Induction of Apoptosis and Inhibition of c-erbB-2 in Breast CancerCells by Flavopiridol1

Yiwei Li, Mahbubur Bhuiyan, Samir Alhasan,Adrian M. Senderowicz, and Fazlul H. Sarkar2

Department of Pathology, Karmanos Cancer Institute, Wayne StateUniversity School of Medicine, Detroit, Michigan [Y. L., M. B.,S. A., F. H. S.], and Developmental Therapeutics Program, NationalCancer Institute, Bethesda, MD [A. M. S.]

ABSTRACTFlavopiridol is a flavone that inhibits several cyclin-

dependent kinases and exhibits potent growth-inhibitoryactivity against a number of human tumor cell lines, bothin vitro and when grown as xenografts in mice. It ispresently being investigated as a novel antineoplasticagent in the primary screen conducted by the Develop-mental Therapeutics Program, National Cancer Institute.Because breast cancer is the most common cancer andsecond leading cause of cancer-related deaths in women inthe United States, we investigated whether flavopiridolcould be an effective agent against a series of isogenicbreast cancer cell lines having different levels of erbB-2expression and differential invasion and metastatic char-acteristics. Flavopiridol was found to inhibit the growthof MDA-MB-435 (parental) and 435.eB (stable transfec-tants) cells that were established by transfectingc-erbB-2cDNA into MDA-MB-435. Induction of apoptosis was alsoobserved in these cell lines when treated with flavopiridol,as measured by DNA laddering, PARP, and CPP32 cleav-ages. We also found modest up-regulation of Bax anddown-regulation of Bcl-2, but there was a significantdown-regulation of c-erbB-2 in flavopiridol-treated cells.Gelatin zymography showed that flavopiridol inhibits thesecretion of matrix metalloproteinase (MMP; MMPs 2and 9) in the breast cancer cells and that the inhibition ofc-erbB-2 and MMPs may be responsible for the inhibitionof cell invasion observed in flavopiridol-treated cells. Col-lectively, these molecular effects of flavopiridol, however,were found to be independent of c-erbB-2 overexpression,suggesting that flavopiridol may be effective in all breastcancer. From these results, we conclude that flavopiridolinhibits the growth of MDA-MB-435 breast cancer cells,induces apoptosis, regulates the expression of genes, and

inhibits invasion and, thus, may inhibit metastasis ofbreast cancer cells. These findings suggest that flavopiri-dol may be an effective chemotherapeutic or preventiveagent against breast cancer.

INTRODUCTIONFlavopiridol[5,7-dihydroxy-8-(4-N-methyl-2-hydroxypyri-

dyl)-69-chloroflavone hydrochloride] is a flavone that inhibitsseveral cyclin-dependent kinases and exhibits potent growth-inhibitory activity against a number of human tumor cell lines,both in vitro and when grown as xenografts in mice (1–5). It hasattracted considerable attention because of its unique cellulartargets and its ability to kill noncycling tumor cellsin vitro (6).Flavopiridol is presently being evaluated in a phase I clinicaltrial at the National Cancer Institute (7). However, the molecularmechanism by which flavopiridol exerts its tumor suppressiveeffect has not been fully evaluated.

Apoptosis is one of the important pathways through whichchemotherapeutic agents inhibit the growth of cancer cells.Thus, it is important to investigate whether the induction ofapoptosis and alterations of apoptosis-related gene expressionare associated with the molecular mechanism by which fla-vopiridol may exerts its biological effects on breast cancer cells.The induction of apoptosis is partly mediated intracellularly byseveral genes, such as Bcl-2 and Bax (8). Bcl-2 functions as asuppressor of apoptotic death triggered by a variety of signals(9), whereas a predominance of Bax over Bcl-2 acceleratesapoptosis upon apoptotic stimuli (10).

