compounds
that detoxify peroxides and are elevated in non
(Richardson and Borsos-Nachtnebel, 1951).
small lung cancer (NSLC) and thyroid cancer,
Multiple plant derived products possess
a predictive factor for disease and associated
chemopreventive effect by inducing Nrf2
with prognosis (Kim et al., 2007; Yanagawa et
activation (Kelloff et al., 2000; Sporn and Suh,
al., 1999). Trx and TrxR collectively form a
2000; Talalay and Fahey, 2001; Yang et al.,
redox couple with a pivotal role in maintaining
2001). Nrf2 activation results in increased
cellular redox status in cellular functions
expression
(Brigelius-Flohe, 2008). Despite its protective
preventing biomolecules from the damaging
role as redox couple, TrxR1 was elevated in
effects of oxidative and xenobiotic stress. Nrf2
gastrointestinal cancer tissues (Arner and
knockout mice studies strengthened the notion
Holmgren, 2006; Iida et al., 2004). TrxR
of Nrf2 serving as a novel chemopreventive
knockdown lung carcinoma cells showed
factor controlling sensitivity to carcinogens
reversal of tumorigenicity and invasion.
(Slocum and Kensler, 2011). Ablation in Nrf2
Enhanced cellular expression of TrxR has been
led to enhanced tissues damage caused by
attributed
cigarette
to
gastrointestinal
esophageal
cisplatin
Biomed Res J 2015;2(1):57-82
resistance,
and
as
chemopreventive
of
smoke,
cytoprotective
hyperoxia,
agents
proteins
ischemic
67
Gambhir et al.
reperfusion, portal vein embolization, and
inhibition (Huang et al., 2015; Ying et al.,
chemical toxins (Chan et al., 1996; Cho and
2014). Nrf2 is also an anti-inflammatory
Kleeberger, 2010; Kudoh et al., 2014;
transcription factor and activation of Nrf2 and
Shirasaki et al., 2014; Zhao et al., 2011). Mice
dependent genes reduce chronic inflammation
with Nrf2 over-expression resulting from
associated
Keap1 knockout shows increased resistance to
pulmonary cancer. A protective role of Nrf2 is
lung cancer cell metastasis (Satoh et al., 2010).
supported by studies in mice with a single-
Nrf2 ablation was associated with enhanced
nucleotide polymorphism (SNP) in the
sensitivity to mutagens and showed increased
promoter region. The polymorphism was
carcinogenesis in bladder, skin, hepatocytes
associated with increased susceptibility to
and colon on exposure to nitrosoamine,
hyperoxia induced lung damage, due to low
ultraviolet, aflatoxin, dextran sulphate sodium
expression of Nrf2 (Cho et al., 2002;
and azoxymethane (Iida et al., 2004; Khor et
Yamamoto et al., 2004).
cancers
like
colorectal
or
al., 2006; Osburn et al., 2007; Saw et al., 2011;
Though higher levels of Nrf2 are observed
Xu et al., 2006; Yates et al., 2006). Curcumin,
in multiple malignancies, the role in initiation,
sulforaphane, oltipraz and CDDO-imidazole
promotion or transformation of normal cells
activate Nrf2 while exerting chemopreventive
remains contentious. Low levels of Nrf2 were
effects and Nrf2 deficiency in mouse models
essential for oncogenic transformation of
abrogated their chemopreventive effects
mesenchymal stem cells (Funes et al., 2014).
(McMahon et al., 2001; Ramos-Gomez et al.,
Epigenetic reactivation of Nrf2 attenuated
2003; Shen et al., 2006; Slocum and Kensler,
skin epidermal cell transformation (Su et al.,
2011; Sussan et al., 2009). Nrf2 has also been
2014). Over-expression of Nrf2 in cancer cells
implicated
ROS
may enable survival under conditions of
dependent genetic lesions that promote
oxidative stress, or detoxify xenobiotics
metastasis (Satoh et al., 2010). Under
leading to better survival. Nrf2 prevented
conditions of increased ROS levels, Nrf2
initiation of lung cancer, but accelerated
induced expression of Kruppel-like factor 9
progression through the Kras signalling
(Klf9), which further enhanced oxidative
pathway. Thus Nrf2 activators may pave the
stress mediated cell death (Zucker et al.,
way for prevention of lung cancer (Satoh et al.,
2014). The anti tumor potential of Klf9 in
2013).
in
protecting
against
different cancer types has been reported with
inhibition of Glioblastoma stemness through
Nrf2
transcriptional
Interventions
repression,
and
induced
apoptosis in prostate cancer cells by Akt
as
Target
for
Therapeutic
Nrf2 activation and enhanced expression of its
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
68
dependent genes associated with redox
oleanane triterpenoids. Administration of
regulating proteins, phase II detoxifying
several Nrf2 activators in clinical trials was
enzymes and transporters are exploited by
well tolerated, resulting in elevated levels of
cancer cells to survive and proliferate.
cytoprotective enzymes (Kensler et al., 2012;
Therefore, agents that inhibit Nrf2 expression
Linker et al., 2011; Palsamy and Subramanian,
in cancer cells may provide a novel strategy for
2011; Scannevin et al., 2012). In a Chinese
therapeutic interventions to enhance efficacy
study, aflatoxin intoxication as a risk factor
of existing chemotherapeutic drugs. Brusatol,
was reduced by oltipraz (Kensler et al., 2003).
Bruceajavanica,
Favourable effects of sulforaphane, a potent
a
plant
extract
from
selectively inhibits Nrf2 by increasing
Nrf2
activator,
were
observed
ubiquitination and degradation. It reduced
promotion or progression phase of cancer, and
resistance towards cisplatin in cultured
sulforaphane inhibited cancers of multiple
xenografts (Ren et al., 2011). 6-Hydroxy-1-
sites including skin, lung, bladder, breast,
methylindole-3-acetonitrile
(6-HMA)
colon and stomach (Conaway et al., 2005;
protected against cisplatin induced oxidative
Dinkova-Kostova et al., 2006; Gills et al.,
nephrotoxicity by inhibiting Nrf2 activation
2006; Hu et al., 2006; Shen et al., 2007).
(Moon et al., 2013). Luteolin, a plant derived
Chemoprevention
flavanoid, inhibited proliferation of tumor
extracts of broccoli are in clinical trials in
cells and reduced toxicity of cisplatin in a mice
China (Egner et al., 2011). Similarly, synthetic
model (Lin et al., 2010; Sun et al., 2012; Tang
oleanane triterpenoids reduced progression of
et al., 2011). All-trans retinoic acid (ATRA)
lung, breast and pancreatic cancers, and
inhibited Nrf2 by activating retinoic acid
delayed onset of tumor driven by Kras, Trp53,
receptor α, which directly interacts with Nrf2
Brca1 and Erbb2 oncogenes (Liby et al.,
and restrain binding to ARE (Wand et al.,
2010).
with
in
the
sulforaphane-rich
2013a). However, the use of Nrf2 inhibitors in
Salutary health effect of phytochemicals
cancer therapy is at a nascent stage and
that induce Nrf2 highlights the role of Nrf2-
requires development of specific agents to
activating foods and spices in human diet.
minimize non-specific off-target effects.
Food products like curcumin from turmeric
Assuming Nrf2 as a target for cancer
root,
sulforaphane
from
broccoli,
and
prevention, several population-based clinical
seaweed-based extracts from green alga Ulva
trials
diverse
lactuca were shown to activate the Nrf2
chemopreventive drugs including phenethyl
pathway in vivo. Extract with sulforaphane
isothiocyanate,
curcumin,
concentration that is achieved by dietary
resveratrol, fumaric acid esters and synthetic
broccoli consumption, offered protection
were
conducted
oltipraz,
Biomed Res J 2015;2(1):57-82
with
69
Gambhir et al.
against particulate pollution in humans
Future perspective and conclusion:
(Boddupalli et al., 2012; James et al., 2012;
Given the dual role of Nrf2 in cancer, the prime
Wang et al., 2013b). Similarly, phytochemical
query is the role of Nrf2 in cancer initiation or
constituents of garlic, tomatoes, grapes, green
cell transformation. Transient activation of
tea, coffee, and berries show Nrf2 activating
Nrf2 by pharmacological activators is safe for
properties, indicating beneficial effects by
the purpose of chemoprevention as the
dietary consumption (Kropat et al., 2013).
activators do not seem to increase the tumor
Numerous dietary supplement companies
burden. A major concern of use of Nrf2
have developed mixtures of known Nrf2
activators is their cytotoxicity and non-
activators to increase the antioxidant system in
specific mechanism of action. The activators
body. Protandim (LifeVantage, Inc, Sandy,
show a tendency to modulate cellular redox
UT, USA), reduced oxidative stress in humans
and are reactive towards cysteine residues
(Nelson et al., 2006).
which may lead to modulation of signalling
Apart
of
pathways. Thus, designing specific Nrf2
phytochemicals and dietary intake, life style of
activators like ETGE and DLG mimetic, based
an individual also plays an important role in
on co-crystal structure of Neh2 domain and
Nrf2 activation. A relationship between
Keap1, may reduce the off target effects.
physical activity and Nrf2 activation was
Further, demonstration of miRNA mediated
established in a mouse model and exercise-
regulation Nrf2 pathway provides a new
induced oxidative stress was higher in Nrf2
conduit to explore additional targets. Multiple
knockout
from
mice
the
(Miller
implication
et
al.,
2012;
studies highlight the cross talk of Nrf2 with
Muthusamy et al., 2012; Zhao et al., 2013).
other signalling pathways imperative for cell
Evidence from several studies provide a strong
survival. Results from our laboratory have
incentive for development of novel Nrf2
demonstrated implication of Nrf2 cross talk
activators as putative cancer chemopreventive
with NF-κB as a prime target for anti-
agents in normal healthy individuals without
inflammatory effect (Gambhir et al., 2014).
affecting pro-survival potential. However,
Thus, novel agents targeting Nrf2 pathway
caution must be exercised as a pro-
specifically needs investigation.
tumorogenic role of Nrf2 in various cancers
indicates dual nature of Nrf2 activation. Nrf2
SUMMARY
activation may provide a survival advantage to
Nrf2 is a redox sensitive transcription factor,
pre-existing cancer cells and also participate in
maintained at low basal levels under normal
resistance to chemotherapy or radiotherapy.
conditions. Upon activation, it mediates
expression of dependent cytoprotective genes,
Biomed Res J 2015;2(1):57-82
Diverging role of Nrf2 in cancer
70
Figure 3: Dual role of Nrf2. The bright side is indicated by Nrf2 functions in normal cells where it acts as cytoprotective
transcription factor inducing expression of an array of cytoprotective proteins, antioxidants and detoxifying enzymes
leading to increased survival and cancer prevention. In tumor cells, constitutive high levels of Nrf2 provides an ambient
niche for cancer cells to grow by reducing toxicity of endogenous ROS and xenobiotics. High levels of Nrf2 may increase
cancer cell survival and imparts chemoresistance leading to poor clinical outcome.
phase II detoxifying enzymes and antioxidant
and makes them refractory to chemotherapy
machinery. Evidences illustrating a positive
and/or radiotherapy. Evidence to directly
role of Nrf2 in cancer prevention has been
implicate Nrf2 in cancer initiation needs
documented. Thus, efforts are underway to
confirmation. However, Nrf2 facilitates a
identify novel agents that can activate Nrf2.
reducing environment through up-regulation
However, constitutive expression of Nrf2 may
of
prevent death of precancerous lesions and
machinery. Thus providing armour for cancer
promote survival of cancer cells under
cell to create an ambient growth niche and
oxidative stress suggesting a dual role (Fig. 3).
resist toxicity of xenobiotics. Hence, Nrf2 may
The transient activation of Nrf2 is beneficial,
serve as an additional target for therapeutic
in countering ill effects of xenobiotics,
interventions, increasing susceptibility of
oxidative
cancer in conjuction with chemotherapy or
stressors,
carcinogens
and
mutagens. Whereas, persistent activation of
Nrf2 in tumor cells confers survival advantage
Biomed Res J 2015;2(1):57-82
the
antioxidant
and
cytoprotective
radiotherapy treatment modalities.
71
Gambhir et al.
ACKNOWLEDGEMENTS
Bioscience Group, Bhabha Atomic Research
The authors would like to thank Dr. Anu Ghosh
Centre.
and Dr. S. Jayakumar for proofreading the
review. We also would like to acknowledge the
CONFLICT OF INTEREST
constant
The authors claim no conflict of interest.
encouragement
Chattopadhyay,
of
Associate
Dr.
S.
Director,
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Review
Physiology of Embryo-Endometrial Cross Talk
Deepak N. Modi* and Pradeep Bhartiya
Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health,Parel, Mumbai,
India
Implantation of the blastocyst stage embryo into the maternal endometrium is a critical determinant and a
rate-limiting process for successful pregnancy. Embryo implantation requires synchronized changes in the
endometrium before and after arrival of blastocyst into the uterine cavity. Extensive cross talks occur
between the fetal and maternal compartments around the time of implantation which are reflected by
morphologic, biochemical and molecular changes in the endometrial cells and the differentiating
trophoblast cells. The embryo induced morphologic changes include occurrence of epithelial plaque
reaction, stromal compaction and decidualization. Embryonic signals also alter the expression of a large
number of transcription factors, growth factors and their receptors and integrins. Thus the embryo
superimposes a unique signature on the receptive endometrium for successful implantation. Functionally,
the embryo-endometrial cross talk is essential for endowing a “selector activity” to the receptive
endometrium to ensure implantation of only a developmentally competent embryo. On selection, the
decidua creates a conducive microenvironment for trophoblast invasion leading to placentation. Clinical
evidences suggest that along with receptivity, a defective “selector” activity of the receptive uterus may be a
cause of infertility and recurrent miscarriages. Defects in trophoblast invasion are associated with
pregnancy complications like preeclampsia and intra-uterine growth retardation. It is envisaged that
understanding of the embryo-endometrial dialogue leading to the “selector” activity, aids in development of
appropriate therapeutic modalities for infertility related disorders and miscarriages. Conversely, it might
also benefit the development of anti-implantation drugs for contraception.
INTRODUCTION
Human reproduction is an inefficient process.
indicate that uterine implantation governs
Epidemiological evidences suggest that only
reproductive
30% of all conceptions get clinically
incompetence at the endometrial level can be a
recognized, and a large number of these are
constraining factor. Thus, understanding the
lost spontaneously. Furthermore, the success
molecular
of assisted reproduction is low, as reasonably
interaction is of interest to reproductive
good quality embryos fail to implant and there
biologists, clinicians and couples affected by
is a high frequency of spontaneous abortions.
infertility. The understanding is also essential
These epidemiologic and clinical evidences
for designing rational management strategies
success
events
of
and
maternal
embryo-maternal
Key words: Endometrium, Decidua, Implantation, Biosensor, Invasion, Trophoblast, Abortion.
