ICANCER R@EARCH 54, 2424-2428, May 1. 19941

ABSTRACT

The effects of the bioflavonoid quercetin (3,3',4',5,7-pentahydroxyflavone) on the growth and cell cycle progression of the human breast cancercell line MDA-MB468 have been studied. Quercetin inhibited cell proliferation with an IC@ (a drug concentration which inhibited growth by

50% following a 3-day exposure) value of 7 @sgIml.In actively growingcultures, the addition of quercetin resulted in the accumulation of cells at

the G2-Mphase. We have correlated these effects on cell proliferation withthe observation that quercetin strongly Inhibited, in a time- and dosedependent fashion, the expression ofthe mutated p53 protein, which is theonly form present at high levels in this cell line. This inhibition takes placeat the translational leveL Quercetin did not affect the steady-state mRNAlevels of PS3, but prevented the accumulation of newly synthesized p53protein. This quercetin action appeared to be somewhat specific for p53because the drug did not alter the amount of other proteins present in

MDA-MB468 cells such as P-glycoprotein and did not prevent the induction of the synthesis of epidermal growth factor receptor in response toepidermal growth factor.

INTRODUCTION

Quercetin (3,3',4',5,7-pentahydroxyflavone), a widely distributedflavonoid, is a common component of the human diet (1). Thiscompound has been shown to inhibit the growth of various cell linesderived from human cancers, such as leukemia and Ehrlich ascitestumors (2), squamous cell carcinoma of head and neck origin (3),gastric (4) and colon (5) carcinoma, and the estrogen receptor positivebreast carcinoma MCF-7 (6). Quercetin treatment arrested humangastric tumor cells in the G1 phase (4) and leukemic T-cells in late G1phase of the cell cycle (7). The growth inhibitory effects of quercetinmay be a consequence of its ability to interfere with enzymaticprocesses involved in the regulation of cellular proliferation. Biological activities known for this flavonoid, such as the inhibition ofprotein tyrosine kinases (8—10),protein kinase C (1 1, 12), and cyclicAMP and cyclic GMP phosphodiesterases (13, 14), the increase ofcyclic AMP levels (15), and the interaction with estrogen type IIbinding sites (6), may explain in part the growth inhibitory effects ofquercetin in the various experimental systems so far examined.

In the present report, we have tested the effects of quercetin on thegrowth of the human breast carcinoma cell line MDA-MB468. Theproliferation of MDA-MB468 cells was markedly inhibited by thepresence of quercetin, and cell cycle progression was altered withaccumulation of cells at G2-M. MDA-MB468 are estrogen receptornegative cells that overexpress a transcriptionally active mutant p53protein (16, 17) which has been recently related to its neoplasticphenotype (18). Since the presence of mutated forms ofp53 has beenimplicated in the etiology of human breast cancer (16, 19), and thefact that mutant p53 protein is required continuously to maintain thetransformed phenotype (20), we have evaluated the effect of quercetinon mutant p53 expression. Our results indicate that p53 protein levels

Received 12/6/93; accepted 3/1/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed, at Georgetown UniversityMedical Center, Lombardi Cancer Center, Room P427, 3800 Reservoir Road NW.,Washington, DC 20007.

were markedly reduced in response to quercetin treatment. This effectshowed a certain degree of specificity and may contribute to explainthe growth inhibitory properties of quercetin on this cell line.

MATERIALS AND METHODS

Cell Culture and Drug Treatmeni MDA-MB468 cells were obtainedfrom the American Type Culture Collection (Rockville, MD), and were grownin improved minimum essential Eagle's medium with 2 mMglutamine (Biofluids, Rockville, MD), 10% fetal bovine serum, 100 units/mi penicillin, and100 @Wmlstreptomycin, at 37°Cin a 5% CO2 atmosphere.

Quercetin was purchased from Sigma (St. Louis, MO). Quercetrin andquercetin-3-rutinoside were the generous gift of Dr. T. Leighton. All flavonoids were dissolved in ethanol. The final concentration of ethanol in theculture medium never exceeded 0.9% (v/v), and the same concentration was

present in control experiments.Growth Rate and Flow Cytometric Analysis. Cells were seeded in 6-cm

plates at 0.5 X iø@cells/plate. Two days after inoculation, the culture mediumwas changed and quercetin was added at different doses. At various experimental intervals, the number ofviable cells was determined by the Trypan bluedye exclusion test.

