From www.bloodjournal.org by guest on December 22, 2014. For personal use only. Quercetin Inhibits the Growth of Leukemic Progenitors and Induces the Expression of Transforming Growth Factor-P1 in These Cells By Luigi M. Larocca, Luciana Teofili, Simona Sica, Mauro Piantelli, Nicola Maggiano, Giuseppe Leone, and Franco 0 . Ranelletti We previously showed that quercetin (3,3',4',5,7 pentahydroxyflavone) inhibits ina dose-dependent manner the growth of acute leukemias and is able to enhance the antiproliferative activity of cytosine arabinoside. We show here that quercetin inhibits the clonogenic activity of 20 of 22 acute leukemias (AL; 4 MI-AML, 3 MS-AML, 2 MS-AML, 3 M4-AML, 3 M5-AML, and 7 ALL). In the present report, we show that the induction of transforming growth factor-PI (TGF-PI) in leukemic blasts is one of the growth-inhibitory mechanisms of quercetin in these cells.This observation was supported by thefollowing data. (1)Quercetin-sensitive leukemic blasts, when treated with quercetin, secrete large F LAVONOIDS AREA class of natural substances widely distributed in the plant kingdom' that display a variety of biologic actions.' Recently, we and others haveshown (3,3',4',5,7-pentahydroxyflathat the flavonoidquercetin vone) inhibits the growth ofhuman leukemic cell lines'^' and of acute myeloid leukemia (AML) and acute lymphoid leukemia(ALL) primary leukemic blasts.' Moreover, we have shown that quercetin is able toinhibit the colonyformation by AML and ALL progenitors.' Furthermore, quercetin enhances the antiproliferative activity of cytosine arabinoside on the leukemic cell line HL60 and on fresh AML and ALL progenitors,'likely by interactingwiththeso-called type I1 estrogen binding sites (type I1 EBS)."' The term of transforming growth factor-p (TGF-P) indicates a family of structurally related proteins with powerful hematopoieticregulatory properties."' TGF-p I stimulates or inhibitstheinvitro growth of normalmyeloidprogenitors9. I?- 15 depending on their stage of differentiation. TGF-01 mightselectivelyact as a negativeregulatoron early hematopoietic progenitors,sparingmore committed Severalstudiesshowed the inhibitoryeffect of TGF-p1 on the growth of primary AML blasts in all samples tested but one."^" Because wehave previously observed that quercetin inhibits the growth of ovarian cancer cell line by inducing the expression of TGF-P 1 ,2' we evaluated whether the modulation of TGF-PI production by quercetin could be a mechanism by whichthisflavonoid exerts itsgrowth-inhibitory effect on AML and ALL blasts. From the lstituti di Anatomia Patologica, Serneiotica Medica and Istologia, Universita Cattolica del Sacro Cuore, Romu, ltuly. Submitted July 13. 1994; uccepted Januar?, 30, 199.5. Supported by grants 40% and 60% from MURST and by Grant No. ACRO 94,01098 PF39from CNR. to LuigiM.Lurocca,MD,lstitutodi Addressreprintrequest Anatomiu,PatologicaUniversit2CuttolicaSC,Largo F. Vito. I , Romu 00168, Italy. The puhlicution costsof this article were defrayed in part page by chargepayment.Thisarticlemustthereforebeherebymarked "advertisement" in accordance with 18 U.S.C. section 1734 solely t o indicate this ,fact. 0 1995 by The American Society o j Hernatology. 