*Corresponding Author: Deepak N. Modi, Molecular and Cellular Biology Laboratory, National Institute for
Research in Reproductive Health, Parel, Mumbai, India.
Email: [email protected], [email protected]
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
84
for implantation failure and treatment of
transformations. At the same time, the
infertility.
fertilized ovum undergoes several rounds of
Our current understanding of the process
cell division and transforms into blastocyst.
of embryo implantation and the determinants
The blastocyst has an outermost layer of
of successful pregnancy have mainly stemmed
specialized cells, trophoblast cells, that
from animal models and in vitro studies using
surround the pluripotent inner cell mass. The
human tissues. Based on the data derived it is
trophoblast cells come in direct contact with
clear that endometrial receptivity and embryo
the receptive endometrium establishing a firm
implantation are complex processes involving
attachment with the endometrial epithelial
a delicately poised balance of maternal
cells termed as apposition. Subsequently, the
hormones, endometrial factors and embryonic
trophoblasts invade the endometrium and
influences. The current review focuses on
establish contact with the maternal circulation
cellular and molecular events associated with
to form the placenta.
endometrial receptivity and implantation to
accomplish
successful
conception.
The endometrium is refractory to embryo
The
implantation throughout the menstrual cycle
embryo-endometrial cross talk at the time of
except for a few days after ovulation.
embryo apposition and implantation mainly in
Approximately, on days 21–24 of the human
the primates will be discussed. The general
menstrual cycle (8–10 days post ovulation),
understanding of the processes of endometrial
the uterus becomes "receptive", enabling
receptivity and implantation has been a subject
blastocysts
of recent reviews (Gellerson and Brosens,
epithelium.
2014; Ozturk and Demir, 2010; Young, 2013;
receptivity”, the achievement of this stage is
Young and Lessey, 2010).
highly dependent on the ovarian steroids,
to
adhere
Termed
to
as
the
luminal
“window
of
estrogen and progesterone. The estrogen in the
follicular phase leads to proliferation of
Endometrial receptivity
A mutual
communication
the
endometrial epithelial cells, the progesterone
blastocyst and the uterus is indispensable for
surge that occurs in response to ovulation leads
implantation. Akin to many developmental
to differentiation of the estrogen primed
processes, it involves an elaborate sequence of
endometrium to endow receptivity. Any
genetic and cellular interactions, to be
disturbance in the levels of these hormones
executed within an optimal temporal frame for
adversely affects endometrial physiology
successful pregnancy. In order to receive a
leading to failure of implantation. The
developing embryo, the endometrium endures
endocrine regulation of the menstrual cycle
a series of morphological and physiological
and role of hormones in endowing receptivity
Biomed Res J 2015;2(1):83-104
between
85
Modi and Bhartiya
to the endometrium has been a subject of
series of cell divisions and reaches uterine
extensive studies (Jabbour et al., 2006; Young,
lumen at the blastocyst stage. At this time the
2013).
trophoblast cells are differentiated and the
Morphologically, the receptive phase
embryo is ready to hatch. From rodents to
endometrium is characterized by presence of
humans, this embryo induces a second round
columnar epithelium with microvilli, an
of differentiation both at the morphologic and
increase in stromal cell proliferation and
biochemical level (Banerjee and Fazleabas,
appearance of pinopod-like structures on the
2009). Distinct from the “receptive” stage
luminal epithelium (Tu et al., 2014).
endometrium,
The
the
embryo
induced
morphological features of the “receptive”
differentiation of the receptive endometrium is
endometrium are associated with expression
rather limited and largely derived from
of a range of biochemical and molecular
experimental studies in the non-human
markers, crucial for endowment of this phase
primates and endometrial biopsies obtained
of
like
from conception cycles in humans. Three
transcription factors, integrins and their
major non-human primate models to identify
ligands, cytokines and growth factors, have
and dissect embryo induced morphological
been associated with the receptive phase (Tu et
and physiological changes in the receptive
al., 2014; Wang and Dey 2006; Zhang et al.,
stage
2013,). A molecular signature of the “receptive
hysterectomies
window”
profiling
endometrium of baboons or rhesus macaques
technologies have been identified that can
sequentially mated with males of proven
phenotype different phases of the menstrual
fertility; 2) Endometria obtained from mated
cycle including the receptive stage to
bonnet monkeys where the presence of the
objectively classify the implantation window
embryo
(Garrido-Gómez et al., 2013; Haouzi et al.,
preimplantation
2012). These findings open the field for the
Endometrial tissue obtained from baboons
diagnosis of the endometrial defects in assisted
where human chorionic gonadotropin (hCG)
reproductive technology programs (Ruiz-
has been infused in the uterine lumen in a
Alonso et al., 2013).
manner that mimics the transit of blastocyst.
the
uterus.
using
Several
global
markers
gene
endometrium
has
and/or
been
factor
are:
1)
biopsies
verified
assay;
Timed
of
the
using
a
and
3)
The models have inherent advantages and
Embryo induced morphologic changes in
disadvantages but are highly complementary
the receptive endometrium
and provide valuable information in terms of
In a conception cycle, the egg that has
identification and deciphering the functional
fertilized in the fallopian tube undergoes a
consequence of embryo induced changes in
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
86
uterine receptivity.
Beyond the plaque reaction, thinning of
the basal lamina and thickening and diffusion
Epithelial changes: The earliest endometrial
of the apical and lateral gap junctions in
response prior to implantation is characterized
luminal epithelial cells has been reported in
by an increased proliferative activity of the
pregnant
luminal and glandular epithelium. Distinct
endometrium (Demir et al., 2002; Rosario et
from the epithelial proliferation observed in
al., 2008). Along with these changes, a few
the follicular phase, this proliferative activity
granulocytic stromal cells are also observed in
in the pregnant uterus is restricted to focal
the luminal epithelium of pregnant bonnet and
areas. In the luminal epithelium, there are large
rhesus monkeys prior to implantation (Ghosh
clump of nuclei with distinct entero-
et al., 1993;
reduplication and poorly packed chromatin
conceivable that these changes occur to
along with loss of basement membrane. These
promote adhesiveness to the trophoblast cells
changes are termed as “epithelial plaque
at the time of apposition and invasion.
human,
bonnet
and
baboon
Rosario et al., 2008). It is
reaction” (Jones and Fazleabas, 2001; Rosario
et al., 2005a). The formation of epithelial
Stromal changes: An almost universal
plaques is restricted to pregnant endometrium
reaction of the endometrial stromal cells in
and reported in a variety of primate species
response to an embryo is decidualization. In its
including humans (Rossman, 1940). While
broadest sense, decidualization is defined as
consistently detected in the conception cycle,
the postovulatory endometrial remodeling
the epithelial plaque reaction is hormonally
which includes secretory transformation of
regulated and does not require presence of an
uterine stroma, influx of specialized uterine
embryo, as infusion of hCG directly in the
natural killer cells, and vascular remodeling. A
uterus leads to formation of epithelial plaques
more restricted definition of decidualization is
similar to those observed in pregnant monkeys
an
(Fazleabas et al., 1999; Jones and Fazleabas,
endometrial
stromal
2001).
specialized
and
The
functional
significance
of
epithelioid
transformation
cell
of
with
distinctive
the
highly
functions.
epithelial plaques is not clear. It is speculated
Decidualization only occurs in species in
that the plaque may provide nutrition by means
which placentation involves breaching of the
of intracellular glycogen (Enders et al., 1985;
luminal epithelium by the trophoblasts. The
Rossman, 1940). The plaque response may
extent of this differentiation process often
stimulate precocious development of the
correlates with the degree of trophoblast
maternal vasculature below the epithelium
invasion (Dunn et al., 2003; Gellersen et al.,
(Enders et al., 1985).
2007).
Biomed Res J 2015;2(1):83-104
87
Modi and Bhartiya
Morphologically, the elongated spindle
functionalis region of the endometrial bed
like stromal cells of the secretory phase
(Rosario et al., 2005a). Increased vascularity
endometrium transform into cobblestone like
and angiogenesis at the implantation site of
enlarged decidual cells with multiple club
rhesus monkeys has been reported (Sengupta
shaped projections arising from the cell
and Ghosh, 2002). A similar increase in the
surface and contain abundant glycogen stores
number of small blood capillaries in the
and lipid droplets (Welsh et al., 1985; Wynn et
stroma of the endometrial functionalis have
al., 1974). In humans, this transformation
been demonstrated in baboons infused with
occurs even in absence of an embryo and is
physiological doses of hCG in the uterine
referred to as the pre-decidual response. In a
lumen (Banerjee and Fazleabas, 2009; Jones
conception cycle, under the continuous
and Fazleabas, 2001). These observations
support of steroid hormones and blastocyst
suggest that maternal tissues initiate neo-
derived signals, decidualization of the entire
vascularization which may be required for
endometrium is observed (Brosens et al.,
immune cell differentiation and infiltration
2002; Gellersen and Brosens, 2014). The
(See below).
decidua forms a dense cellular matrix that
allows coordinated trophoblast invasion while
Immune cell infiltration:
simultaneously protecting the conceptus from
population in the endometrium consists of T
maternal and environmental insults (Kliman,
cells,
2000; Redhorse et al., 2004).
In the non-
lymphocytes. T cells and macrophages
human primates, decidualization is observed
account for a substantial proportion of the
in conception cycle or on treatment with hCG
leukocyte population in human endometrium
(Jones and Fazleabas, 2001; Rosario et al.,
throughout the menstrual cycle (Jones et al.,
2005a). These observations suggest that unlike
1998; King, 2000). The largest leukocyte
in humans, embryo/embryonic factors are
population in the human endometrium are the
required for the endometrial stromal cells to
large granulated lymphocytes which express
undergo decidualization in monkeys.
natural killer (NK) cell antigen CD56. The
macrophages
and
The leukocyte
large
granular
uterine NK (uNK) cell population is distinct
Vascularization: A characteristic feature of
from peripheral blood NK cells in phenotypic
the endometrium in a conception cycle is the
and molecular characteristics (Cooper et al.,
enhanced microvasculature. In the pregnant
2001; Fukui et al., 2011; King et al., 1991;
bonnet monkeys, a large number of small
Lysakova-Devine and O'Farrelly, 2014).
blood vessels are detected in the stroma
Around the time of implantation, uNK cells
underlying luminal epithelium and the
comprise 70–80% of the leukocyte population
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
88
in the endometrium and numbers increase if
during early pregnancy. The following section
conception occurs (King, 2000; Kodama et al.,
reviews the in situ molecular changes
1998). It remains to be identified whether the
occurring in the primate endometrium in
increase in cell number is solely the result of in
response to embryonic signals.
situ proliferation or homing from the
peripheral circulation. In a conception cycle,
Estrogen receptors (ER) and Progesterone
the uNK cells differentiate into decidual NK
receptors (PR)
(dNK) cells, functionally distinct from non-
Sex steroids exert their effects through their
pregnant uterine counterparts (Kodama et al.,
receptors,
1998). The functional significance of uNK and
progesterone receptor (PR). As compared to
dNK cells in the primate endometrium is
non-conception cycle, both ER and PR
largely speculative. Based on mouse studies
expression is higher in the conception cycle
and clinical observations, it appears that NK
around implantation in bonnets, baboons and
cells are crucial for pregnancy and failure of
rhesus (Ghosh and Sengupta, 1988, Rosario et
uNK transformation to dNK cells leads to
al., 2008). Post apposition ER expression is
pregnancy loss (Fukui et al., 2011; Gong et al.,
lost in the epithelium and stroma but retained
2014; Quenby and Farquharson, 2006).
in the wall of spiral arteries, blood vessels, and
Whether
myometrial smooth muscle cells (Hild-Petito
this
transformation
occurs
exclusively in response to embryo derived
signals or due to decidualization of the stromal
cells, needs to be investigated.
estrogen
receptor
(ER)
and
et al., 1992; Perrot-Applanat et al., 1994).
While PR is most abundantly expressed in
the uterine glands and stroma in the receptive
phase, expression of PR is down-regulated in
Embryo
induced
molecular
trans-
the glands but present in the stroma
formations in the receptive endometrium
surrounding the glands and spiral arteries, wall
The molecular dialogue between the embryo
of spiral arteries, blood vessels, and smooth
and endometrium involves a complex network
muscle cells of the myometrium (Ghosh and
of signaling molecules that mediate cell–cell
Sengupta, 1988; Hild-Petito et al., 1992;
or
Perrot-Applanat et al., 1994).
cell–extracellular
matrix
(ECM)
interactions, and include factors such as
cytokines,
growth
factors,
cell-adhesion
Homeobox genes HOXA10 and HOXA11
molecules and matrix metalloproteinases.
HOX genes are essential for endometrial
There is some evidence indicating that the
growth, differentiation and receptivity by
levels of steroid receptors, growth factors and
mediating some functions of progesterone.
cytokines are modulated in the endometrium
Both HOXA10 and HOXA11 are expressed in
Biomed Res J 2015;2(1):83-104
89
Modi and Bhartiya
human endometrial epithelial and stromal
transcriptome in luminal epithelium may be
cells, and their expression is significantly
inhibitory for implantation, and hence may be
higher in mid- and late-secretory phases,
down regulated by embryonic stimuli. As
coinciding with time of embryo implantation
transcription factors, HOX genes regulate
and high levels of estrogen and progesterone
other downstream target genes leading to
(Daftary and Taylor, 2006; Godbole et al.,
proper development of endometrium and
2007; Modi and Godbole, 2009; Taylor et al.,
receptivity to implantation. A number of
1998; Xu et al., 2014).
molecular and morphological markers specific
Unlike steroid receptors, the expression of
to the implantation window are regulated by
HOXA10 is induced in the endometria of
HOX genes, including pinopodes, β3 integrin
bonnet monkeys in the conception cycle.
and
Abundant expression of HOXA10 protein is
protein-1 (Daftary and Taylor, 2006; Modi and
detectable in stromal and glandular cells of the
Godbole, 2009). All the HOX targets are also
pregnant bonnet monkeys (Godbole et al.,
modulated in the endometria in response to the
2007). Interestingly, treatment of endometrial
embryo (Nimbkar-Joshi et al., 2009).
insulin-like
growth
factor-binding
cells with spent blastocyst culture medium
and/or hCG resulted in increased transcription
Cytokines and growth factors
of HOXA10 (Blitek et al., 2011; Fogle et al.,
Leukemia inhibitory factor (LIF), Interleukin-
2010; Sakkas et al., 2003). However, unlike
6 and -11 (IL-6 and IL-11) are members of a
the glands and the stroma, in luminal
single family of cytokines that share the signal
epithelium of the conception cycle, HOXA10
transducer receptor unit gp130 in target cells to
expression is reduced and expression is
elicit biologic effects. All these three cytokines
virtually absent in the pre-epithelial plaques
play key roles in implantation. First identified
(Godbole et al., 2007; Modi and Godbole,
in the mouse where targeted disruption of the
2009) These observations are surprising, as in
LIF
the mouse, suppression of HOXA10 in
(Stewart
epithelial cells leads to inhibition of embryo
expression/secretion has been reported
implantation; overexpression leads to increase
infertile women with defects in implantation
in litter size in mouse (Bagot et al., 2000).