The effect of quercetin on cell cycle was studied on actively growing cells:4 X 10@cells were seeded in 10-cm plates, and after 24 h the drug was addedat different concentrations. Cultures were replenished with fresh medium andquercetin after the third day of treatment. Samples were collected for up to 6days after the addition of quercetin, and cell cycle distribution was determined.

Cell cycle analysis was performed as described (21) using a flow cytometerFACSIar Plus (Becton Dickinson FACS System).

mRNA Analysis. Cells were grown for 24 h, then treated for 2 and 6 h withdifferent doses of quercetin (30 and 75 @g/ml).At the end of the treatments,the cells were washed with phosphate buffered saline and lysed with guanidineisothiocyanate. Total RNA was extracted as described (22). Total RNAS (20@.Lg)from each treatment were denatured in 1.1 M formaldehyde and 50%formamide and electrophoresed in 1% agarose gels under denaturing condi

tions. RNAS were then blotted and fixed to nitrocellulose membranes. Prehybridization and hybridization were carried out using the Wahi buffer (23), for

6 and 24 h, respectively, at 42°C.Filters were hybridized with the full-lengthhuman p53 complementary DNA. The probe was labeled with [a32P]dCTP byrandom priming using the Megapnme DNA labeling system (Amersham,Arlington Heights, IL), and purified through Sephadex G-50 spin columns.Specific activity was usually about 5 x 108 cpm/@g. Blots were washed 3times at room temperature in 2X standard saline-citrate, 0.1% SDS2 and twiceat 42°Cin 0.lX standard saline-citrate, 0.1% SDS. The same blot washybridized with a f3-actin probe (Clontech, Palo Alto, CA) to ensure equalloading of the samples.

Immunoblotting Analysis. After treatment, cells (5 X 1O@/6-cmplate)were washed with phosphate buffered saline and disrupted by addition of 150@tlof lysing buffer (10 mM sodium phosphate, pH 7.5, 100 mr@tNaC1, 1%

Triton X-100, 0.5% sodium deoxycholate, 1 m@iphenylmethylsulfonyl fluoride, 16 @.&g/mlaprotinin)/dish. Equal amounts of protein (40 ,@g)were subjected to 12.5% SDS-polyacrylamide gel electrophoresis. Samples were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore,Bedford, MA) in a transfer buffer containing 25 mMTris, 192 mMglycine, and20% (v/v) methanol. Membranes were blocked overnight with 1% low fat drymilk in Tris-buffered saline containing 0.05% Tween-20. Immunodetection ofp53 was performed using 3 @g/mlof a commercial monoclonal anti-p53antibody (Ab-1; Oncogene Science, Uniondale, NY). The same concentration

2The abbreviationsusedare:SDS,sodiumdodecylsulfate;EGF,epidermalgrowthfactor; EGF-R, epidermal growth factor receptor.

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Quercetin Mediates the Down-Regulation of Mutant p53 in the Human BreastCancer Cell Line MDA-MB468

MatIas A. Avila, Juan A. Velasco, JoséCansado, and Vicente Notarlo'

Division of Experimental Carcinogenesis, Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 2@XJO7

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INHIB@ON OF MUTANT p53 PROTEIN BY QUERCETIN

with 7 p@g/mlof quercetin (Fig. 2A), and 61% in those treated with 30@g/ml(Fig. 2B).Regulation of Mutant p53 Protein Levels: Dose Dependency

andTimeCourseof the QuercetinEffect.Becausep53expressionhas been correlated with the growth of MDA-MB468 cells (18, 24),and quercetin proved to inhibit the proliferation of this cell line, weanalyzed the effect of quercetin treatment on the steady state p53levels. As shown in Fig. 3A, quercetin induced a significant decreasein the level of p53 over an 8-h treatment. The effect was dosedependent and, within this period of time, it was already apparent ata dose of 30 p@g/ml.Higher concentrations of quercetin had a dramaticeffect, bringing p53 to nearly undetectable levels at 75 @Wml.