0006-497//95/R512-0027$3.00/0 3654 amounts of TGF-P1 in the medium and show positivity for TGF-PI-immunoreactive material in thecytoplasm. (2) At a concentration of 8 pmol/L, antisense TGF-P1 oligonucleotides prevent the growth-inhibitory action of quercetin. (3) Anti-TGF-p1 neutralizing monoclonal antibodies can prevent almost completely the growth-inhibitory activity of quercetin. The analysis of quercetin-resistant casesconfirmed as well the central role of TGF-P1 in the growthinhibitory activity of quercetin. In conclusion, quercetin can act as a cytostatic agent for leukemic cells by modulating the production of TGF-PI. 0 1995 by The American Society of Hematology. MATERIALS AND METHODS Isolation cf leukemic blasts rrnd clonogenic as.say. Fresh leukemic blastswereisolatedfromheparinizedbone marrow aspirates obtained from 20 leukemic patients ( l 4 AML and 6 ALL) at diagnosis and from 2 patients in relapse ( I AML and I ALL). All samples were obtained after informed consent was obtained. Diagnoses were established according to the French-American-British recommendations?'.:" after cytologic examinations and cytochemical assays (peroxidase and naphtyl acetate esterase). Immunologic phenotype was defined in ALLpatients.Aspiratesdiluted with Hank'sBalanced SaltSolution(HBSS)werelayeredontoFicoll-Hypaquedensity gradient ( I ,077 g/mL,pH 7.6,292 mOsm/L) and centrifugedat 400g for 30 minutes. Light-density mononuclear cells containedmore than 70% blast cells. In AML and in non-T ALL, T lymphocytes were removed by rosetting with neuraminidase-treated sheep erythrocytes. After T-cell depletion, the percentage of blasts in the resulting cell population was greater than 90%, as assessed by morphologic, cytoenLymatic. andimmunophenotypicanalysis.Blastsweresuspended at I X 10' cells/mL in Iscove's modified Dulbecco's medium (IMDM: Flow Laboratories, Irvine, UK) containing 0.9% methylcelx lo-' mol/L2-mercaptoethanol lulose (Sigma, St Louis, MO). 2 (Sigma), 20% heat-inactivated fetal calf serum (HI-FCS; Flow Labs), and 10% phytohemagglutinin (PHA)-leukocyte-conditioned medium, prepared accordingtothe method of Aye ct al." Aliquots of 0 . 1 mL were plated in 96-microwell flat-bottom plates (Becton Dickinson, Lincoln Park. NJ). All plates were incubated at 37°C i n a fully humidified S% C02-9S% air atmosphere. Quercetin (Aldrich. Steinheim,Germany)wasadded at the indicated concentration at the start of cultures and then every 2 days from an absolute ethanol stock solution. Ethanol (vehicle) concentration never exceeded I % (vol/vol) in untreated and treated cultures. Each samplewas cultured in quadruplicate. After 6 to 10 days, aggregates consisting of 10 o r more cells were counted as colony-forming unit-leukemic (CFU-L). Result are expressed as the number of colonies (mean of 4 plates) per IO'cells plated. To verify the leukemic natureof CFU-ALL, cells i n coloniesfromALLpatientswerestained with Wright-Giemsa. peroxidase, and nonspecific esterase. TGF-81 u r d TGF-82 NSSCZJ. Purified ( > 9 5 % ) primary leukemic blasts (2 X 10" cells/mL) were cultured, for various lengths of time. i n 35 X 10 mm Petri dishes (Falcon; Becton Dickinson) in RPM1 1640 medium (GIBCO Biocult, Paisley, UK) supplemented with 2 mmolL L-glutamine, 1 0 0 U/mL penicillin, and 2% HI-FCS i n the presence of l wmol/L quercetin. An equivalent amount of ethanol was added to cells as a control.At the end of incubation the medium was collected with siliconized pipet tips, combined with Apoprotin (final concentration, 0.04 trypsin inhibitor units/mL: Sigma), centrifuged at 8001: for 15 minutes in siliconized tubes to remove cellular Blood, Vol 85, No 12 (June 15). 1995: pp 3654-3661 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. QUERCETININDUCESTGF-P1 3655 IN LEUKEMICBLASTS Table l.Quometin InhibitoryCapacity of Colony Formation and Clonogonic Efficiency in Acute Leukemic Subtype8 Case NO. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Leukemic Cell Subtype ID 50%. ipmoVLl Clonogenic Efficiencyt M1 M1 M1 M1 M2 M2 M2 M3 M3 M4 M4 M4 M5 M5 M5 ALL ALL ALL ALL ALL ALL 8.0 30.0 4.0 Resistant 10.0 Resistant 3.0 0.3 0.5 0.6 0.1 0.06 0.2 0.08 0.8 0.01 0.04 0.07 0.4 91 87 154 100 15 8 * The ID 50% was 65 186 125 132 53 46 13 6 18 12 41 23 80 12 21 0.03 0.01 calculatedfrom the analysis ofthe dose-response curves. t Number of CFU-L perlo'cells plated. Results shownare the mean of quadruplicate cultures in the presence of vehicle alone (1% ethanol, vol/vol). The standard deviationfor each value was less than 10% and was omitted. debris, and then stored at -80°C until use. To evaluate the concentration of TGF-p1 and TGF-p2 in conditioned medium, we useda sensitive radioimmunoassay (New England Nuclear Research products, Dupont, Boston, MA) and an immunoenzymatic method (R & D System, Minneapolis, MN) strictly specific for TGF-p1 and TGFp2 detection, respectively. Each assay was performed according to the instructions of the manufacturer. To accurately quantify TGF-p1 , samples were activated using a two-step acidificatiodneutralization method. Briefly, 100 pL samples were incubated in the presence of 10 pL 1.2 NHCl for 15minutes at m m temperature &er vortexing. Then, 20 pL of 0.5 m o m HEPEWO.72 m o m NaOH was added to neutralize. The measured TGF-p1 value was multiplied by1.3 to correct for the dilution. This procedure allowes the quantitation of total (active + latent) TGF-PI. Immunohistochemical analysis. Primary leukemic blasts (2 X lo6celldmL) were cultured for 12hours in 35 X 10 mm Petri dishes (Falcon; Becton Dickinson) in RPM1 1640 medium supplemented with 2 mmom L-glutamine, 100 U/mL penicillin, and 2% HI-FCS at 37°C in a fully humidified 5% C02-95% air atmosphere in the presence of 1 p m o m quercetin. An equivalent amount of ethanol was added to control cells. At the end of culture, cells were incubated for 10 minutes in Caz+/MgZc-freecold PBS containing 0.02%ethylenediaminetetracetic acid, harvested by gently pipetting, and washed three times with phosphate-buffered saline (PBS) supplemented with 2 mg/mL ofbovine serum albumin (Flow Labs). Cytospins prepared with Shandon Cytospin (Shandon, Cheshire, UK) were fixed with 4% para-formaldehyde in PBS for 10 minutes at room temperature (RT) and permeabilized with cold (-20T) methanol for 10 minutes. Cells were then washed with PBS and incubated for 1 hour at RT with an anti-TGF-p1 mouse monoclonal antibody (MoAb; R & D System; clone TB 21; 10 pg/mL) or with an unrelated mouse MoAb isotype-matched control. Hydrogen peroxide, normal goat blocking serum, biotinylated Igs, avidin-biotin complex, and3-amino-9ethylcarbazole substrate solutions were used according to the manufacturer (ABC ELITE detection system; Vector, Burlingame, CA). Cells were lightly counterstained with Mayer's hematoxylin and mounted with CrystallMount (Biomeda, Foster City, CA). In the negative controls, no immunostaining was detectable. The positivity of the reactions were independently assessed in blind fashion by two pathologists; 100 consecutive cells in three or more fields were counted. The overall intensity of the staining was arbitrarily scored as negative or positive. The results were expressed as the percentage of positively stained cells regardless of intensity. Inhibition of TGF-p1 synthesis by antisense oligonucleotide. The sequences of the phosphorothioate oligonucleotides were as follows: TGF-p1 antisense, 5'-CCCGGAGGGCWCGGGGGA3'; TGF-p1 sense, 5'-TCCCCCmCGCCCTCCGGG-3'; TGFp 1 missense, 5'-GGCGAGCGAGTGAGCGCGCGG-3"4(ATG ini- n A Fig 1. Tim.cwrw of TGF-p1 production by AML"4 (AI and ALL IB) blnta culturedinthe presence (0)or absence (0) of 1 pmol/L