(Mikolajczyk et al., 2006; Tawfeek et al.,
While this might reflect the fundamental
2012). While LIF seems to be a critical
differences in the mechanisms associated with
requirement for implantation, the expression
implantation
primates,
is not modulated by embryonic signals as the
observations in the monkey indicates that
levels do not alter in the implantation phase
products
endometria of bonnet monkeys in conception
in
of
rodents
and
HOXA10-modulated
gene
showed
et
al.,
implantation
1992),
failure
reduced
LIF
in
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
90
as compared to non-conception cycle (Rosario
conception of rhesus and bonnet monkeys
et al., 2005b). However, in rhesus the
(Rosario et al., 2005b; Sengupta et al., 2003).
expression of LIF and its receptors are
Several growth factors like Tumor Growth
increased in the endometria of pregnant
Factor (TGF) beta, Epidermal Growth Factor
monkeys as compared to non-pregnant
(EGF) and Tumor Necrosis Factor (TNF)
controls (Sengupta et al., 2003). LIF is crucial
alpha are pro-inflammatory cytokines that
for implantation in primates as administration
have emerged to be critical mediators for
of an antagonist for LIF receptor or antibody
implantation owing to their direct effects on
against LIF directly in to the uterine cavity of
immune cells (Dimitriadis et al., 2005;
monkeys and mice, results in failure of
Omwandho et al., 2010). In the window of
pregnancy (Sengupta et al., 2006; Terakawa et
implantation,
al., 2011; White et al., 2007). The results
endometrial TGF beta and its receptor occurs
indicate that LIF is essential in the process of
in the glandular epithelium of animals in the
blastocyst implantation. LIF is also a promotor
conception cycles as compared to non-
of trophoblast invasion (Suman et al., 2013a;
conception cycles (Rosario et al., 2005b;
2013b).
Sachdeva et al., 2001). TNF alpha and its
IL-6 and IL-11 are pleiotropic cytokines
receptor
a
significant
population
increase
increases
in
in
the
required for implantation. IL-11 expression is
endometria of animals in the conception
increased during decidualization (Godbole
cycles as compared to non-conception cycles
and Modi, 2010), recombinant IL-6 and IL-11
(Nimbkar-Joshi et al., 2009; Rosario et al.,
promote
human
2005c). EGF and its receptors are detected in
endometrial cells in vitro (Dimitriadis et al.,
both the glands and stromal compartments of
2005; Menkhorst et al., 2010). IL-6 and IL-11
the receptive phase endometrium; expression
are also promoters of trophoblast invasion
is increased mainly in the stromal cells of the
(Champion et al., 2012; Modi et al., 2011;
pregnant animals (Slowey et al., 1994). An
Suman et al., 2009; 2013). IL-11 and the
increase in endometrial LIF, EGF, TGF and
receptor IL-11Rα are detected in the decidua
TNF by endometrial cells in presence of
mainly at implantation sites in cynomolgus
embryonic stimuli prior to apposition suggests
and rhesus monkeys (Champion et al., 2012;
induction of an inflammatory like condition in
Dimitriadis et al., 2005). It is also detected in
the implantation phase endometrium, which
the vascular endothelial cells and epithelial
may be a requirement for initiation of
plaques. Likewise, the expression of IL-6 is
pregnancy; the increase in expression in
significantly higher in endometria of animals
stromal compartment implies involvement
in the conception cycles as compared to non-
with decidualization.
decidualization
Biomed Res J 2015;2(1):83-104
of
91
Modi and Bhartiya
pregnancy using the bonnet model. The results
Integrin and their ligands
revealed that expression of alpha v increases in
Integrins are heterodimeric glycoproteins
luminal epithelial cells of pregnant animals,
which undergo dynamic temporal and spatial
show a shift in localization at the site of
changes
the
attachment (Nimbkar-Joshi et al., 2012). At
endometrium during the menstrual cycle in
the non-attachment pole, the alpha(v) integrin
women (Lessey and Arnold, 1998; Reddy and
is mainly in the basal zone of the luminal
Mangale, 2003). Likewise the extracellular
epithelial cells. However, at the attachment
matrix (ECM) ligands for these receptors are
pole, alphav is redistributed and also detected
likely to play a role in the establishment of a
in the apical pole. The differential subcellular
receptive endometrium. The integrins and
distribution of integrin is directed by
their cognate ligands show dynamic changes
embryonic stimuli as treatment of epithelial
in levels of expression and polarization during
cultures with conditioned medium of human
early pregnancy. In the baboon, the collagen
embryos obtained at IVF leads to increased
receptor alpha1beta1 and fibronectin receptor
distribution of alpha v on the apical membrane
alpha4beta1 expressed in glandular epithelium
(Nimbkar-Joshi
during window of receptivity are lost with the
observations imply that embryonic stimuli not
establishment of pregnancy. The vitronectin
only directs cellular reprogramming by
receptor, alpha4 beta3 is expressed in the
changing gene expression, but also controls
glandular epithelium in pregnant animals. The
intracellular protein trafficking leading to
osteopontin
preferential sorting of proteins.
in
their
distribution
receptor,
alphav
in
beta3,
is
et
al.,
2012).
These
expressed in both glandular epithelium, and
From the above studies it is clear that
decidualizing stromal cells of pregnant
embryo induces distinct changes in the
animals (Fazleabas et al., 1997; Mangale and
receptive stage endometrium and affects
Reddy, 2007). In the mouse decidua,
almost all the compartments in preparation of
interactions between integrin alphav beta3 and
pregnancy. These changes seem to be induced
vitronectin is required to maintain a balance
in response to secretions by the embryonic
between cell proliferation and apoptosis, along
cells and are highly localized in nature. A
with modulation of inflammatory responses
summary of the morphological and molecular
(Mangale et al., 2008).
changes
Recently
the
dynamics
of
integrin
expression mainly alphav beta3 in the uterine
that
occur
in
the
receptive
endometrium in presence of an embryo are
shown in Fig 1.
epithelium has been detailed in early
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
92
Figure 1: Morphological and molecular changes in the endometrium in response to embryonic signals. The
receptive endometrium senses the endometrium and undergoes extensive biochemical and morphological remodeling.
The molecular changes that occur in the stromal and epithelial compartments are highlighted.
Functional
Consequence
of
the
designed to decipher functional consequences
Endometrial-Embryo Cross-Talk
of embryo induced changes in endometrial
From the discussion above it is clear that
cells (Weimar et al., 2012). While it would be
remarkable changes occur in the molecular
beyond the scope of this article to review these
profile of endometrium at the time of
studies, the data derived from these studies,
apposition and implantation, distinct from
combined with changes seen in vivo, it appears
those during the window of receptivity. The
that the embryo signals the endometrial bed
changes seem to be induced in response to
prior to implantation making it competent for
secretions by the embryonic cells and are
embryo quality control and trophoblast
localized in nature. However, the functional
invasion.
connotations of such observations remain far
from clear. This is mainly due to our inability
Decidua as a “Selector” for embryo quality
to perform genetic manipulations in the
control
endometria of primates. Nevertheless, in
The exceptional rate of early pregnancy loss
recent years, elegant in vitro models have been
may be due to the high prevalence of
Biomed Res J 2015;2(1):83-104
93
Modi and Bhartiya
chromosomal abnormalities in the embryo.
decidual cells challenged with conditioned
Genetic analysis of blastomeres taken from
medium from good and poor quality embryos,
good quality embryos obtained at in vitro
identified 449 decidual genes deregulated in
Fertilization (IVF) showed that around 70%
response to medium conditioned by poor-
harbor complex chromosomal abnormalities
quality embryos (Brosens et al., 2014). One of
(Chow et al., 2014; Mertzanidou et al., 2013).
the down regulated genes in response to
Such observations raise the question of how to
signals in conditioned media derived from
safeguard the mother against prolonged
poor quality embryos was HSPA8. The protein
investment in potentially developmentally
functions in protein assembly and folding,
abnormal embryos. One school of thought
clatherin-mediated endocytosis, assembly of
believes that abnormal embryos by themselves
multiprotein complexes, transport of nascent
are
implantation
polypeptides, and regulation of protein folding
resulting in pregnancy failure. In recent years
(Stritcher et al., 2013). The observation
however, experimental evidence indicate that
suggests
spontaneous decidualization of endometrium
developmentally impaired human embryos
coupled to menstruation is a judicious strategy
induce endoplasmic reticulum (ER) stress
to meet the challenge. The decidua may play a
response in decidualizing cells. An in vivo
key role in discriminating normal and
proof for the in vitro observations came from
abnormal blastocysts to allow pregnancy.
studies in uteri of mice flushed with
Evidences to support this hypothesis were
conditioned
obtained from co-culture of decidual cells with
developmentally competent and incompetent
morphologically normal and abnormal human
embryos.
embryos obtained at IVF. While morpho-
transcriptome revealed that medium derived
logically normal embryos had no major effects
from competent embryos evoked a supportive
on production of a selected set of cytokines;
intrauterine environment, whereas medium
media derived from decidual cells co-cultured
derived from poor quality embryos led to ER
with morphologically arrested or abnormal
stress (Brosens et al., 2014). Thus, it implies
blastocysts led to down-regulation of IL-1b, -
that the endometrium not only senses signals
6, -10, -17, -18, eotaxin and heparin-binding
derived from the embryo and responds to
EGF-like growth factor (Teklenburg et al.,
create a pro-implantation condition, but is also
2010a). Such down-regulation is associated
capable of terminating the window of
with closure of endometrial competence for
endometrial receptivity to enable the mother to
implantation and menstruation (Evans and
dispose of compromised embryos. The
Salamonsen, 2014). Microarray analysis of
observation adds another dimension to the
incompetent
towards
that
soluble
culture
Analysis
signal
medium
of
the
from
of
uterine
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
94
potential of decidualizing endometrial stromal
and decidual cells or spent medium increases
cells as sensors of embryo quality during
trophoblast invasion (Godbole et al., 2011;
implantation.
Menkhorst
Thus, we propose a dual-phase response of
the
endometrium.
The
steroid
et
al.,
2012).
We
have
demonstrated that decidual cell secretome
primed
enhances invasion of trophoblast cells through
receptive phase endometrium responds to the
altered expression of matrix metalloproteases
incoming embryo creating an obligatory
(MMPs) and tissue inhibitors of matrix
environment for implantation. At the same
metalloproteases (TIMPs) (Godbole et al.,
time the decidua gains a 'selector' activity to
2011).
recognize developmental competence of the
trophoblasts, the decidual cells also gain a
implanting embryo. Based on the blastocyst
migratory and invasive phenotype (Gellersen
competence as judged by the decidua, either
et al., 2010; Weimar et al., 2013). Thus,
pregnancy is continued or the maternal
decidualization and embryo driven changes in
response is aborted and culminates in
the uterine cells creates a microenvironment
menstruation.
favorable for implantation and placentation.
Conversely,
in
response
to
Numerous growth factors that regulate the
proliferation and invasion of trophoblast cells
Regulation of trophoblast invasion
Once
the
endometrium
a
have been identified at the fetal-maternal
developmentally competent blastocyst and
interface. The various factors secreted by the
decides to continue with pregnancy, the
decidual cells and/or the associated cell types
embryo apposes and trophoblast cells begin to
and their influence on trophoblast invasion has
breach the luminal epithelium and invade in to
been recently reviewed (Knöfler, 2010; Modi
the maternal decidua to establish placentation.
et al., 2012). Amongst the various factors, IL-
Trophoblast cells are inherently invasive and
6, LIF and IL-11 are abundantly produced by
can invade any tissue. However, in the
the endometrial stromal and decidual cells,
pregnant endometrium the invasion is highly
and play a key role in trophoblast invasion
controlled. It is believed that the decidualized
(Fitzgerald et al., 2008; Modi et al., 2012;
stromal cells secrete a complex array of
Suman et al., 2013a; Suman and Gupta, 2014).
molecules
controlled
IL-6 and LIF stimulates invasion of primary
migration/invasion. While several of the
trophoblast and JEG-3 choriocarcinoma cells
molecules are already expressed by the
via the STAT3 signaling pathway (Jovanović
receptive endometrium, others are induced
and Vićovac, 2009; Suman and Gupta, 2014).
post decidualization and receiving of the
The role of IL-11 in trophoblast invasion is
embryonic signals. Co-culture of trophoblast
less clear as it inhibits the invasion of primary
that
permit
Biomed Res J 2015;2(1):83-104
encounters
the
95
Modi and Bhartiya
trophoblast and HTR-8/SVneo cells, but
placenta formation, a major cause of
increases invasion of the choriocarcinoma
pregnancy related complications like fetal
JEG-3 cells (Suman et al., 2009; 2012; 2013b).
growth
The
hypertension leading to preeclampsia.
discrepancy
may
originate
from
differences in the transcription factor content
In
restriction
converse,
and
impaired
gestational
endometrial
of the two cell lines. However, the data
selectivity can result in superfertility. The
suggests that locally produced IL-6, LIF and
hypothesis stems from the observations that
IL-11 act to finely tune invasion. While the
women with recurrent miscarriages are highly
cumulative effects of various factors and their
fecund and time to pregnancy is reduced in
roles
under
in
vivo
need
those women with a history of five or more
investigations, the observations together
miscarriages (Teklenburg et al., 2010b). Since
suggest
developmentally
that
conditions
decidualization
driven
incompetent
blastocyst
transformation of endometrial stromal cells
implant (due to failure of selectivity), these
creates a uterine microenvironment that
would lead to late first trimester abortions.
controls trophoblast invasion.