The time course of quercetin action on p53 protein was alsofollowed. Cells were treated with 50 p.g/ml of quercetin for differenttime intervals. As indicated in Fig. 3B, changes in p53 levels werealready detectable as early as 2 h after the addition of the drug. Longerincubations, up to 8 h, resulted in a progressive decrease of the signal.

These results show that quercetin is able to down-regulate thesteady-state levels of mutated p53 in a time and dose dependentmanner, and that this effect takes place within a range of concentrations similar to that described above to regulate the proliferation ofMDA-MB468 cells.

Specificity of the Quercetin Effect on p53 Levels. Quercetin hasbeen reported to inhibit the synthesis of macromolecules in Ehrlichascites tumor cells (15). To rule out the possibility that quercetineffects on p53 protein were only a consequence of an overall inhibition of protein synthesis, we studied the levels of other proteins before

Incubation Time (Days)

Fig. 2. Effect of quercetin on cell cycle progression. Two days after inoculation of 4 X@ cells, quercetin was added to the medium at a concentration of 7 paJml (A ) and 30

gi@g/ml(B). Cultures were monitored for up to 6 days. The percentage of cells in G, (A),S 4, andG2-M(C) phasewasdeterminedby quantifyingtheDNAhistograms.Points,mean of duplicate experiments.

control

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Fig. 1. Growth inhibition of MDA-MB468 cells treated with different doses of quercetin. Cells (0.5 x 10°)were seeded and, after 2 days, quercetin was added to the medium.The number of viable cells was determined at different time intervals after the onset of thetreatment. Results are the average of 3 experiments. SEM were in the range of ±10%.

of anti-MDR-1 polyclonal antibody or anti-EGF-R antibody (OncogeneScience) was used for immunodetecting P-glycoprotein and EGF-R. Cellextracts were incubated for 4 h with each antibody, being then incubated for1.5 h in the presence of an alkaline phosphatase-conjugated secondary antibody. Proteins were visualized with nitroblue-tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate following manufacturer's instructions (Promega,Madison, WI).

Cell Labeling and Immunoprecipitation. Cells (5 X 10@)were seeded in10-cm plates and grown as described for 24 h. Before labeling, the cells werestarved for 30 mm in methionine-free Dulbecco's modified Eagle's medium(Gibco-Bethesda Reasearch Laboratories, Gaithersburg, MD) in the absence ofserum. Then 400 p@Ciof [35Sjmethionine (1175 Ci/mmol; New EnglandNuclear, Boston, MA) were added per plate in 3.5 ml of methionine-freemedium, and the incubation was continued for 5 h, in the presence or absenceofquercetin. Radiolabeled cells were disrupted with 400 pi of lysing buffer perplate. Lysates were clarified at 12,000 X g for 30 mm, and the radioactivityincorporated was determined by scintillation counting of an aliquot from eachlysate. Aliquots of cell lysates with the same amount of radioactivity wereincubated with anti-p53 antibody Ab-2 (Oncogene Science) for 2 h at 4°C.Immune complexes were collected with protein A bound to Sepharose-4Bbeads (Pharmacia, Piscataway, NJ), washed extensively with lysis buffer, andanalyzed by 12.5% SDS-polyacrylamide gel electrophoresis. Immunoprecipitated polypeptides were visualized by autoradiography.

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RESULTS

Effects of Quercetin on Cell Growth and Cell Cycle Progression. As shown in Fig. 1, quercetin induced a dose-dependent inhibition of the growth of MDA-MB468 cells that was already evidentafter 24 h of treatment. Three days after the addition of 5, 10, 15, and30 @Wmlof quercetin, viable cell number decreased to 62, 38, 27, and20% of the control value, respectively. The drug concentration whichinhibited growth by 50% following a 3-day exposure was estimated tobe about 7.0 @g/ml.