quercetin. Blasts(2x W d l r l m L ) were cultured for the indicated time with or without quercetin; at tho end of the culture period, tho conditioned m a dia were hanrosted and the amount of total (activated Iatent) TGF-p1 was evaluatedby a radioimmunoassay er repotted in Materials and Methoda. + s e Sa W m 0 8 16 24 32 40 48 (hr) B1 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. LAROCCA ET AL 3666 tiation codon or its complement underlined in the sense and antisense sequences). Phosphorothioate oligonucleotides were purchased high performance liquid cbmatography (HPLQ-puriiied from MedProbe (Oslo, Norway). Cells were incubated in the presence of oligonucleotides at the final concentration of 8 pmol/L, essentially as described by others for evaluation of differentgenesexpresion.'^^^^ Oligonucleotides or control medium were added to the cells in IMDM in the absence of serum. Two hours later, 10% HIFCS was added to the liquid culture. After 15 to 17 hours, oligonucleotides or control medium were added again andcells were plated as described abovein a semisolid culture medium containing quercetin at various concentrations(0.001 to 10 pmol/L) or vehicle alone. Cm-Ls were scored after 6 to 10 days. [email protected] neutralizing experiments. To test the prevention of the growth inhibitory effectof quercetin by a monoclonal antiTGF-01, $2,-83 neutralizing antibody (anti-TGF-ps MoAb; Genw e ) , leukemic blasts were plated, as reported above in semisolid culture medium containing the anti-TGF-PS MoAb andor quercetin at the indicated final concentrations.Matched control cultures contained unrelated MoAb. CFU-Ls were scored after 6 to 10 days. RESULTS Inhibition of CFU-L by quercetin. As shown in Table 1, thecolonyformationbyleukemic cells was inhibited by quercetin in all but 2 cases (patients no. 4 and 6)that were resistant to quercetin at a concentration up to 100 pmol/L. Confirming previous reports:2o blasts with high clonogenic efficiency were less sensitive to quercetin than those with a low clonogenic capacity.In fact, the quercetin concentration inhibiting growth by 50% (ID 50%) positively correlated with the clonogenic efficiency of leukemic cells ( r = .81; n = 19; P < .001). A"M3, "4, and -M5 and commonALL were highly sensitive to quercetin, with an ID 50% ranging from 0.1 to 0.01 pmol/L. &-M1 and -M2 were less sensitive to quercetin, including the 2 resistant cases. Secretion of TGF-p1 by leukemic blasts treated with quercetin. To ascertain whether quercetin is able to stimulate the productionof TGF-B1 and TGF-p2, the amount of these cytokines were measured at various time points during 48 hours of culture in 1 &-M4 case (patientno. 10; Fig 1A) and in 1ALL case (patientno. 16; Fig 1B).A " M 4 blasts constitutively released total (active latent) TGF-p1 that was evaluable in the medium after 24 hours of culture. In the presence of 1 pmol/L quercetin, a peak of TGF-p1 release was evident after 4 hours (Fig lA), followedbya decrease at 12 hours to about 42% of the peak value. The concentration of TGF-B1 did not further change until the end of culture period. The cell number was stable during the culture period both in quercetin-treated and in control cultures. ALL blasts did not show constitutiverelease of total (active latent) TGF-p1 during the 48-hourperiod of culture. + + Fig 2. Irnrnunolocalization of TGF-p1 in AML-M5 (A and B) and in ALL (C and D) blasts culturedfor 12 hours with (B and D) or without (A and C) 1 prnollL quercetin. Original magnification x 800. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. QUERCETININDUCESTGF-P1 3657 IN LEUKEMIC BLASTS Table 2. Effect of Quercetin on TGF-B1 Expression in Blast-Enriched Bone Marrowa From AML (baeNo. 14) and ALL (Care No. 20) Leukemic Patianta ALL AML-M5 Cell Lineage (3%)t Granulocytic Monocytic Erythroid (-1 (2%) (95%) Blast Untreated" Treated 51 t W 95 t 5 20 t 4 20 2 4 60 t 9 85 t 8 - - Untreated Treated (2%)t (1%) 60t5 823 98 t 4 40 t 7 (1%) 36 5 4 0 56 2 5 82 t 6 (96%) Cells were cultured as outlined in Materials and Methods for 12 hours with vehicle alone (ethanol 0.1%, vol/vol) or 1 pmol/L quercetin. t Percentage of each celltype as assessed by cytoenzymatic and immunophenotypic analysis. Percentage of TGF-01 immunoreactivecells. Results are expressed as the mean t SD of two independent quadruplicate counts. * In the presence of 1 pmol/L quercetin, ALL blasts showed a peak of TGF-P1 release after 12 hours of culture (Fig lB), followed by a decrease at 24 hours to about 50% of the peak value. The number of ALL blasts after 48 hours of culture was 80% of the initial number of cells plated both in quercetin-treated and in control cultures. The concentration of TGF-P2 was below the detection levels of the enzyme-linked immunosorbent assay (ELISA) sensitivity in all samples tested (data not shown). Immunolocalization of TGF-PI. To determine if quercetin enhances the intracellular levels of TGF-P1 , immunohistochemical assays using an anti-TGF-Dl MoAb were performed on blasts from 1 patient with AML-M5 (patient no. 14) and 1 with ALL (patient no. 20) cultured for 12 hours with or without 1 pmol/L quercetin. As shown in Fig 2, in both cases quercetin enhances the intracellular content of anti-TGF-Bl immunoreactive materials. The immunoreac- 100m 0 1 tive product was localized in the cytoplasmic compartment. The blasts of the patient with AMGM5 expressed higher basal levels of TGF-P1 than the blasts of the patient with ALL. The presence of vehicle (ethanol) in the culture medium did not modify the basal level of TGF-p1 expression (data not shown). As shown in Table 2, the percentage of blasts in leukemic bone marrow preparations was 295%. With due caution given the low number of cells observed, it could be noted that in the remaining contaminating population (1) most segmented and immature granulocytes showed constitutive expression of TGF-P1 not modified by quercetin treatment and (2) in both monocytic and erythroid cell lineages quercetin increased the percentage of TGF-P1 immunoreactive cells. Effects of antisense TGF-PI oligonucleotide and of antiTGF-PI, -P2, $3 neutralizing MoAbon the inhibition effect of CFU-L by quercetin. The aim of our study was to evaluate the role of TGF-P1 in quercetin-dependent inhibition of CFU-L formation. To inhibit TGF-P1 production we used an antisense oligonucleotide. An anti-TGF-PS MoAb was used to neutralize TGF-PS produced by leukemic blasts. A 80 - 60- rw 0 g 4020 - - 0 10-'O10-~IO-*10" io-' None Q concentration (M) Fig 3. CFU-ALL inhibition byq u a d n alone (0)or in the preaonce of 8 pmol/L antisenae (A)or miaaonae (0) TGF-B1 oligonucleotides is dependent onthe concentration of quercetin. Cdla 110' cella) were treeted with quercetin and TGF-B1 oligoa as reported in Matoriala and Methods.R w l t a ahown ora the mean of four replicatea for aach variable. Standarddoviatbns were baa than 10% and were omitted. The number of control CFU-ALL was 41 k 3 for 10' cells plated. Q Q+antisense Treatments Fig 4. TGF-B1 production bythe r m e ALL bloats shown in Fig 3 cultured for 12 houra in the pr#snco of vehicle alono Inona) or 1 pmol/L quercetin (a) or lpmol/L quercetin plua8 pmol/L antiunae TGF-B1 oligoa (Q a n t i r n r ) . At the end of