Thus lack the “selector” activity in the
endometrium may be a causative factor
Clinical Repercussions of the Embryo-
towards compromised pregnancy. Indeed, a
Endometrial Cross-Talk
loss of selection sensing has been observed in
Endometrial receptivity is a major rate limiting
endometrial stromal cells derived from
step and bottleneck for the success of assisted
women experiencing recurrent miscarriages
reproductive technologies. The discovery that
(Salker et al., 2010). Furthermore, when
embryonic signals potentiate the already
flushed through the mouse uterus, secreted
primed uterus has opened several avenues for
factors from decidualizing cultures of stromal
understanding of the process of implantation
cells derived from patients with recurrent
and initiation of pregnancy. Given the
miscarriages prolonged the window of
experimental evidence demonstrating the
receptivity and also increased the incidence of
embryo-endometrial cross talk plays a key role
pathological implantation sites, immune
in endowing receptivity as well as selectivity
defects and fetal demise (Brosens et al., 2014).
to the endometrium, a logical consequence of a
Additionally, endometrial stromal cells from
reduced ability to recognize embryonic signals
patients with recurrent miscarriages show
is implantation failure and/or miscarriage.
altered responses to hCG, and failure to
Suboptimal response to signals of high quality
discriminate between high and low quality
embryos
suboptimal
human embryos (Salker et al., 2010; Weimar
environment for subsequent development and
et al., 2012). Thus, the “selector” activity of
will
result
in
a
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
96
the decidua may be a key to successful
al., 2012). It is believed that 'poor placentation'
pregnancy and defects in the process may
along
cause recurrent miscarriages.
fetoplacental
Once the embryo has implanted, the
trophoblast
cells
invade
to
establish
with
hypoxic
site,
micromilieu
shear
stress
of
of
uteroplacental blood flow, and aberrantly
secreted proinflammatory substances into
placentation. Multiple stages of placentation
maternal
could be compromised that can lead to
contribute to progression of preeclampsia and
diseases. Pre-eclampsia affecting 3–5% of
IUGR (Furuya et al., 2008; Gourvas et al.,
pregnancies, which is characterized by
2012; Ji et al., 2013; Saito and Nakashima,
gestational
2014).
hypertension
and
severe
circulation,
Since
trophoblast
proteinuria, and is a major cause of fetal and
placentation
maternal deaths.
decidualization,
While pre-eclampsia is
are
synergistically
invasion
dependent
defective
on
and
proper
embryo-
detected later in gestation (20 weeks onwards),
endometrial cross talk can lead to improper
its pathogenesis is established early in
decidual response thereby causing poor
gestation where trophoblast invasion is
trophoblast invasion, shallow placentation and
defective. It has been shown that in women
hence preeclampsia. Preliminary evidence
with preeclampsia shallow placental invasion
suggests that decidualization defects might
and inadequate plugging of the spiral artery
exist in decidua of women with pregnancies
affects blood supply into the intervillous space
complicated with preeclampsia (Saito and
and alters the consistency of the blood flow.
Nakashima, 2014).
This can lead to fluctuations in the supply of
Thus, there is a need for better
oxygen to the placenta (Ji et al., 2013; Saito
understanding of the basic processes of
and Nakashima, 2014), triggering a maternal
placentation and mechanisms that go awry in
response by increasing blood pressure and
women with preeclampsia and IUGR for
compromising fetal development (Furuya et
effective therapeutic approaches to these
al., 2008). Another disorder caused by defects
common disorders.
in trophoblast invasion is intrauterine growth
restriction (IUGR). IUGR arises as a result of
CONCLUSIONS
inadequate blood supply and/or inadequate
The embryo-endometrial cross talk has
transport of nutrients across the placenta to the
evolved as a delicately poised mechanism to
fetus, resulting in a range of mechanisms
respond initially to the hormonal trigger to
including reduced uteroplacental blood flow,
achieve receptivity and then to amplify the
compromised feto-placental angiogenesis and
decision under embryonic signals to permit
subsequent villous development (Gourvas et
pregnancy. During this critical period, the
Biomed Res J 2015;2(1):83-104
97
Modi and Bhartiya
Figure 2: Biosensor activity of the implantation stage endometruim. A) In presence of a normal good quality embryo
the endometrium undergoes extensive biochemical and molecular transformation allowing the apposition of the embryo
to the luminal epithelium. Consequently, the secretory factors from the decidua promote invasion of trophoblast cells
allowing plancetation. B) In presence of a poor quality embryo the endometrium responds by activating a strong
inflammatory cascade thereby triggering closure of receptivity leading to menstruation.
receptive endometrium gains the ability for
survival ability. Once the receptive and the
selectivity, where response of the luminal
selective competence of the endometrium are
epithelium serves to transduce and amplify
ensured and the right blastocyst implants, the
signals coming from competent embryos
decidua creates a local microenvironment that
renders the underlying decidual layer more
is conducive for trophoblast invasion and
receptive to invasion (Fig. 1). This permits
placentation (Fig. 2).
embryo apposition followed by trophoblast
The significance of such bimodal and
invasion to establish placentation (Fig. 2). In
biphasic
the event the endometrium experiences
implanting embryo is potentially far reaching.
presence of a poor-quality embryo, the
To date, treatment of recurrent miscarriages
supportive network is not activated, but the
and implantation failure are inefficacious and
decidua mount a stress response, leading to
highly empirical. The recent understanding of
withdrawal
in
the dual processes has revealed that recurrent
menstruation and failure of pregnancy (Fig. 2).
implantation failure may be caused by defects
Such a selector mechanism of the decidua is
in endometrial embryo cross talk. It will be
highly desirous to avoid investing energy in
necessary to unravel the molecular processes
pregnancy with abnormal fetuses which may
that control the timely transition of the
not survive till term or have compromised
receptive uterus to a selective decidua which
of
receptivity
resulting
endometrial
response
to
the
Biomed Res J 2015;2(1):83-104
Embryo-endometrial cross talk
98
subsequently permits trophoblast invasion to
establish
placentation.
Once
elucidated,
ACKNOWLEDGEMENTS
effective approaches to modulate implantation
The authors acknowledge Indian Council of
and treat pregnancy complications will be
Medical Research (ICMR), New Delhi, for
feasible proposition. The knowledge acquired
financial assistance.
from such studies, is envisaged to assist in the
development of specific therapeutics for
CONFLICT OF INTEREST
infertility disorders, and may also lead to the
The authors claim no conflict of interest.
development of new and improved methods
for endometrium based contraception.
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Research Article
Human EGFR-2, EGFR and HDAC Triple-Inhibitor
CUDC-101 Enhances Radiosensitivity of GBM Cells
Cody D. Schlaff1, W. Tristram Arscott1,2,3, Ira Gordon1, Kevin A. Camphausen1 and Anita Tandle1*
1
Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda MD, USA
2
University of Vermont College of Medicine, Burlington, VT, USA
3
Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD, USA
Radiotherapy remains the standard treatment for glioblastoma multiforme (GBM) following surgical
resection. Given the aberrant expression of human epidermal growth factor receptor 2 (HER2) and
epidermal growth factor receptor (EGFR) which may play a role in therapeutic resistance to receptor
tyrosine kinase inhibitors, and the emerging use of histone deacetylase (HDAC) inhibitors as
radiosensitizers, we defined the effects of CUDC-101, a triple inhibitor of HER2, EGFR and HDAC on the
radiosensitivity of GBM cells. Clonogenic survival was used to determine the in vitro radiosensitizing
potential of CUDC-101 on GBM, breast cancer, and normal fibroblast cell lines. Inhibitory activity was
defined using immunoblots and DNA double strand breaks were evaluated using γH2AX foci. Effects of
CUDC-101 on cell cycle and radiation-induced cell kill were determined using flow cytometry and
fluorescent microscopy. CUDC-101 inhibited HER2, EGFR and HDAC and enhanced in vitro
radiosensitivity of both GBM and breast cancer cell lines, with no effect on normal fibroblasts. Retention of
γH2AX foci was increased by CUDC-101 alone and in combination with irradiation for 24 h. Treatment with
CUDC-101 increased the number of cells in G2 and M phase, with only increase in M phase statistically
significant. An increase in mitotic catastrophe was seen in a time-dependent fashion with combination
treatment. The results indicate the tumor specific CUDC-101 enhanced radiosensitization in GBM, and
suggest that the effect involves inhibition of DNA repair.
INTRODUCTION
The
development
of
hematological malignancies; or using a single
molecularly targeted radiosensitizers is crucial
agent to target a specific pathway, such as
for improving the efficacy of radiotherapy as a
erlotinib,
cancer
main
selectively inhibit receptor tyrosine kinases
approaches exist regarding the incorporation
(RTKs). The latter has historically been the
of
incorporating
more popular approach in solid malignancies
multiple drugs known to target multiple
for the vast majority of newly investigated
distinct pathways, such as, in the treatment of
cancer treatment regimens, particularly when
treatment
molecular
and
application
modality.
inhibitors:
Two
gefetinib
or
lapatinib
which
Key words: Glioblastoma multiforme, Radiosensitization, HDAC, Multi-target therapy, HER2, EGFR.
*Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive
Magnuson Clinical Center Room B3-B100, Bethesda MD 20892, USA.
Email: [email protected]
Biomed Res J 2015;2(1):105-119
CUDC-101 induced tumor cell radiosensitization
106
combined with radiotherapy. Single-target
agents,
despite
their
CUDC-101 has been shown to be an
pharmacokinetic
effective agent as a monotherapy for treatment
simplicity, lower cost and reduced risk of
of various tumor cell lines in vitro including
adverse effects, are often clinically limited due
non-small cell lung cancer, pancreatic, breast,
to the genetic heterogeneity and myriad
prostate, brain, and liver cancers (Lai et al.,
dysregulated pathways that exist not only
2010), and is currently undergoing phase I and
among different malignancies, but from cell to
Ib clinical trials as a monotherapy or in
cell variations within the same histology (Lai
combination with radiotherapy for multiple
et al., 2010).
cancers. As an initial step in evaluating the
Recently, single small molecular inhibitors
potential of CUDC-101 as a clinically
have been designed to simultaneously target
applicable radiosensitizer, we investigated the
multiple critical cellular pathways to induce
effects of CUDC-101 in a panel of cancer and
cell death. The drug, CUDC-101, was
normal cell lines. The data indicated that
designed to target two members of the ERBB
CUDC-101 selectively enhances tumor cell
family, human epidermal growth factor
radiosensitivity in vitro. Moreover, the
receptor 2 (HER2) and epidermal growth
mechanism appears to involve inhibition of
factor receptor (EGFR), as well as function as
DNA double strand break (DSB) repair and
a pan-histone deacetylase inhibitor (HDACi)
modulation of the cell-cycle.
(Lai et al., 2010). The overexpression of these
two
RTKs
has
been
associated
with
MATERIALS AND METHODS
tumorigenesis and aggressiveness in many
Cell lines and treatment
cancers, including glioblastoma multiforme
The human GBM cell line U251, breast
(GBM) (Cancer Genome Atlas Research,
carcinoma cell line MDA-MB-231 and
2008; Kesavabhotla et al., 2012; Lal et al.,
normal human lung fibroblast cell line MRC9
2002; Mittapalli et al., 2013; Pierga et al.,
were obtained from American Type Culture
2013; Wang et al., 2013). Additionally, histone
Collection (ATCC). U251 and MDA-MB-231
acetyltransferase
cells
inactivation
has
been
were
grown
and
maintained
in
associated with oncogenesis, yet it is the
Dulbecco's Modified Eagle Medium (DMEM;
aberrant HDAC activity that is considered a
Invitrogen) supplemented with 10% Fetal
potential target for cancer therapy. Depending
Bovine
on the experimental system, HDACi has been
maintained at 37ºC, 5% CO2. MRC9 cells were
reported to induce tumor cell differentiation,
grown and maintained in minimum essential
apoptosis, and/or growth arrest, putatively via
medium (MEM; Invitrogen) supplemented
modulation of gene expression (Shabason et
with 10% FBS, non-essential amino acids
al., 2011).
Biomed Res J 2015;2(1):105-119
Serum
(FBS;
Invitrogen)
and
107
Schlaff et al.
(NEAA), and sodium pyruvate (Invitrogen).
Cell cycle analysis
Lyophilized CUDC-101 (MW: 434.49) was
The evaluation of the cell cycle phase
purchased from Selleck chemicals and 3.43
distribution was performed using the BD
mg of CUDC-101 was reconstituted in 1 mL of
FACSCalibur. Treatment protocols were
DMSO (solubility: 20 mg/mL) and stored at -
identical to the clonogenic treatment regimen
20 ºC at a concentration of 10 mM. DMSO was
and cells were seeded into 10 cm petri dishes.
not shown to have an effect and subsequent
Samples were stained with propidium iodide
experiments were done with a media only
(PI) and analyzed using flow cytometry. To
control unless otherwise noted. Cultures were
determine the activation of the G2 cell cycle
irradiated using the Pantak source at a dose of
checkpoint, mitotic cells were distinguished
2.27 Gy/min.
from G2 cells as previously reported by Xu et
al. (2002) utilizing the mouse monoclonal
antibody (Cell Signaling) against phospho-H3
Clonogenic Assay
Cultures
were
dissociated
with
0.25%
histone (S10) (6G3) followed by staining with
trypsin EDTA (Invitrogen) to create a single
a Alexa Fluor-488® F(ab`)2 fragment of goat
cell suspension and a specified number of cells
anti-mouse conjugated secondary antibody
were seeded into each well of a six well tissue
(Invitrogen) (Xu and Kastan, 2004; Xu et al.,
culture plate. After allowing cells time to
2002). In this assay the increase of M phase
adhere (24 h) cultures were treated with
reflects the abrogation of the G2 checkpoint.
varying doses of radiation: 0, 2, 4, 6, and 8 Gy,
The data represents the mean ± SEM of
followed by CUDC-101 (0.5 µM for U251 and
minimum of three independent experiments.
1.0 µM for MDA-MB-231). The drug was not
removed for duration of the assay. Ten to
Apoptotic Cell Death
fourteen days after seeding, colonies were
The BD Annexin V: FITC Apoptosis
stained with crystal violet, and the number of
Detection Kit (Catalog Number: 556547) was
colonies (≥ 50 cells) were determined.
performed
Surviving fractions were calculated and
instructions. In brief, cells were washed twice
survival curves generated by normalizing for
with cold PBS and resuspended in 1X binding
the amount of CUDC-101-induced cell death.
buffer.
The data represents the mean ± standard error
suspension was incubated for 15 minutes at
of mean (SEM) of minimum two independent
room temperature (25ºC) in the dark after
experiments.
adding 5 µL of FITC-Annexin V and PI.
as
per
Subsequently,
the
100
manufacturer's
µL of
cell
Samples were brought to a final volume of 500
Biomed Res J 2015;2(1):105-119
CUDC-101 induced tumor cell radiosensitization
108
µL and run on the FACSCalibur capturing
limiting criteria. For each condition 00 cells
10,000 events.
were scored. The data represents the mean ±
SEM
Immunofluorescent staining for γH2AX
of
minimum
three
independent
experiments.