The effect of quercetin on cell cycle progression was also studied.Actively growing cells were treated with 7 or 30 @Wmlof quercetin,and the distribution through the cell cycle was analyzed at differentintervals. As depicted in Fig. 2, quercetin treatment induced a progressive accumulation of cells in the G2-M phase. The percentage ofcells in G1 phase decreased in a time- and dose-dependent fashion.The percentage of cells in S phase did not change significantly; therewas some accumulation for up to 3 days, decreasing thereafter (Fig.28). G2-M cells gradually increased, and by the sixth day of treatmentthey represented about 46% of the total population in cultures treated

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—97Fig. 3. Western blot analysis of quercetin effect onp53 protein levels. A, exponentially growing MDAMB468 cells were treated with increasing doses of 68—quercetin for 8 h. Lane 1, control; Lane 2, 10 pg/mI;Lane 3, 30 pg/ml; Lane 4, 50 pg/mI; Lane 5, 75@Lg/ml.B, time course of quercetin effect on p53

protein at 50 @.tWml.Lane 1, control; Lane 2, 1 h;Lane 3, 2 h; Lane 4, 4 h; Lane 5, 6 h; Lane 6, 8 h.

43—

and after quercetin treatment. The expression of the plasma membraneP-glycoprotein, responsible for the multidrug resistance phenotype,has been found to be increased in biopsy samples from most types ofhuman tumors and tumor cell lines (25), including the MDA-MB468cells. As shown in Fig. 44, the presence of P-glycoprotein was readilydetectable by Western blot, and the treatment of cells with 30 or 75

@g/mlof quercetin for either 5 or 24 h did not alter P-glycoproteinlevels.

An additional approach was taken to ascertain the specificity of thequercetin action on p53 down-regulation. EGF treatment of MDAMB468 cells has been shown to induce the expression of its ownreceptor (26, 27). As shown in Fig. 4B, a dramatic increase in thelevels of EGF-R protein were detected by Western blot when cellswere incubated for 7 h with 10 ng/ml of EGF. The synthesis of EGF-Rwas not altered when this treatment was performed in the presence of30 or 75 ,.Lg/ml of quercetin. These data demonstrate that downregulation of p53 by quercetin is not caused by a general nonspecificinhibition of protein synthesis in MDA-MB468 cells.

To rule out the possibility that the down-regulation of mutant p53may be a cell cycle dependent phenomenon, cells were arrested by

Fig. 4. Quercetin had no effect on either the cxpression of P-glycoprotein or the induction ofEGF-R by EGF treatment of MDA-MB468 cells. A,Western blot analysis of P-glycoprotein in cellstreated with quercetin. Lane 1, control; Lanes 2 and3, 5-h treatment with 30 and 75 pg/mI; Lanes 4 and5, 24-h treatment with 30 and 75 @Wml,respectively. B, quercetin effect on EGF stimulation ofEGF-R expression in MDA-MB468 cells; the extentof EGF-R induction was analyzed by Western blot.Lane 1, control; Lane 2, EGF; Lane 3, EGF plus 30pg/mi of quercetin; Lane 4, EGF plus 75 pg/mI ofquercetin.

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serum starvation (48 or 72 h) and then treated with the same range ofquercetin concentrations described above for actively proliferatingcultures. Western blot analyses demonstrated that cell cycle arrestedcells contain p53 levels as high as those in proliferating cells, and thatquercetin treatment after cell cycle arrest also results in down-regulation of p53 (data not shown), thus indicating that the effect ofquercetin on p53 is independent of the cell cycle status of the cultures.

The effect on p53 protein levels of other molecules structurallyrelated to quercetin was also analyzed. Cells were treated for differentperiods oftime with 50 or 100 @Wmlof either quercetrin or quercetin3-rutinoside. None of these compounds had significant effects on thesteady state levels of p53, even at concentrations higher than thatrequired for quercetin maximal effect (data not shown).

Mechanism of Quercetin Inhibition of p53 Protein Expression.To gain some insight into the mechanism by which quercetin reducedthe steady state levels of p53 protein, we analyzed the effects of thisdrug on the steady state levels ofp53 mRNA. Cells were treated fordifferent periods in the presence of 30 or 75 @&Wmlof quercetin, andmRNA levels were measured by Northern blot hybridization. Asshown in Fig. 5, quercetin treatment did not decrease p53 mRNA