the Culture period, the conditioned mediawere hanreated and the amount of total (ectivatod + Iatont) TGF-B1 waa evaluated bya radioimmunoasaayaa reported in Materiala end Methods. + From www.bloodjournal.org by guest on December 22, 2014. For personal use only. LAROCCA ET AL 3658 120 ] 1 120 100 AI 100 B( 4 E 80 2 2 60 rcl 80 S8 60 % 40 40 0 20 20 0 0 0 0.1 1 10 Q concentration (PM) 120 g 4 cl 100 120 1 2 4 80 100 80 360 3 60 %l E 9 0 0.1 l 10 Q concentration (PM) +4 40 O 8 20 0 (m), 40 20 0 0 0.1 1 10 Q concentration (PM) preliminary dose/response curve showed that oligonucleotide concentrations greater than 10 pmol/L were toxic (data not shown). A final concentration of 8 pmol/L was used in all experiments. To showthat antisense TGF-PI oligonucle&dab could inhibit quercetin-induced cytokine production, 1 case of flavonoid-sensitive ALL (ID SO% -0.4 pmolL) in which antisense TGF-P1 oligonucleotides prevented the action of quercetin (Fig 3) was analyzed for the production of TGF-P1 in the presence of TGF-P I antisense oligonucleotides. As shown in Fig 4, TGF-P1 antisenseoligonucleotides could prevent the quercetin-induced cytokine production. Figure S shows the effectof TGF-P1 antisense oligonucleotideand neutralizing anti-TGF-psMoAb on quercetindependent inhibition of leukemic blasts growth. In I AMLM4 (patient no. IO, Fig 5A) and in 1 ALL (patientno. 19, Fig SB) the addition of antisense but not sense(data not shown) or missense TGF-P1 oligonucleotides prevented the inhibitoryaction of quercetin. The effect of the TGF-P1 antisense oligonucleotides was proportional to the flavonoid concentrations. The prevention of quercetin effects by antiTGF-PS MoAb was concentration dependent, resulting in a total prevention at an antibody Concentration of 100 pg/mL. In the quercetin-resistant AML-M2 case (patient no. 6, Fig SC), TGF-/3 1 antisense oligonucleotides produced an inhibition greater than SO% on CFU-L formation that was independent of the quercetinconcentrationused. Moreover, antiTGF-PS MoAb inhibited in a dose-dependent manner CFULformation, in this case with a maximum activityat 100 pg/mL anti-TGF-0s MoAb. In the quercetin-resistant AMLM1 case (patient no. 4, Fig SD), neither TGF-P1 antisense oligonucleotides nor anti-TGF-Ps MoAb produced any evident effect on CFU-L. An isotype-matched MoAb was used Fig 5 . Effect of antisense TGF-p1 oligos and neutralizing anti-TGF-psMoAb on quercetin-mediated inhibition of leukemic blasts. (AI AML-M4 (patient no. 10 of Table 1); (B) ALL (patient no. 19of Table ll;(Cl AMLM 2 (patient no. 6 of Table 1); (Dl AML-M1 (patient no. 4 of Table 1). Cells were plated atlo5 cells/ mL, as reported in Materialsand Methods, in the presence of vehicle alone (ethanol l % , vol/voll or quercetin (.l at the indicated concentrations. Eight micromoles of antisense (0) and missense oligonucleotides ( B ) were used alone or in combination with various quercetin concentrations. Anti-TGF-ps MoAb were added at 25 pg/mL (€4). 50 pg/mL and 100 pg/mL (0) with or without quercetin at various concentrations. Thenumber of control CFU-L are reported in Table 1. Results were expressed as the mean of quadruplicate cultures. Standard deviations were less than 10% and were omitted. 0 0.1 1 10 Q concentration (PM) as a control for anti-TGF-0s MoAb; no effects on CFU-L formationwas observed even at the highestconcentration used (100 pg/mL; data not shown). The addition of TCF-Pl antisense oligonucleotides prevented thc inhibitory effect of 1 pmollL quercetin in all t.he quercetin-sensitive cases (Fig 6). The extent of the effect of TGF-P1antisense oligonucleotidesvaried amongthe patients and did not seem to be related to the sensitivity of the leukemic blasts to quercetin. DISCUSSION Although originally described as a negative regulator of normal myeloid progenitor cell growth, recent studies have shown that TGF-01 is a bifunctional regulator of hematopoietic cells. TGF-P1 can either or stim~late'~.''