Immunofluorescent staining and counting of
γH2AX nuclear foci was performed as
Immunoblotting and antibodies
previously described (Xu and Kastan, 2004;
Cells were seeded onto 60-mm dishes, lysed in
Xu et al., 2002). Cells were seeded on four
radioimmunoprecipitation
well chamber slides and treated with 2 Gy
buffer (Sigma) containing phosphatase and
irradiation followed by treatment with CUDC-
protease inhibitors (Roche, Indianapolis, IN).
101. Slides were examined on a Leica upright
Protein concentrations were quantified using a
fluorescent
were
DC Protein Assay kit (Bio-Rad Laboratories,
imported into ImageJ image software for
Hercules, CA). Thirty micrograms of protein
analysis. For each treatment condition γH2AX
was resolved on 4% to 20% Tris-Glycine gels
foci were counted in 50 cells. The data
and transferred to nitrocellulose membranes
represents the mean ± SEM of minimum three
(Bio-Rad Laboratories) and probed with the
independent experiments.
indicated antibodies. Primary antibodies were:
microscope.
Images
assay
(RIPA)
actin, CD9 (Millipore, Germany), phosphoMitotic Catastrophe
/total EGFR, and acetyl-/total histone H3 (Cell
The presence of fragmented nuclei was used to
Signaling Technology, Danvers, MA) and
define cells undergoing mitotic catastrophe.
phospho-/total HER2 (Upstate Cell Signaling
Cells were seeded on cover slips and treated
Solutions, Lake Placid, NY). Blots with
with 2 Gy irradiation followed by treatment
phosphorylated targets were visualized with
with
nuclear
Super-Signal West Femtoluminol substrate
fragmentation cells were fixed with a 10%
(Thermo Scientific, Rockfold IL) and ECL
neutral buffered formalin solution and
Prime luminol reagent (GE Health-care,
incubated with α-tubulin followed by anti-
Pittsburgh, PA) was used for total protein.
mouse Alexa-555 and mounted with Prolong
Actin was used as a loading control.
CUDC-101.
To
visualize
gold antifade reagent containing DAPI.
Normal cells and cells undergoing mitotic
Statistical Analysis
catastrophe were manually counted as cells
In vitro experiments were repeated minimum
with nuclear fragmentation. For cells to be
twice and Student's t-test was used for
considered positive for mitotic catastrophe,
statistical analyses. Data are presented as
cells with greater than 2 lobes were used as the
mean ± SEM. A α value of p < 0.05 was
Biomed Res J 2015;2(1):105-119
Schlaff et al.
109
considered significant. Analyses were done in
assay was performed initially to determine
GraphPad version 6 (Prism).
appropriate dosage with minimal toxic effects.
For this study, cells were plated at clonogenic
RESULTS
density, allowed to attach overnight, and
CUDC-101 can effectively target intended
treated with 4 Gy irradiation followed
ligands
immediately by treatment with CUDC-101
To assess the effects of CUDC-101 on the
using a dose range including the previously
radiosensitivity of tumor cells, a cytotoxicity
reported average IC50 values of U251 and
Figure 1: CUDC-101 effectively targets the intended ligands. The U251 was radiated (0 or 4 Gy) and incubated with
0.1 or 0.5 µM of CUDC-101 for 24 h. (A) Levels of phosphorylated HER2 and EGFR, and acetylated histone H3 were
analyzed via immunoblot analysis. (B) Results show a decrease in phosphorylated HER2 and total EGFR in the presence
or absence of radiation at 0.5 µM; however no effect was seen on pHER2 and pEGFR at 0.1 µM. Furthermore, acetyl-H3
is maintained and increased in the presence of CUDC-101 with/without radiation at both concentrations tested,
supporting the effect seen by clonogenic survival.
Biomed Res J 2015;2(1):105-119
CUDC-101 induced tumor cell radiosensitization
110
Figure 2: CUDC-101 radiosensitivity is tumor cell specific. Cells were radiated with increasing doses of radiation
(closed circles) or post radiation incubated with various concentrations of CUDC-101 for 10 to 14 days (open circles).
Colony-forming ability was assessed and survival curves generated after normalizing for the cytotoxicity of CUDC-101.
(A) U251 cells were given 0.5 µM CUDC-101 and (B) MB231 cells were given 1.0 µM. (C) Normal fibroblast cell line
MRC9 was irradiated with increasing doses of radiation (closed circles) or 0.5 µM CUDC-101 (open circles) or 1.0 µM
CUDC-101 (closed triangles) following irradiation. Survival curves show that the radiosensitive activity of CUDC-101 is
tumor cell specific, whereas MRC9 cells were not affected. Dose enhancement factors (DEFs) were assessed at
surviving fractions (SFs) 0.1 and 0.01 where applicable. Data represents three-independent experiments (A-B) and twoindependent experiments (C) with points representing mean, and error bars SEM.
MDA-MB-231 cells. U251 and MDA-MB-
molecule's intended RTK targets, CUDC-101
231 cells were kept in drug containing media
was given alone or immediately following
for duration of the assay (10 to 14 days). Cells
irradiation (4 Gy) to U251 cells and RTK
were then stained with crystal violet and
inhibition was analyzed via immunoblot.
assessed for inhibition of colony formation.
Results confirmed that CUDC-101 targeted
Dosing CUDC-101 at 0.5 μM and 1.0 μM
the intended ligands (Fig. 1). CUDC-101
immediately post-radiation for the duration of
treatment
the entire assay was most effective in
phosphorylated
inhibiting colony formation while having
dependently, irrespective to the addition of
minimal single agent toxic effect on U251 and
irradiation, while pEGFR levels were only
MDA-MB-231 respectively (Data not shown).
modulated at 0.5 µM and no further decrease
These concentrations were used to further
in ligands level with combination treatment
investigate the radiosensitive effects of
was
CUDC-101.
however, the levels of total EGFR decreased
To confirm that CUDC-101 inhibited the
Biomed Res J 2015;2(1):105-119
reduced
observed
the
HER2
(Fig.
activation
(pHER2)
1A).
of
dose
Interestingly
dose dependently in both single agent and
111
Schlaff et al.
combination treatment cohorts. Also, as
approximate
value
for
the
degree
of
expected of an HDACi, treatment with
cytotoxicity for the evaluation of CUDC-101
CUDC-101 increased levels of acetylated H3
in combination with radiation. As shown in
in a dose dependent manner. The protein levels
Fig. 2A and B, a dose enhancement factor
are quantitated and normalized against a
(DEF) of 1.42 in U251 and 1.37 in MDA-MB-
loading control, actin (Fig. 1B).
231 was observed at a SF of 0.1.
Accordingly, the potential for tumor-
CUDC-101 treatment inhibits clonogenic
specific
survival tumor specifically
radiosensitivity was determined. The normal
The hallmark of radiosensitivity is reduced
fibroblast cell line MRC9 was treated with
clonogenicity. In both the tumor cell lines,
both 0.5 μM and 1.0 μM using the same
treatment
with
treatment schedule and colony formation was
irradiation
resulted
CUDC-101
following
actions
of
CUDC-101
on
in
assessed as previously mentioned. CUDC-101
radiosensitivity as assessed by colony forming
treatment reduced the MRC9 SF to 0.79 ± 0.15
ability 10 to 14 days later. U251 cells treated
and 0.87 ± 0.007 respectively; in contrast to
with 0.5 μM CUDC-101 yielded a surviving
the tumor cell lines, CUDC-101 had little
fraction (SF) of 0.67 ± 0.12; while treatment of
effect on the radiosensitivity (1.13 at 0.5 μM;
MDA-MB-231 cells with 1.0 μM gave a SF of
1.25 at 1.0 μM) of MRC9 (Fig. 1C). Albeit
0.65 ± 0.07. These values indicate an
some sensitivity was induced it was not
in
an
increase
Figure 3: Treatment with CUDC-101 plus radiation impairs the DNA damage repair response. U251 cells seeded in
chamber slides were exposed to 2 Gy irradiation followed by 0.5 μM CUDC-101 and fixed at specified times for
immunoflourescent analysis of nuclear γH2AX foci retention. Foci were evaluated in ≥ 50 nuclei per treatment per
experiment. (A) Representative images obtained from media at 24 h (top left panel), 2 Gy irradiation at 24 h (top right
panel), 0.5 μM CUDC-101 treatment after 24 h exposure (bottom left panel), and 0.5 μM CUDC-101 immediately
following 2 Gy irradiation at 24 h exposure (bottom right panel). Significant retention of γH2AX foci occurs with drug alone
and combination therapy after 24 h with the combinatory effect significantly increased over drug only. (B) Data represents
three independent experiments. Columns represent the mean and error bars are the SEM. * p< 0.05 ** p< 0.001.
Biomed Res J 2015;2(1):105-119
112
CUDC-101 induced tumor cell radiosensitization
Figure 4: CUDC-101 increases the percentage of cells in more sensitive phases of the cell cycle. U251 cells were
seeded in 10-cm petri dishes, stained with PI and pH3 to differentiate mitotic cells and analyzed by flow cytometry. (A)
Representative histogram of cell cycle distribution of U251 cells treated with drug or irradiation alone or combination
treatment from an independent experiment. (B) Representative dot plots of cell cycle distribution of U251 cells from an
independent experiment, gating and analysis was done using FloJo analysis software. (C) Treatment with 0.5 µM CUDC101 in the absence of irradiation significantly increased the number of cells in M-phase; however this effect was not
augmented by the addition of irradiation (2 Gy). Additionally an increase in the G2 phase was observed for both CUDC101 alone and in combination with irradiation but was not statistically significant. Data represents three independent
experiments. Columns represent the mean and error bars are the SEM. *p<0.05; **p< 0.001.
Figure 5: CUDC-101 increases mitotic catastrophe. U251 cells were grown on cover slips and were irradiated (2 Gy)
and exposed to CUDC-101. At 24, 48 and 72 h after treatment cells were fixed for immunocytochemical analysis of mitotic
catastrophe. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was
evaluated in at least 150 cells per cohort. Representative fluorescent micrographs are of cells fixed at 72 h after treatment
for (A) medium, (B) 0.5 µM CUDC-101, (C) 2 Gy irradiation and (D) 0.5 µM CUDC-101 following 2 Gy irradiation. (E)
Graph shows percent mitotic catastrophe; *p< 0.05; ***p< 0.0001. Analysis was done using a two-way ANOVA with
Bonferroni multiple comparisons post-test.
Biomed Res J 2015;2(1):105-119
113
Schlaff et al.
statistically significant. Since the effect seen in
treated with combination radiation and 0.5 μM
GBM was significant compared to drug alone
CUDC-101 (14.40 ± 0.92; p = 0.0003). As
and the DEF was greater than that seen in the
expected at 24 h almost complete resolution of
breast cancer cell line, we focused on this
DNA DSBs was seen in cells that were given
histology. Further experiments to investigate
irradiation only, as the average number of foci
the radiosensitization effects of CUDC-101
per cell returned to near baseline levels. The
used U251 cells.
significant inhibition of γH2AX resolution at
24 h suggests that CUDC-101 inhibits repair
CUDC-101 treatment increased retention
of DNA DSBs.
of γH2AX foci
The most lethal form of injury to cells is DNA
CUDC-101 post irradiation redistributes
damage. Thus drugs inhibiting or retarding
GBM cells within cell cycle
DNA repair have the potential of effective
Progress through the cell cycle depends on
radiosensitizers. Therefore, the rate of DNA
regulated set of checkpoints, which become
repair via resolution of γH2AX foci was
activated in the event of DNA damage. It is
measured. The γ-variant of the H2A family
known that radiation has an effect on inducing
was shown to be a biomarker of DNA double
G2/M delay
stranded
delayed
Therefore, the distribution of tumor cells
resolution of the foci indicate inhibition of
within the cell cycle was assessed by flow
DNA repair mechanisms (Kuo and Yang,
cytometry (Fig. 4A-B). As shown in Fig. 4C, at
2008). CUDC-101 was added following
24 h an increase of cells in G2 was observed,
irradiation (2 Gy) and at 1, 6 and 24 h and U251
although the increase was not significant (25.8
nuclei were visualized via immunofluorescence
± 3.5% to 40.5 ± 7.0%; p = 0.134). Addition-
for
resolution.
ally, at 24 h a significant increase in M phase
Representative images at 24 h are shown in
from 2.30 ± 0.24% to 6.70 ± 1.1% was
Fig. 3A. At 1 h and 6 h a significant difference
observed with CUDC-101 alone (p = 0.02).
in the average number of foci per cell was not
The addition of radiation to CUDC-101,
observed (Fig. 3B). However, there was a
however, did not augment this increase in M
significant increase in foci between cells
phase. In fact, a slight decrease in M phase in
treated with drug only compared to no
cells treated with combination treatment (8.37
treatment at 24 h in U251 (6.33 ± 0.73; p =
± 1.1% to 6.86 ± 0.08%) was observed,
0.003) was observed. Furthermore, at 24 h
however this difference was not statistically
there was a significant difference in the
significant (p = 0.247). Based on these results,
average number of foci per cell in those cells
it may be possible that treatment with CUDC-
breaks
inhibition
(DSBs)
of
and
γH2AX
(Hall and Giaccia, 2006).
Biomed Res J 2015;2(1):105-119
CUDC-101 induced tumor cell radiosensitization
114
101 may push cells into M phase by having off-
involves
an
target effects in altering the activity of critical
catastrophe. Cells with nuclear fragmentation,
regulators of this checkpoint (e.g. ATM and
defined as the presence of 2 or more distinct
Chk2) regardless of the amount of DNA
nuclear lobes within a single cell, were
damage, and may partially account for CUDC-
classified
101-mediated enhancement in radiation-
catastrophe. As shown in the representative
induced cell killing.
fluorescent micrograph in Fig. 5A, cells
as
enhancement
going
of
mitotic
through
mitotic
undergoing mitotic catastrophe could be
CUDC-101 increases mitotic catastrophe as
distinguished after treatment of irradiation (2
mode of cell death
Gy), CUDC-101, and combination treatment.