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osteosarcoma cell line lacking endogenous p53 protein expression,cell growth is stimulated and saturation density is increased, demonstrating a gain of function for mutant p53 that can promote cellulartransformation (31). Although this p53 mutant is less efficient thanother mutants in the cooperation with ras in primary rat cell transformation, its expression has been linked to the maintenance of thetransformed phenotype of MDA-MB468 cells (18, 24). Our findingthat quercetin inhibited the proliferation of MDA-MB468 cellsprompted us to investigate the quercetin action on p53 expression. Ourresults showed that this flavonoid efficiently reduced the steady-statelevels of p53 in a time- and dose-dependent fashion. Quercetin hasbeen previously shown to inhibit the expression of unidentified proteins that have been related to the transformed phenotype of humancolon cancer cells and human leukemic T-cells (5, 7). In this paper, wedemonstrate for the first time that this flavonoid specifically inhibitsthe expression of a mutant p53 implicated in cellular transformation.This effect can be directly related to the growth inhibitory properties

of quercetin in this cell line, because the continuous expression ofmutant p53 gene is required to maintain the transformed phenotype ofMDA-MB468 cells (20).

We have also analyzed the action of quercetin-related molecules onp53 expression in MDA-MB468 cells. Quercetrin and quercetin-3-rutinoside had no detectable effect on p53 protein. This could be aconsequence of their more polar nature, preventing them from reaching intracellular targets otherwise accessible to quercetin. To rule outthe possibility that quercetin effect on p53 protein levels could be theconsequence of an overall inhibition of protein synthesis, we evaluated the effects of this drug on other cellular proteins. Although it hasbeen described that quercetin inhibits the expression of the multidrugresistant gene, which encodes P-glycoprotein, in a human hepatocarcinoma cell line (Hep 02) (32), our results showed that P-glycoprotein, present at high levels in MDA-MB468 cells, was not affected byexposure to quercetin. The specificity of quercetin effect on p53expression was further supported by our finding that quercetin did notprevent the stimulation of EGF-R synthesis in MDA-MB468 cells byEGF treatment. These results support the notion that quercetin doesnot interfere randomly with ongoing protein synthesis.

Quercetin has been shown to inhibit the synthesis of heat-shockproteins at the level of mRNA accumulation (33). Our data demonstrate that this is not the case for p53, since the levels of mRNA didnot change in cells exposed to quercetin. However, when p53 proteinsynthesis was analyzed, quercetin treatment resulted in a clear reduction of immunoprecipitable newly synthetized protein. Although quer

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Fig. 6. Autoradiography of (35Slmethionine-labeled p53 immunoprecipitates fromMDA-MB468 cells treated with different concentrations of quercetin for 5 h. Lane 1,control untreated cells; Lane 2, cells treated with 10 pg/mI of quercetin; Lane 3, 30 pg/mI;Lane 4, 50 pg/mI.

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Fig. 5. A, p53 mRNA levels in quercetin-treated and untreated cells. Lane 1, control;Lanes 2 and 3, cells treated for 2 and 6 h with 30 @.tg/ml;Lanes 4 and 5, cells treated for2 and 6 h with 75 @Wmlof quercetin. B, same blot hybridized with @3-actinprobe.

levels, demonstrating that there was no alteration of p53 transcriptionor mRNA processing. This result led us to examine whether quercetincould prevent the synthesis of p53 protein. This issue was addressedby labeling the cells with [35Slmethionine in the presence or absenceof various doses of quercetin. After a period of 5 h, cells were lysedand aliquots with equal amounts of radioactivity were immunoprecipitated. Fig. 6 shows that quercetin treatment produced a dosedependent reduction in the amount of newly synthesized p53. Thisresult strongly indicates that the effect of quercetin on p53 steady statelevels is mediated through the inhibition of p53 mRNA translation.

DISCUSSION

We have examined the effects of quercetin, a dietary flavonoid, inthe growth of the estrogen independent MDA-MB468 human mammary tumor cells. Our data show that quercetin was able to inhibitproliferation of this cell line in a dose dependent fashion, with a drugconcentration which inhibited growth by 50% following a 3-dayexposure of 7 @g/mland a cytostatic effect at about 30 @g/ml.Theprogression of actively growing cells through the cell cycle wasanalyzed, and the presence of quercetin resulted in the accumulationof cells at G2-M. Similar results have been obtained when the humangastric cancer cell line HGT-27 was treated with the flavonoidgenistein (28). On the other hand, when MDA-MB468 cells werearrested in G1 by serum starvation, and the cell cycle was reinitiatedby serum addition, the presence of quercetin prevented the transitfrom G1 to cycle in a reversible fashion (data not shown). Theseresults indicate that quercetin blocked MDA-MB468 cell growthregardless of the cell cycle status of the culture, and that the targets ofits effect should be active or present throughout the cell cycle topromote cellular proliferation.