.'~ the growth of murine and human hematopoietic progenitor cells; in particular, TGF-P1 is a potent inhibitor of primitive hematopoietic progenitors, whereas the growth of more committed progenitor cells is either not affected or stimulated by TGF-PI."' Previous studiessuggested that the growth of most human leukemic celllinesand of the vast majority ofcells from AML patients was inhibited by TGF-P1.'y-27 The datapresented here show that the bioflavonoid quercetin inhibits the growth of AML and ALL blasts in vitro by the induction of TGF-P1 production. This finding is based on the following observations: (1) quercetin enhances the intracellular content and the secretion of TGF-01 by sensitive leukemic blasts; (2) the inhibitory action of quercetin can be partially blocked by TGF-01 antisenseoligonucleotidesandcan be almost totallyabolished by neutralizing anti-TGF-PI, $2, -03 MoAb. From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 3659 QUERCETININDUCES TGF-Dl IN LEUKEMICBLASTS 100 M3 - M5 M1 - M2 ALL 0 0 80 60 40 20 0 I ~~ I I I O-2 I I m m 6 8 4 0 A I I I v3 E Fig 6. Antisense TGF-p1 oligonucleotides prevent the inhibitory actionof quercetin (1 pmol/L) on CFU-L. Cells were plated at lo6 cells/ mL, as reported in Materials and Methods,in the presence of quercetin elone(Q) or in combinationwith 8 pmol/L antisense (as) or missense (mSI oligonucleotides. The number of control CFU-L were reportedin Teble 1. Results were expressed as the mean of quadruplicate cultures. Standerd deviations wereless than 10% and were omitted. Patient nos.: panelM1-M2, (0)1, (0)2, (0)3, (D)4, (A) 5, (A)6, ( 0 ) 7; panel M3M5, (0)8, IO) 9,101 10, (D) 11, (A) 12, (A)13, ( 0 ) 14, ( + l 15; panel LLA, (0)16, (0)17, ( 0 )18,(D) 19, (A) 20, (AI 21. Because it has been reported that retinoic acid, which also induces TGF-P1 production, is able to upregulate TGF-01 receptor expression on the HL-60 cell line,33it seems interesting to investigate whether quercetin can act in a similar fashion. The differences between TGF-01 antisense oligonucleotides and anti-TGF-Ds MoAb in preventing the effect of quercetin could be explained as follows. (1) Neutralizing antibodies but not antisense oligonucleotides can block the effect of TGF-01 already present in quercetin untreated cells as shown by immunohistochemistry. If this is true, quercetin could stimulate the secretion in addition to the synthesis of TGF-01. (2) TGF-Pl antisense oligonucleotides are not able to completely inhibit TGF-01 synthesis, as shown in the case of the ALL presented in Fig 4. (3) The neutralizing anti-TGF-0s MoAb used in this study is able to neutralize TGF-/33 in addiction to TGF-01. It isthen possible that quercetin could stimulate the release of TGF-P3 as well. TGF-02 is not involved in the quercetin-dependent growth inhibition because it is not produced by quercetin-treated leukemic blasts. Two patients (nos. 4 and 6) behaved differently from all other patients. Particularly, in both cases leukemic blasts were resistant to the growth-inhibitory effect of quercetin. In patient no. 4, because quercetin induces TGF-P1 produc- tion in leukemic blasts (data not shown), this resistence could be dependent on the unresponsiveness of these cells to TGFD l . The growth of leukemic blasts from patient no. 