Lai et al. (2010) reported that treatment with
There was a time dependent increase in the
CUDC-101
number
induced
expression
of
of
cells
undergoing
mitotic
proapoptotic and antiproliferative proteins in
catastrophe after the treatment with either
breast and colon cancer cells lines. To
radiation or CUDC-101 up to 72 h. In cells
determine
receiving the combination treatment, a
whether
the
increase
in
radiosensitivity resulting from CUDC-101
significantly
treatment was due to an enhancement of
undergoing mitotic catastrophe was detected
radiation-induced
V
at 48 and 72 h, 61.6 ± 11.1% and 70.3 ± 6.2%
staining 24 h after treatment was measured. As
respectively. Furthermore, this increase in
expected for a solid tumor cell line, radiation
mitotic catastrophe was greater than additive
induced little apoptotic cell death; treatment
as compared to irradiation and CUDC-101
with CUDC-101 yielded essentially identical
alone. These data suggest that the CUDC-101
levels of apoptosis, and the combination of
mediated radiosensitization is achieved by an
irradiation (2 Gy) and CUDC-101 had no
inhibition in DNA DSB repair resulting in an
effect on the frequency of apoptotic cell death
increase
events, indicating that the CUDC-101-
catastrophe. Additionally, the data supports
mediated radiosensitization of U251 glioma
the observations seen in cell cycle analysis
cells does not involve enhanced susceptibility
indicating that the increase in M phase seen
to apoptosis. (Data not shown)
with drug alone, maintained when combined
apoptosis, Annexin
in
greater
cells
number
undergoing
of
cells
mitotic
The apparent inhibition of DSB repair,
with irradiation at 24 h, was indicative of an
increase of cells in M phase, and no increase in
increase in mitotic catastrophe at later time-
radiation-induced apoptosis suggests that
points.
CUDC-101
induced
Biomed Res J 2015;2(1):105-119
radiosensitization
115
Schlaff et al.
DISCUSSION
in 40–50% of tumors, often followed by gene
GBM (WHO Grade IV) is the most common
rearrangement
malignant central nervous system tumor with
independent, constitutively phosphorylated
an incidence of 0.4 to 2.8 per year per 100,000
and cell surface localized receptor tyrosine
persons, and typified by nuclear atypia,
kinases (RTK) that enhances tumorgenicity
mitosis, endothelial proliferation and necrosis
(Heimberger et al., 2005; Lal et al., 2002;
(Mineo et al., 2006). Despite aggressive
Lopez-Gines et al., 2010; Sugawa et al., 1990;
therapies, prognoses for GBM remains poor,
Wikstrand et al., 1997). Furthermore, recent
and average overall survival remains 12-16%
preclinical and clinical studies showed that
(Stupp et al., 2009). A majority of these
EGFR may play a role in radioresistance
patients
will
at
some
in
a
ligand-
undergo
through activation of downstream signaling
radiotherapy typically combined with a
cascades such as, PI3K/Akt/mTOR, and its
radiosensitizing agent. Attempts to develop
involvement
clinically
have
(Palumbo et al., 2014). Targeting HER2 is a
cytotoxic
well established target for breast cancer
chemotherapeutic agents (Camphausen et al.,
therapy and a negative prognostic factor for
2005).
cancers of breast, lung and brain (Hiesiger et
relevant
traditionally
point
resulting
radiosensitizers
been
limited
to
Recently, agents targeting HDAC have
in
regulating
autophagy
al., 1993; Tateishi et al., 1991).
gained in popularity, as they are shown to
In regards to GBM, however, HER2 is not
enhance the radio response by relaxing the
expressed in adult glial cells, but its expression
chromatin, leaving it more susceptible to DNA
has been shown to increase with the degree of
damage, among other mechanisms. Lai et al.
astrocytoma degeneration
(2010) showed the potent inhibition of
2006). Taken with the fact that the major
multiple oncogenic pathways with CUDC-101
action of HER2 is the heterodimerization of
as
currently
HER2 with other tyrosine kinase family
investigating the effects of this compound in a
members like EGFR; and this HER2
dose escalation study for various cancers as a
heterodimerization, produces a more potent
single
in
RTK with higher ligand affinity and tyrosine
combination with radiation and cisplatin for
kinase activity, and a lower internalization and
locally advanced head and neck cancer
degradation rate, inhibiting HER2 may not
(NCT01384799).
the
only decrease HER2 activity, but could also
investigation to assess the radiosensitizing
affect the activity of its dimerization partners
potential of CUDC-101 in GBM.
(EGFR), thus helping to explain the effects
a
monotherapy,
agent
and
are
(NCT01171924),
We
and
extended
In GBM, EGFR is genomically amplified
(Mineo et al.,
observed here with CUDC-101. Limited
Biomed Res J 2015;2(1):105-119
CUDC-101 induced tumor cell radiosensitization
116
evidence exists showing the role of HER2 in
been shown to correlate with amount of DNA
radio response; however, a study by Duru et al.
DSBs (Kuo and Yang, 2008). When X-rays
(2012) showed that a pro-survival network
induce DNA damage γH2AX is recruited to
initiated by HER2 was responsible for
the damage site and repair proteins such as
radioresistance in breast cancer stem cells.
ATM or DNA-PK phosphorylate γH2AX, and
Additionally, irradiation of breast cancer cell
p53BP, MRN and BRCA1 are recruited to
lines has been associated with increased
complete the repair complex (Mah et al.,
expression of EGFR and HER2, which augments
2010). We have previously shown that the
the response to HER2-targeted therapy with
HDACi valproic acid induces retention in
trasztuzumab (Wattenberg et al., 2014).
γH2AX foci on the sites of DNA DSBs yet, the
Interestingly, we also saw an increased
sites of damage are being repaired as observed
expression of HER2 and EGFR in U251 cells
by comet assay (Camphausen et al., 2005). A
treated with irradiation alone.
similar effect may occur with CUDC-101.
We show CUDC-101 inhibited HER2,
Additionally, based on the increase of retained
EGFR and HDAC in U251 and MDA-MB-
foci at 24 h and cells in the M-phase of the cell
231 cell lines, which was accompanied by
cycle, CUDC-101 may be inducing DNA
enhancement in radiosensitivity, while no
damage as a single agent, and therefore
significant radiation-induced cell kill was seen
causing an abrogation of the G2/M checkpoint.
in a normal fibroblast cell line. Although
Alternatively CUDC-101 may show indirect
0.1µM CUDC-101 effectively maintained Ac-
effects on critical regulators of the checkpoint.
H3, it did not affect HER2 and EGFR
Therefore, this conflict between the DNA
deactivation or reduce colony formation. 0.5
repair complex remaining on the DNA damage
µM CUDC-101 maintained or increased Ac-
sites after repair, and the cells still being
H3 levels, decreased levels of pHER2 and total
pushed into M-phase, may explain the
EGFR, and achieved a significant DEF. Thus
increase in mitotic catastrophe and the CUDC-
the question arises, if some combinatorial
101-induced radiosensitization.
permutation of these targets is essential for
In order for a putative radiosensitizing
inducing radiosensitization, or is it solely an
agent to be effective clinically, the effects seen
HDACi effect? Albeit an important question,
in vitro must be replicated under in vivo tumor
it was beyond the scope of this study, and
xenograft models. Aside from defining tumor
needs further investigation.
radioresponsiveness, the ability of the agent to
The apparent mechanism of CUDC-101-
target its intended ligands is critical. The
induced radiosensitization appears to involve
radiosensitizing ability seen in vitro by
an inhibition of DNA DSB repair. γH2AX has
clonogenic survival with a DEF of 1.42 was
Biomed Res J 2015;2(1):105-119
117
Schlaff et al.
not replicated in vivo (DEF 1.2) due to a high
The evaluation of the radiosensitizing
degree of toxicity and substantial weight loss
potential of CUDC-101, provides the basis for
(data not shown). Various other agents have
additional preclinical exploration of the
shown promise in vitro, but their effects were
radiosensitizing
unable to be translated to in vivo models or
investigation and understanding of the
clinically, limited by the toxicity (Camphausen
specific molecular mechanisms addressing the
et al., 2005). Much of the biological
necessity to target all three ligands to achieve
consequences and mechanisms of action
clinically
explaining this phenomenon are unclear.
warranted.
potential.
applicable
Further
sensitization,
is
Additionally, in the case of CUDC-101, whose
targets are nuclear (HDAC) and surface
ACKNOWLEDGEMENTS
receptors (EGFR and HER2), CUDC-101 may
This research was supported by the Intramural
not effectively reach and inhibit the target(s)
Research Program of the NIH, National
responsible for enhancing radiosensitization
Cancer Institute, National Institutes of Health.
in sufficient number of tumor cells in vivo to
The authors acknowledge NIH CCR Confocal
achieve a significant response. It may be
Microscropy Core Facility, Radiation Biology
feasible to combine multiple drugs together as
Branch and Dr. Deedee Smart for use of the
single agents (i.e. lapatinib + erlotinib +
equipment.
vorinostat) with radiotherapy, as reported in
various haematologic cancers.
CONFLICT OF INTEREST
The authors claim no conflict of interest.
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Biomed Res J 2015;2(1):105-119
Research Article
Phenotypic and Functional Characterization of a
Marrow-derived Stromal Cell Line, M210B4 and its
Comparison with Primary Marrow Stromal Cells
Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale*
Stem Cell Laboratory, National Centre for Cell Science, NCCS Complex, University of Pune Campus, Ganeshkhind,
Pune, India
In vitro co-culture system consisting of bone marrow stromal cells (BMSCs) or mesenchymal stromal cell
lines of marrow origin has provided important clues about the regulation of hematopoietic stem cells (HSCs)
by their microenvironment or niche. In the current studies, we have compared phenotypic and functional
characters of a marrow-derived mesenchymal stem cell line, M210B4, with BMSCs. We demonstrate that
M210B4 resembles BMSCs in terms of phenotypic characters. Unlike the BMSCs, M210B4 differentiated
only towards adipogenic lineage, and was refractory towards osteogenic differentiation. However, M210B4
cells exhibited a higher HSC-supportive ability as assessed by flow cytometry analyses of the output cells
from co-cultures. We observed that M210B4 cells show a constitutively higher activation of p44/42 and p-38
MAPK pathways compared to BMSCs, contributing to their higher HSC-support in vitro. Overall, the results
show that M210B4 forms a suitable in vitro system to study HSC regulation in vitro.
INTRODUCTION
Hematopoiesis takes place in bone marrow, a
various cytokines and extra-cellular molecules
complex micro-environment comprised of
(ECMs).
various cells like osteoblast, endothelial cells,
emanating from these cells also contribute
CXCL12-abundant reticular (CAR) cells,
actively in the HSC fate decision (Blank et al.,
adipocytes,
cells
2008; Eckfeldt et al., 2005). Mesenchymal
(MSCs), pericytes, etc. (Calvi et al., 2003;
stromal cells (MSCs) form a very important
Ding et al., 2012; Kiel et al., 2005; Mendez-
part of the HSC microenvironment. Although
Ferrer et al., 2010; Omatsu et al., 2010; Park et
these cells have been used as feeder layers for
al., 2012; Sacchetti et al., 2007; Sugiyama et
several years (Jang et al., 2006), their precise
al., 2006; Zhang et al., 2003). Hematopoietic
participation
stem cells (HSCs) occur in close contact with
documented recently (Jing et al., 2010;
the cells of the microenvironment, which
Mehrasa et al., 2014; Walenda et al., 2010).
control the fate of the HSCs via secretion of
Subsequent studies have demonstrated that
mesenchymal
stromal
Various
in
signaling
the
HSC
mechanisms
niche
was
Key words: Mesenchymal stromal cells, M210B4, HSC.
*Corresponding Author: Vaijayanti Kale, National Centre for Cell Science, NCCS complex, University of Pune Campus,
Ganeshkhind, Pune, India.
Email: [email protected], [email protected]
Biomed Res J 2015;2(1):120-133
Singh et al.
121
MSCs actively participate in regulation of
whether they express MSC-like phenotype and
hematopoiesis and play an important role in
support hematopoiesis with efficiency at par
homing and engraftment of transplanted HSCs
with BMSCs. In the present study, we have
(Bensidhoum et al., 2004; Sohni et al., 2013).
compared M210B4 cell line with the primary
In vitro co-culture of HSCs with stromal
feeder layers forms an excellent model to
bone marrow-derived stromal cells (BMSCs)
using phenotypic and functional parameters.
study molecular mechanisms involved in the
regulation of hematopoiesis in general, and
MATERIALS AND METHODS
HSC fate in particular. Murine stromal cells
Cells
were isolated and characterized by Tropel et
The protocols used in animal experimentation
al. (2004) and constituted an important in vitro
were approved by the institutional animal
tool to study stromal cell biology. To have a
ethics committee (IAEC). The C57BL/6J
constant supply of feeder cells, several stromal
(CD45.2)
cell lines were generated. Some of the clonal
(Ptprc;
stromal cell lines that have been established
Laboratory, Bar Harbor, USA) were housed
are PA6 (Piacibel et al., 1996), M210B4
and bred in our experimental animal facility
(Sutherland et al., 1991), S17 (Winwman et
(EAF). BMSCs were isolated from bone
al., 1993), and MS5 (Tordjman et al., 1999).
marrow of 6–8 weeks C57BL6/J mice by
These cells have been successfully used as
flushing the femurs with complete medium,
feeder layers and possess hematopoiesis-
constituting Iscove's modified Dulbecco's
supportive ability in vitro.
medium (IMDM) (HiMedia, Mumbai, India)
and
B6.SJL-PtprcaPepcb/BoyJ
CD45.1)
mice
(The
Jackson
The M210B4 cell line is a clone derived
supplemented with 20% mesenchymal stem
from bone marrow stromal cells from
cell FBS (Mesen-FBS; Stem Cell Technology,
(C57BL/6J × C3H/HeJ) F1 mouse (Lemoine
Vancouver, British Columbia, Canada) and
et al., 1988), and supports hematopoiesis when
plated in a petridish. After 8–10 days of
used as feeder layer for long-term culture-
incubation, with intermittent removal of non-
initiating cell (LTC-IC) assay (Burroughs et al
adherent cells and addition of fresh medium,
1994). Our group has used this cell line to
the adherent cells were used for the
study various aspects of hematopoiesis (Bajaj
experiments (Anjos-Afonso et al., 2008).
et al., 2011; Hinge et al., 2010).
M210B4 cell line was purchased from ATCC
In spite of its extensive use in LTC-IC
and maintained in RPMI1640 (HiMedia,
assays, these cells have not been critically
Mumbai, India) supplemented with 10% FBS
evaluated
primary
(GIBCO, Invitrogen, Carlsbad, California,
marrow-derived stromal cells. It is not known
USA). Lineage negative (lin-) cells were
in
comparison
with
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
122
isolated from bone marrow mononuclear cells
irradiated (8000 rads of gamma ray, Co60)
(MNCs) of Ptprc mice by using biotin-labelled
feeders for 7 days in either IMDM
anti-mouse lineage antibody cocktail prepared
supplemented with 10% MSC-FBS (MSC
from Biotin Mouse Lineage panel (BD
qualified
Pharmingen, San Diego, California, USA) and
California, USA) or Myelocult medium (Stem
®
Dynabeads
biotin
binder
(Invitrogen,
Calrsbad, California, USA).