Quercetin has been shown to inhibit the growth ofthe human breastcancer cell line MCF-7 (6). In this case, the inhibitory effects ofquercetin were related to its capacity to interact with type II estradiolbinding sites. Since MDA-MB468 cells are estrogen receptor negative, this mechanism of action could not account for quercetin effects.

One of the main characteristics of the MDA-MB468 cell line is thepresence of a single p53 allele that harbors a point mutation at codon273 (p53-273H) (16). This mutant p53 has been demonstrated tocooperate with ras in transforming primary cells (29, 30), and to retaintranscriptional activity for the p53 consensus sequence (p53CON)(17). When this mutant allele is introducedinto SAOS-2 cells, an

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12. Grunicke, H., Hofmann, J., Maly, K., Uberall, F., Posch, L., Oberhuber, H., andFiebig, H. The phospolipid- and calcium-dependent protein kinase as a target in tumorchemotherapy. Adv. Enzyme Regul., 28: 201—216,1989.

13. Beretz, A., Anton, R., and Stoclet, J. C. Flavonoid compounds are potent inhibitors ofcyclic AMP phosphodiesterase. Experientia, 34: 1054—1055, 1978.

14. Ruckstuhl, M., Beretz, A., Anton, R., and Landry, Y. Flavonoids are selective cyclicGMPphosphodiesteraseinhibitors.Biochem.Pharmacol.,28: 535—538,1979.

15. Graziani, Y., and Chayoth, R. Regulation of cyclic AMP level and synthesis of DNA,RNA and protein by quercetin in Ehrlich ascites tumor cells. Biochem. Pharmacol.,28: 397—403,1979.

16. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hosteter, R., Cleary, K.,Bigner, S. H., Davidson, N., Baylin, S., Devilez, P., Glover, T., Collins, F. S., Weston,A., Modali, R., Harris, C. C., and Vogeistein, B. Mutations in the p53 gene occur indiverse human tumor types. Nature (Lond.), 342: 705—708,1989.

17. Chen, J. Y., Funk, W. D., Wright, W. E., Shay, J. W., and Minna, J. D. Heterogeneityof transcriptional activity of mutant p53 proteins and p53 DNA target sequences.Oncogene, 8: 2159—2166, 1993.

18. Wang, N. P., To, H., Lee, W., and Lee, E. Y. Tumor suppressor activity of RB andp53 genes in human breast carcinoma cells. Oncogene, 8: 279—288,1993.

19. Barket, J., Bartkkova, J., Vojtesek, B., Staskova, Z., Rejthar, A., Kovarik, J., andLane, D. P. Patterns of expression of thep53 tumor suppressor in human breast tissuesand tumors in situ and in vitro. tnt. J. Cancer, 46: 839—844, 1990.

20. Zambetti, G. P., Olson, D., Labow, M., and Levine, A. J. A mutant p53 protein isrequired for the maintenance of the transformed cell phenotype in p53 plus rastransformed cells. Proc. Nati. Acad. Sci. USA, 89: 3952—3956,1992.

21. Vindelov, L. L., Christensen I. J., and Nissen, N. I. A detergent-trypsin method forpreparation of nuclei for flow cytometric DNA analysis. Cytometry, 3: 323—327,1983.

22. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156—159, 1987.

23. WahI, G. M., Stern, M., and Stark, G. R. Efficient transfer of large DNA fragmentsfrom agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by usingdextran sulfate. Proc. Natl. Acad. Sci. USA, 76: 3683-3687, 1979.

24. Casey, G., Lo-Hsueh, M., Lopez, M. E., Vogelstein, B., and Stanbridge, E. J. Growthsuppression of human breast cancer cells by the introduction of a wild type p53 gene.Oncogene, 6: 1791—1797,1991.