6 was stimulated by [email protected], antisense oligonucleotides, blocking the synthesis of TGF-Pl, induced a reduction of more than 50% of the clonogenic activity in this case. Furthermore, 100 p g / d of neutralizing [email protected] MoAb inhibited almost abolished CFU-L activity (Fig 5C). In this case, quercetin did not significantly increase the release of TGF-P1 above background (data not shown) and was unable either to stimulate or inhibit the leukemic cell growth. These data are in accordance with a recent report’’ about heterogeneous responses of leukemic cell lines and primary leukemic blasts to the growth-regulatory action of [email protected] Although the mechanism of the antiproliferative activity of quercetin remains to be fully clarified, there is evidence suggesting that the action of this substance is probably mediated by its interaction with the so-called type II EBS.3*6.34-36 Indeed, our data indicate that the quercetin ID 50% in sensitive leukemic cells is compatible with the dissociation constant (kd) of type I1 EBS in leukemic blasts! This possibility is also supported by the observation that the antiestrogen tamoxifene, which binds to type I1 EBS,37 induces TGF0 1 production in estrogen-receptor-positive MCF-7 human breast cancer cells3’ as well as in estrogen-receptor-negative From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 3660 LAROCCA ET AL human fetal fib rob last^.'^ For these reasons, it seems interesting to investigatewhether tamoxifene could inhibit leukemic cell growth as well. REFERENCES 1. Kilhnau J: The flavonoids, a class of semi-essential food com- ponents: Their role in human nutrition. World Rev Nutr Diet 24: 117, 1976 2. Gabor M: Szent-Gyorgyi and the bioflavonoids: New results and perspectives of pharmacological research into benzo-pyrone derivatives, in Cody V, Middleton E Jr, Harborne JB, Beretz A (eds): Plant Flavonoid Biology and Medicine 11: Biochemical, Cellular, and Medical Properties. New York, NY, Liss, 1988, p 1 3. Scambia G , Ranelletti FO, Benedetti Panici P, Piantelli M, Rumi C, Battaglia F, Larocca LM, Capelli A, Mancuso S: Type I1 estrogen binding sites in a lymphoblastoid cell line and growth inhibitory effect of estrogen, antiestrogen and bioflavonoids. Int J Cancer 46: 11 12, 1990 4. Yoshida M, Yamamoto M, Nikaido T: Quercetin arrests human leukemic T-cells in late G, phase of the cell cycle. Cancer Res 52:6676, 1992 5. Post JFM, V m a RS: Growth inhibitory effects of bioflavonoids and related compounds on human leukemic CEM-Cl and CEM-C7 cells. Cancer Lett 67:207, 1992 6. Larocca LM, Piantelli M, Leone G, Sica S , Teofili L, Benedetti Panici P, Scambia G, Mancuso S , Capelli A, Ranelletti FO: Type I1 oestrogen binding sites in acute lymphoid and myeloid leukaemias: Growth inhibitory effect of oestrogen and flavonoids. Br J Haematol 75:489, 1990 7. Larocca LM, Teofili L, Leone G, Sica S , Pierelli L, Menichella G , Scambia G, Benedetti Panici P, Ricci R, Piantelli M, Ranelletti FO: Antiproliferative activity of quercetin on normal bone marrow and leukemic progenitors. Br J Haematol 79:562, 1991 8. 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For personal use only. 1995 85: 3654-3661 Quercetin inhibits the growth of leukemic progenitors and induces the expression of transforming growth factor-beta 1 in these cells LM Larocca, L Teofili, S Sica, M Piantelli, N Maggiano, G Leone and FO Ranelletti Updated information and services can be found at: http://www.bloodjournal.org/content/85/12/3654.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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