Cell
FBS,
Invitrogen,
technology,
Columbia,
Carlsbad,
Vancouver,
Canada). The
British
medium
was
supplemented with 25 ng/ml murine IL-6, 25
Differentiation towards adipocytes and
ng/ml murine SCF and 10 ng/ml murine IL-3
osteoblasts
(Peprotech, Rocky Hill, USA). We compared
For adipocytic differentiation, BMSCs or
the HSC-supportive ability of M210B4 and
M210B4 cells were treated with standard
BMSCs
adipogenic
conditions:
differentiation
medium
under
four
A)
different
Non-irradiated
culture
BMSCs/
comprising insulin (4 μg/ml), 3-isobutyl, 1-
M210B4 in (IMDM + 10% Mesen-FBS),
methyl
B) Irradiated BMSCs/M210B4 in (IMDM +
xanthine
(IBMX)
(500
µM),
dexamethasone (0.25 μM), indomethacin (200
10%
Mesen
FBS),
C)
Non-irradiated
µM) and 2 µg/ml insulin for 15-18 days. The
BMSCs/M210B4 in Myelocult medium and
adipogenic differentiation was confirmed by
D) Irradiated BMSCs/M210B4 in Myelocult
staining lipid droplets with Oil Red O dye
medium. After 7 days of co-culture, the cells
osteoblastic
were harvested and analyzed for LSK (lin-
differentiation, BMSCs or M210B4 cells were
Sca1+ c-Kit+) stem cell population using flow
treated with β glycerophosphate (10 nmol/L),
cytometry. The LSK population was further
dexamethasone (100 nmol/L) and ascorbic
analyzed as long-term HSCs (LT-HSCs) or
acid (0.05 nmol/L) for 15–18 days. To confirm
short-term HSCs (ST-HSCs) based on CD34
osteoblastic differentiation, cells were stained
expression. The absolute numbers were
with Alizarin Red S to detect calcium deposits
calculated based on % of cells obtained on
(Sila-Asna et al.,2007).
flow cytometer and total yield, while the fold
(Bajaj
et
al.,
2011).
For
increase
was
calculated
by
comparing
Co-culture assay
absolute numbers of input population to that of
BMSCs or M210B4 cells were seeded in
the output population.
collagen-coated (50 µg/ml) 24-well plate as
feeder layer. After 24 h, 1 × 105 lin- cells
Flow Cytometry
isolated from mouse bone marrow were co-
For the phenotypic characterization, BMSCs
cultured
and M210B4 cells were stained with APC-
with
either
non-irradiated
Biomed Res J 2015;2(1):120-133
or
Singh et al.
123
conjugated anti-mouse CD44, PE-conjugated
membranes (Biorad, California, USA). The
anti-mouse CD73, APC-conjugated anti-
blots were incubated with specific primary
mouse CD90.1, PE-conjugated anti-mouse
antibodies
CD105, APC-conjugated anti-mouse CD106
(Thr202/Tyr204), anti-p38, and anti-p-p38
(eBioscience, San Diego, California, USA),
(Thr180/Tyr182); and horseradish peroxidase-
PE-conjugated anti-mouse Sca-1, FITC-
conjugated
conjugated anti-mouse CD45 and FITC-
Signaling Technology, Danvers, Massachu-
conjugated
(BD
settes, USA). The signals were detected using
Pharmingen, San Diego, California, USA).
Lumiglo reagent (Cell Signaling Technology,
For LSK profiling, cells were stained with
Danvers, Massachusettes, USA) and the
APC-conjugated mouse lineage antibody
signals were captured on X-Ray films.
cocktail,
anti-mouse
PECy7-conjugated
CD117(c-Kit),
Sca-1
CD34
(BD
PE-conjugated
anti-p44/42,
secondary
anti-p-p44/42
antibodies
(Cell
anti-mouse
anti-mouse
Data were analyzed by Sigma Stat software
California, USA) and FITC-conjugated anti-
using one-way repeated measure analysis of
mouse CD34 (eBioscience, San Diego,
variance (One-Way RM ANOVA; Jandel
California, USA). The isotype-stained cells
Scientific Software, California, USA). The
were used as controls. The stained cells were
plots represent the values as mean ± standard
acquired
error of mean (SEM), and p value ≤ 0.05 was
FACS
Canto
San
Statistical analysis
Diego,
on
Pharmingen,
–
II
(Becton
Dickinson, New Jersey, USA) and analyzed
considered significant.
using BD FACS-DIVA SOFTWARE version
5.0.
RESULTS
M210B4 cell line is comparable to BMSCs
Western blots
at phenotypic level
Whole cell lysates were prepared using RIPA
BMSCs are phenotypically characterized by
lysis buffer supplemented with Phosphatase
surface markers as Sca1+CD44+ CD73+
Inhibitor-1(PI-1), Phosphatase Inhibitor-2 (PI-
CD90.1+ CD105+ CD106+ CD45- CD34-
2), Protease Inhibitor Cocktail (PIC), 1 mM
(Chamberlain et al., 2007; Boxall SL et al.,
Phenyl Methyl Sulphonyl Fluoride (PMSF), 1
2012; Mabuchi et al., 2013). When M210B4
mM Sodium-orthovanadate and 1 mM
cells
Sodium Fluoride (NaF) (Sigma-Aldrich, St.
phenotypic analyses using the standard
Louis, Missouri, USA). The samples having
markers, we observed that both BMSCs as
equal protein concentration were separated by
well as M210B4 are highly positive for CD44,
9% SDS-PAGE and transferred to PVDF
CD106 and Sca-1; and moderately positive for
and
BMSCs
were
subjected
to
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
124
Table 1: Percent and mean fluorescence intensity (MFI) of different surface markers in M210B4 and BMSCs.
S. No.
Surface Markers
1
CD44
2
CD73
3
CD90.1
4
5
CD105
CD106
6
Sca-1
7
CD34
8
CD45
M210B4
BMSCs
a)
% population
99.99
73.89
b)
MFI
35445
1680
a)
% population
8.92
25.84
b)
MFI
216.43
178.41
a)
% populati1on
16.17
28.73
b)
MFI
253.06
173.34
a)
% population
31.48
56.75
b)
MFI
230.91
595.31
a)
% population
99.96
93
b)
MFI
4872
2432
a)
% population
98.99
86.51
b)
MFI
24088
5751
a)
% population
Nil
Nil
b)
MFI
Nil
Nil
a)
% population
Nil
Nil
b)
MFI
Nil
Nil
CD73, CD90.1 and CD105 (Fig. 1A). In terms
differentiation potential. We observed that
of percentages, more number of BMSCs
M210B4 cells differentiated into adipocytes
expressed CD73, CD90.1 and CD105. Both
(Fig. 1B), and not towards the osteogenic
cells were negative for CD34 and CD45. The
lineage. The data showed that M210B4
data showed that M210B4 cells compare well
efficiently differentiates towards adipogenic
with BMSCs at phenotypic level, albeit with
lineage, but not towards the osteoblastic one.
minor numerical differences (Table 1).
M210B4 cells expand long-term HSCs in
M210B4 cell line differentiates towards
vitro
adipocytic lineage
The results showed that in type 'A' (Non-
The marrow-derived Mesenchymal stromal
irradiated feeders in IMDM supplemented
cells are expected to differentiate towards
with 10% MSC-FBS) and type 'B' (irradiated
osteoblastic, and adipocytic lineages under
feeders in IMDM supplemented with 10%
appropriate stimuli (Prockop DJ., 1997;
MSC-FBS) co-cultures, the total number of
Pittenger MF et al., 1999; Dominici et al.,
cells harvested was significantly low in
2006). However, when these cells are cultured
M210B4 set (Fig. 2A-a, 2B-a). The absolute
for long periods they lose their differentiation
numbers of LT-HSCs (LSK CD34-) were
capacity. So we investigated whether M210B4
significantly increased, whereas the absolute
cells possess both adipogenic and osteogenic
numbers of LSK HSC (Lin-Sca-1+ c-Kit+), and
Biomed Res J 2015;2(1):120-133
Singh et al.
125
Figure 1: Characterization of murine bone marrow stromal cells and M210B4 cell-line. A) Flow cytometric analysis
for the MSC surface markers expressed on BMSCs versus M210B4 cell-line; B) The panel illustrates images of
adipocytes formed from M210B4 cells and BMSCs stained with Oil Red O. The panel shows that BMSCs differentiate
towards the osteoblastic lineage as evident by Alizarin Red S staining; M210B4 cells do not show such differentiation.
(Original magnification 100X).
Biomed Res J 2015;2(1):120-133
126
Characterization of a marrow-derived stromal cell line, M210B4
Figure 2: Co-culture of murine hematopoietic stem cells with BMSCs versus M210B4 cell-line. Total numbers of
hematopoietic cells obtained in various co-cultures are depicted (a). Quantitative data showing absolute number of
HSCs (b) and fold change over input cells (c) with respect to total cells harvested, LSK, LT-HSC and ST-HSC, at 4
different conditions (2A) Non-irradiated BMSCs/M210B4 in (IMDM+10%MSC-FBS), (2B) Irradiated BMSCs/M210B4 in
(IMDM+10%MSC-FBS), (2C) Non-irradiated BMSCs/M210B4 in Myelocult medium and (2D)Irradiated BMSCs/M210B4
in Myelocult medium. The data of one representative experiment are depicted and are represented as mean ± SEM. N=3.
ST-HSC (LSK CD34+) were significantly
(Fig. 2A-c and 2B-c). There was no significant
decreased in the M210B4 set as compared to
difference in M210B4 set versus BMSCs set in
the BMSCs set (Fig. 2A-b and 2B-b; Table 2).
type 'C' (non-irradiated feeders in Myelocult
In terms of fold increase over input, the result
medium) co-cultures with respect to LSK-
was the same as that of the absolute numbers
HSC, LT-HSC and ST-HSC populations in
Biomed Res J 2015;2(1):120-133
Singh et al.
127
Table 2: The absolute numbers (mean ± SEM, n=3) and fold increase over input from one representative experiment
have been tabulated.
3
Absolute numbers × 10
Culture
condition
LSK
LT-HSC
ST-HSC
BMSCs
M210B4
BMSCs
M210B4
BMSCs
M210B4
A
30.53±0.85
13.81±0.93
3.22±0.25
10.59±1.26
26.84±1.02
2.11±0.57
B
19.15±1.12
12.67±0.61
1.10±0.07
4.40±0.52
17.94±1.06
7.95±0.10
C
9.69±1.06
13.38±3.86
7.57±1.03
11.63±3.52
1.31±0.07
0.90±0.23
D
3.27±0.15
10.42±0.44
1.88±0.16
7.99±0.34
1.17±0.06
1.75±0.30
Fold increase over input
Culture
condition
LSK
LT-HSC
ST-HSC
BMSCs
M210B4
BMSCs
M210B4
BMSCs
M210B4
A
10.60±0.29
4.80±0.32
1.62±0.12
5.33±0.63
26.62±1.01
2.10±0.56
B
6.65±0.39
4.40±0.21
0.55±0.03
2.22±0.26
17.80±1.05
7.88±0.09
C
3.36±0.37
4.65±1.34
3.81±0.52
5.85±1.77
1.29±0.07
0.89±0.23
D
1.14±0.05
3.62±0.15
0.95±0.08
4.02±0.17
1.16±0.06
1.74±0.29
terms of absolute numbers and fold increase
A, B and D culture conditions used in the
over input (Fig. 2C-b–c; Table 2), with the
experiments, with respect to the primitive LT-
exception of significantly lower total cells
HSC population (Table 2). Under the culture
harvested in M210B4 as compared to that of
condition C, however, both BMSCs and
BMSCs (Fig. 2C-a). Type 'D' co-cultures
M210B4 gave comparable output of various
(irradiated feeders in Myelocult medium) did
HSC populations (Fig. 2C).
not show any difference in the total cells
harvested in M210B4 and BMSCs (Fig. 2D-a),
Signaling pathways operative in M210B4
but the absolute numbers and fold increase
vs. BMSCs
over input with respect to LSK and LT-HSC
Earlier data showed that M210B4 exhibits a
were significantly higher in M210B4 (Fig. 2D-
better HSC-supportive ability compared to the
b–c; Table 2). The absolute number and fold
BMSCs. To examine whether this was related
increase over input of ST-HSC between
to the difference in their signaling gamut, we
M210B4 and BMSCs was non-significant.
subjected the lysates of these cells to western
Flow cytometry graphs for all the 4 sets of co-
blot analyses to detect phosphorylation of p38
culture conditions (A–D) are depicted in
and p44/42 MAPK. The four different culture
supplementary Fig. S1. The data showed that
conditions described above were used.
M210B4 cells have a better HSC-supportive
We observed that under most of the culture
potential in vitro as compared to BMSCs under
conditions the levels of phospho p-38
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
128
Figure 3: Western blot analyses of phosphorylated forms of p38 and p44/42 in BMSCs versus M210B4 cell-line.
Total cell lysate of 48 h cultured BMSCs and M210B4 at different conditions, as shown in the figure were subjected to
Western blot analyses. The blots were probed with (i) anti p-p38 and total p38 and (ii) anti p-p44/42 and total p44/42
antibodies. Equal input of proteins was ensured by probing the blots with antibody to α-tubulin. The intensity of the bands
was quantitated by densitometry using ImageJ software.
(Thr180/Tyr182) were higher in M210B4 as
regulation of HSC fate. The cell line models
compared to the BMSCs (Fig.3A). The levels
become especially useful, when one needs to
of phospho p44/42 (Thr202/Tyr204) were
use a genetic approach to over-express or
higher in M210B4 under culture conditions A,
silence any particular gene. Primary cells can
B and D as compared to BMSCs (Fig. 3B).
also be genetically modified, but need viral
Under the culture condition C, the level of p-
vectors to get sufficient numbers of modified
p38 was high in M210B4, whereas the level of
cells. Secondly, being primary cells, the
p-p44/42 was comparable to BMSCs.
modified cells wither off, necessitating their
Collectively, the data suggests that
reestablishment. Cell lines give a distinct
increased levels of phospho p38 and phospho
advantage of unlimited supply of cells, and
p-44/42 in M210B4 may have conferred upon
also allow use of simple plasmid-based system
M210B4 a better HSC-supportive ability
for gene manipulations. They also facilitate
under culture conditions A, B and D.
generation of independent clones showing
stable expression.