25. Goldstein, L J., Galski, H., Fojo, A., Willingham, M., Lai, S. L, Gazdar, A., Pirker,R., Green, A., Crist, W., Brodeur, G. M., Lieber, M., Cossman, J., Gottesman, M. M.,and Pastan, I. Expression of a multidrug resistant gene in human cancers. J. Natl.Cancer Inst., 81: 116—124,1989.

26. Bjorge, J. D., and Kudlow, J. E. EGF receptor synthesis is stimulated by phorbol esterand epidermal growth factor. J. Biol. Chem., 262: 6615—6622,1987.

27. Bjorge, J. D., Kudlow, J. E., Mills, G. B., and Paterson, A. J. Inhibition of stimulusdependent epidermal growth factor receptor and transforming growth factor-a mRNAaccumulation by the protein kinase C inhibitor staurosporine. FEBS Lett., 243:404—408,1989.

28. Matsukawa, Y., Marui, N., Sakai, T., Satomi, Y., Yoshida, M., Matsumoto, K.,Nishino, H., and Aoike, A. Genistein arrests cell cycle progression at G2-M. CancerRes., 53: 1328—1331, 1993.

29. Hinds, P. W., Finlay, C. A., Quartin, R. S., Baker, S. J., Fearon, E. R., Vogeistein, B.,and Levine, A. J. Mutant p5.3 DNA clones from human colon carcinomas cooperatewith ras in transforming primary rat cells: a comparison of the “hotspot―mutantphenotypes. Cell Growth & Differ., 1: 571—580,1990.

30. Slingerland, J. M., Jenkins, J. R., and Benchimol, S. The transforming and suppressorfunctions ofp53 alleles: effects of mutations that disrupt phosphorylation, oligomerization and nuclear translocation. EMBO J., 12: 1029—1037,1993.

31. Chen, P. L., Chen, Y., Bookstein, R., and Lee, W. H. Genetic mechanisms of tumorsuppression by the human p53 gene. Science (Washington DC), 250: 1576—1580,1990.

32. Kioka, N., Hosokawa, N., Komano, T., Hirayoshi, K., Nagata, K., and Ueda, K.Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene(MDR1) expression caused by arsenite. FEBS Lett., 301: 307—309,1992.

33. Hosokawa, N., Hirayoshi, K., Nakat, A., Hosokawa, Y., Marui, N., Yoshida, M.,Sakai, T., Nishino, H., Aoike, A., Kawai, K., and Nagata, K. Flavonoids inhibit theexpression of the heat shock proteins. Cell Struct. Funct., 15: 393—401, 1990.

34. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 mutations inhuman cancers. Science (Washington DC), 253: 49—53,1991.

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cetin alteration of p53 protein stability leading to enhanced degradation cannot be excluded, the inhibition of its mRNA translation byquercetin seems to be a major determinant for the reduction of p53levels in MDA-MB468 cells.

Experiments to examine the effect of quercetin on wild-type p53were performed with the human breast carcinoma cell line MCF-7.

Results demonstrated that, although normal p53 was down-regulatedalso, higher concentrations of quercetin were required to attain inhibitory effects of the same magnitude as those observed for MDAMB468 cells (data not shown). Whether this is due to reduced uptakeof quercetin by MCF-7 cells or to a direct differential effect onwild-type p53 remains to be elucidated. Our observation that quercetindown-regulates more efficiently the expression of mutant p53 might

explain the growth inhibitory properties of this flavonoid, providingnew insights about its mechanism of action. Because the mutation inthe p53 protein present in MDA-MB468 cells, converting codon 273arginine to histidine, is frequent in human tumors (34), further studieswill examine the applicability of quercetin in the control of theproliferation of tumors containing similar p53 mutant species.

ACKNOWLEDGMENTS

The authors would like to thank Terrance Leighton for providing thequercetin structural analogues, Owen C. Blair for cell cycle analyses, andAnatoly Dritschilo for his support during the development of this project.

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1994;54:2424-2428. Cancer Res   Matías A. Avila, Juan A. Velasco, José Cansado, et al.   Human Breast Cancer Cell Line MDA-MB468Quercetin Mediates the Down-Regulation of Mutant p53 in the

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