Several cell lines like PA6 (Piacibel et al.,
DISCUSSION
In vitro co-culture system using non-irradiated
1996), M210B4 (Sutherland et al., 1991), S17
or irradiated feeder layers formulated with
(Winwman et al., 1993), and MS-5 (Tordjman
primary
mesenchymal
et al., 1991) have been established and
stromal cells or cell lines, is a useful tool to
successfully used in co-culture studies. Our
understand
group has mainly used M210B4 cell line in our
marrow
the
derived
stromal
Biomed Res J 2015;2(1):120-133
cell-mediated
Singh et al.
129
previous work (Hinge et al., 2010; Bajaj et al.,
differentiate towards chondrocytic, and also
2011). Presently, our group is involved in
towards other lineages like neural, muscular,
genetically modifying these cells using
etc.
plasmid vectors expressing various molecules
We subjected both M210B4 and primary
involved in regulation of hematopoiesis.
stromal cells in co-culture studies. Since the
When we initiated these studies, we noticed
feeder cells can be used in non-irradiated or
that though this cell line has been frequently
irradiated form, we employed both the
used in experiments (Sutherland et al., 1991),
conditions and used two different kinds of
its phenotype in comparison with BMSCs has
media typically used in such experiments. The
not
its
data showed that though under most of the
hematopoiesis support in comparison with
culture conditions the total output of
BMSCs has not been examined.
hematopoietic cells in M210B4 set was low,
been
established.
Secondly,
Therefore we compared phenotype of
except in co-culture condition 'D', the fold
M210B4 with BMSCs. We showed that these
increase of LT-HSCs over the input was
cells are phenotypically comparable to
consistently high in M210B4 set, except under
primary BMSCs. On examination of the
condition 'C'. The data suggest that M210B4
differentiation
and
cells possess better ability to support the LT-
adipocytic lineages, we observed that the
HSCs. In culture condition 'C', the absolute
M210B4 cells differentiated into adipocytes
number and the fold increase over input for LT-
(Fig. 1B), but were refractive for osteoblastic
HSCs were higher than in the BMSCs set, but
differentiation. BMSCs are most commonly
the data did not reach significance. This could
known to differentiate into osteoblastic,
be related to milder activation of p44/42
adipocytic and chondrogenic lineages. We
MAPK activation under this culture condition
have shown such tri-lineage differentiation of
(Fig. 3B). Adipocytes are considered as
placental MSCs (Sharma et al 2012).
negative
Adipocytes and osteoblasts form important
(Naveiras et al., 2009), but M210B4 cells in
components of the HSC niche and thus the
spite of being able to differentiate to
differentiation of BMSCs towards these two
adipocytes supported HSC proliferation. This
lineages is relevant in the context of
shows that differentiated adipocytes, but not
hematopoiesis. Therefore, in the present study
pre-adipocytes, may exert negative effect on
we examined the ability of M210B4 to
the HSCs. The overall low output of
differentiate
and
hematopoietic cells thus appears to be
osteoblastic lineages. However, it will be
primarily due to low proliferation of
interesting to see whether these cells
committed progenitors.
towards
towards
osteoblastic
adipogenic
modulators
of
hematopoiesis
Biomed Res J 2015;2(1):120-133
Characterization of a marrow-derived stromal cell line, M210B4
130
The signaling mechanisms operative in
the stromal cells are known to affect HSC fate.
that alteration of p44/42 and p38 pathways in
the stromal cells can affect HSC fate.
Our data showed that constitutively activated
In summary, our study shows that
p44/42 and p38 pathways in M210B4 cells
M210B4 cell line shows phenotypic similarity
under all culture conditions except for the
with the primary BMSCs and has higher HSC-
levels of p-p44/42 under the culture condition
supportive properties by the virtue of the
'C,' may be responsible for their better HSC
signaling gamut present in them. This cell line
support. Stromal cells regulate the HSC fate
thus forms a suitable model to examine the
via secretion of various cytokines and ECM
stromal cell-mediated regulation of stem cell
molecules (Scadden, 2006; Baraniak et al.,
fate. Similarly, this cell line is a suitable model
2010). Cell–cell interactions are also known to
to study adipogenesis (Bajaj et al., 2011).
play important role in this process. In our
future experiments we propose to examine
ACKNOWLEDGEMENTS
whether
show
The authors acknowledge Department of
differential ECM and adhesion molecule
Biotechnology (DBT), Government of India,
profile.
New Delhi (Grant
M210B4
and
BMSCs
BT/PR14036/MED/31/
Our group has shown that p44/42 and p38
101/2010), Director, NCCS, FACS core
pathways are coupled and inversely regulated
facility, and Council of Scientific and
in primitive stem cells (Kale 2004; Kale et al.,
Industrial Research (CSIR) for fellowship to
2004;
SS, SG and MRD).
Kale
autonomous
2005). Although
the
role
signaling
of
MAPK
cell
pathways in the regulation of hematopoiesis is
CONFLICT OF INTEREST
known (Geest et al., 2009), our study showed
The authors claim no conflict of interest.
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follows: Objectives, Materials and Methods, Results,
Discussion and Conclusion.
The abstract should not be structured for review articles.
The abstract should state briefly the purpose of the research, the
principal results and major conclusions. An abstract is often
presented separate from the article, so it must be able to stand
alone.
Keywords
After the abstract provide a maximum of six keywords, to be
chosen from the Medical Subject Headings from Index
Medicus. These keywords will be used for indexing purposes
Abbreviations
Define abbreviations or acronyms that are not standard in this
field at their first occurrence in the article; in the abstract and
also in the main text after it. Ensure consistency of abbreviations
throughout the article.
Text
This should start on the third page and should be subdivided into
the following sections: Introduction, Patients or Materials and
Methods, Results, Discussion and Conclusions,
Acknowledgements.
References
Responsibility for the accuracy of bibliographic citations lies
entirely with the authors. Please ensure that every reference
cited in the text is also present in the reference list (and vice
versa). Any references cited in the abstract must be given in full.
"Unpublished data" and "Personal communications" are not
allowed. As an alternative, say in the text, for example, '(data not
shown)' or '(Dr D. Saranath, School of Science, NMIMS
(Deemed-to-be) University, Mumbai)'. Citation of a reference as
"in press" implies that the item has been accepted for publication
and a copy of the title page of the relevant article must be
submitted.
Indicate references by (first author, year) in the text.
Examples:
Kulkarni J, Khanna A. Functional hepatocyte-like cells derived
from mouse embryonic stem cells: A novel in vitro
hepatotoxicity model for drug screening. Toxicol In Vitro
2006;20:1014-1022.
Bhatnagar R, Dabholkar J, Saranath D. Genome-wide disease
association study in chewing tobacco associated oral
cancers. Oral Oncol 2012;48(9):831-835.
Molinolo AA, Hewitt S, Amornphimoltham PI, Keelawat S,
Saranath D, Gutkind JS et al. Dissecting the Akt/mTOR
signaling network: emerging results from the head and
neck cancer tissue array initiative. Clin Cancer Res
2007;13:4964-4973.
Saranath D. Integrated Biology and Molecular Pathology of
Oral Cancer. In: Saranath D, editor. Contemporary Issues
in Oral Cancer. Oxford Press, 2001:30-71.
List all authors if the total number of authors is seven. For more
than seven authors, first six authors should be listed, followed by
"et al." For further details you are referred to "Uniform
Requirements for Manuscripts submitted to Biomedical
Journals" (J Am Med Assoc 1997;277:927-934).
Figure Captions, Tables, Figures and Schemes
Present these, in the given order, at the end of the article. They
are described in more detail below. High-resolution graphics
files must always be provided separate from the main text file.
Footnotes
Footnotes should be used sparingly. Number them
consecutively throughout the article, using superscript Arabic
numbers. Many word processors build footnotes into the text,
and this feature may be used. Should this not be the case,
indicate the position of footnotes in the text and present the
footnotes themselves on a separate sheet at the end of the article.
Do not include footnotes in the Reference list.
Table footnotes
Indicate each footnote in a table with a superscript lowercase
letter.
Tables
Number tables consecutively in accordance with their
appearance in the text. Place footnotes to tables below the table
body and indicate them with superscript lowercase letters.
Avoid vertical rules. Be sparing in the use of tables and ensure
that the data presented in tables do not duplicate results
described elsewhere in the article.
Nomenclature and Units
Follow internationally accepted rules and conventions: use
the international system of units (SI). If other quantities are
mentioned, give their equivalent in SI.
Preparation of Electronic Illustrations
·
Make sure you use uniform lettering and sizing of your
original artwork.
·
Save text in illustrations as "graphics" or enclose the font.
·
Only use the following fonts in your illustrations: Arial or
Times Roman.
·
Number the illustrations
sequence in the text.
·
Use a logical naming convention for your artwork files.
·
Provide all illustrations as separate files and as hardcopy
printouts on separate sheets.
captions
to
according
to
their
·
Provide
·
Produce images near to the desired size of the printed
version.
illustrations separately.
Formats
Regardless of the application used, when your electronic
artwork is finalised, please "save as" or convert the images to
one of the following formats (Note the resolution requirements
for line drawings, halftones, and line/halftone combinations
given below):
EPS: Vector drawings. Embed the font or save the text as
"graphics".
TIFF: Colour or greyscale photographs (halftones): always use
a minimum of 300 dpi.
TIFF: Bitmapped line drawings: use a minimum of 1000 dpi.
TIFF: Combinations bitmapped line/half-tone (colour or
greyscale): a minimum of 500 dpi is required.
DOC, XLS or PPT: If your electronic artwork is created in any of
these Microsoft Office applications please supply "as is".
Please do not
·
Supply embedded graphics in your wordprocessor
(spreadsheet, presentation) document;
·
Supply files that are optimised for screen use (like GIF,
BMP, PICT, WPG); the resolution is too low;
·
Supply files that are too low in resolution;
·
Submit graphics that are disproportionately large for the
content.
If, together with your accepted article, you submit usable colour
figures then it will be ensured that at no additional charge these
figures will appear in colour on the Web (e.g., ScienceDirect and
other sites) in addition to colour reproduction in print.
proportionate dimensions, so as not to become illegible or
unclear after possible reduction; in general, the figures should
be designed for a reduction factor of two to three. The degree of
reduction will be determined by the Publisher. Illustrations will
not be enlarged.
Do not use any type of shading on computer-generated
illustrations.
Photographs (halftones)
Remove non-essential areas of a photograph. Do not mount
photographs unless they form part of a composite figure. Where
necessary, insert a scale bar in the illustration (not below it), as
opposed to giving a magnification factor in the caption. Note
that photocopies of photographs are not acceptable.
Preparation of supplementary data
Electronic supplementary material to support and enhance your
scientific research is accepted as supplementary file.
Supplementary files offer the Author additional possibilities to
publish supporting applications, movies, animation sequences,
high-resolution images, background datasets, sound clips and
more. Supplementary files supplied will be published online
alongside the electronic version of your article. In order to
ensure that your submitted material is directly usable, please
ensure that data is provided in one of our recommended file
formats. Authors should submit the material in electronic format
together with the article and supply a concise and descriptive
caption for each file.
Proofs
when your manuscript is received by the Publisher it is
considered to be in its final form. Proofs are not to be regarded as
"drafts". One set of page proofs in PDF format will be sent by email to the corresponding author, to be checked for
typesetting/editing. No changes in, or additions to, the accepted
(and subsequently edited) manuscript will be allowed at this
stage. Proofreading is solely your responsibility.
The corrected article will be published as quickly and accurately
as possible. In order to do this we need your help. When you
receive the (PDF) proof of your article for correction, it is
Captions
Ensure that each illustration has a caption. Supply captions
separately, not attached to the figure. A caption should comprise
a brief title (not on the figure itself) and a description of the
illustration. Keep text in the illustrations themselves to a
minimum but explain all symbols and abbreviations used.
Line drawings
The lettering and symbols, as well as other details, should have
important to ensure that all of your corrections are sent back to
us in one communication. Subsequent corrections will not be
possible, so please ensure your first sending is complete. Note
that this does not mean you have any less time to make your
corrections just that only one set of corrections will be accepted.
About School of Science
School of Science was started in 2007 with a view
to provide undergraduate and post graduate
students an opportunity to be a part of the unique
learning methodology of the university, which lays
emphasis on academic excellence combined with
industry oriented training. With the boom in
information technology and more and more
sophistication in instrumentation techniques, there
is now a very thin dividing line between the various
disciplines of science. Therefore, there is a greater
need for flexibility in scientific thought as well as
training manpower on an interdisciplinary plane.
With this thought in view, the SVKM's NMIMS
introduced, highly innovative and unique
interdisciplinary courses at the School of Science
from the academic year 2007-2008. The goal of the
School of Science is to be a Center of Excellence in
the domain of Pure and Applied Science by
providing quality education and research.
Courses Offered
Ph.D. in Biological Sciences, Chemistry (Regular and Professional) and Physiotherapy
Integrated M.Sc.-Ph.D. in Biological Sciences and Chemistry
M.Sc. in Biological Sciences, Chemistry (Analytical and Organic) and Statistics
Master of Physiotherapy [In collaboration with Nanavati Super Speciality Hospital, Mumbai, India]
Post-Graduate Diploma in Physician Assistance (2 years), Operation Theatre
Technology (1 year), Non-Invasive Cardiology (1 year) and Central Sterile Services (1
year) [In collaboration with Asian Heart Institute and Research Centre, Mumbai, India]
Diploma in Clinical Research (Part time: 1 year) [In collaboration with C. B. Patel Research Centre,
Mumbai, India]
Certificate Courses in Molecular Medicine and Molecular Oncology (Part time: 6
months) [for medical/science graduates]
Advanced Course in Clinical Data Management (Part time: 3 months) [In collaboration with
C. B. Patel Research Centre, Mumbai, India]
Salient Features
Research constitutes a major thrust in all the courses offered at the School
Courses oriented to fulfill needs/demands of Research Institutions/Industry
Thrust Areas in Research
Cell Biology, Stem Cell Biology, Molecular Oncology, Reproductive Biology, Microbiology,
Immunology, Pharmacology, Phytochemistry, Nanosciences, Applied Chemistry, Colloidal
Chemistry and Applied Statistics
For More Information Please Contact:
School of Science, NMIMS (Deemed-to-be) University
Tel: 91-22-4219 9943/50; Fax: 91-22-2611 4512; E-mail: [email protected];
Visit us at: http://science.nmims.edu
SVKM’s
Narsee Monjee Institute of Management Studies
Deemed to be UNIVERSITY
V. L. Mehta Road, Vile Parle (W), Mumbai-400 056, INDIA.
Tel: 91-22-4235555 | Fax: 91-22-26114512
Email: [email protected] | Website:www.nmims.edu