Breast cancer is the most common cancer in women in theUnited States, and it remains the second leading cause of cancer-related female deaths in this country (11). Several genes havebeen implicated in breast cancer aggressiveness.c-erbB-2 is akey molecule for breast cancer metastasis. Overexpression ofthec-erbB-2 gene has been found in;20–30% of human breastcancers and has been positively correlated with invasion andmetastasis in cancers (12, 13). It has been found that transfectionof c-erbB-2 into breast cancer cells increased their ability forinvasion and metastasis and that an increase of MMPs wasdetected inc-erbB-2 transfected cells (14). MMPs are believedto be key molecules for cancer invasion and metastasis (15–17).Furthermore, overexpression ofc-erbB-2 has been shown to becorrelated with an increase in MMP secretion and metastaticpotential of breast cancer cells in experimental metastasis assays(14).

In this study, we investigated whether flavopiridol couldinhibit the growth of breast cancer cell lines, MDA-MB-435 and435.eBs (established by transfectingc-erbB-2 cDNA intoMDA-MB-435), and whether c-erbB-2 overexpression may af-fect the sensitivities of these cells to flavopiridol. In addition, wewere interested in elucidating the molecular mechanism bywhich flavopiridol may induce apoptotic cell death in theseisogenic cell lines. We also investigated the effect of flavopiri-

Received 8/3/99; revised 10/1/99; accepted 10/4/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was partly funded by the George Puschelberg foundation.2 To whom requests for reprints should be addressed, at Department ofPathology, Wayne State University School of Medicine, 9374 Scott Hall,540 East Canfield Avenue, Detroit, MI 48201. Phone: (313) 966-7279; Fax:(313) 577-0057 or (313) 966-7558; E-mail: fsarkar@med.wayne.edu.

223Vol. 5, 223–229, January 2000 Clinical Cancer Research

dol on MMP3 secretion and invasion, which was previouslyshown to be affected by c-erbB-2 transfection. Our data showthat flavopiridol inhibits the growth of breast cancer cells irre-spective of c-erbB-2 overexpression, induces apoptosis, regu-lates the expression of genes, and inhibits invasion and, thus,may inhibit metastasis of breast cancer cells.

MATERIALS AND METHODSCell Lines and Culture. Human breast cancer cell line

MDA-MB-435, the 435.eB transfectant cell lines (eB1 andeB4), and the control 435.neo cell line were kindly provided byDr. Dihua Yu at the University of Texas M. D. Anderson CancerCenter. All of the cells were cultured in DMEM/F12 medium(Life Technologies, Inc., Rockville, MD) supplemented with10% FBS, 1% penicillin/streptomycin in a 5% CO2 atmosphereat 37°C. The 435.eB transfectants were generated by transfec-tion of the pCMVerbB-2 plasmid containing the 4.4-kb full-length human normalc-erbB-2 cDNA and the pSV2-neo plas-mid carrying the neomycin-resistance selection marker geneinto MDA-MB-435 cells (14, 18). 435.eB1 and 435.eB4 cellsexpress 258-fold and 165-foldc-erbB-2 compared to parentalMDA-MB-435 (14, 18). The control 435.neo cell line wasestablished by transfecting the pSV2-neo plasmid alone intoMDA-MB-435 cells (14, 18).

Cell Growth Inhibition. The MDA-MB-435 cells,435.neo cells, and 435.eB cells were seeded at a density of 53104/well in a six-well culture dish. After 24 h, the cells weretreated with 70 nM, 150 nM, and 300 nM of flavopiridol orDMSO (vehicle control). Cells treated with flavopiridol orDMSO for 1–3 days were harvested by trypsinization, stainedwith 0.4% trypan blue, and counted using a hemocytometer.

Protein Extraction and Western Blot Analysis. Thebreast cancer cells (MDA-MB-435, 435.neo, and 435.eB) wereplated and cultured in complete medium and allowed to attachfor 24 h followed by the addition of 150 nM or 300 nM flavopiri-dol and incubation for 24, 48, and 72 h. Control cells wereincubated in the medium with DMSO using the same timepoints. After incubation, the cells were harvested by scrapingthe cells from culture dishes using a scraper and collected bycentrifugation. Cells were resuspended in Tris-HCl buffer, son-icated for 23 10 s, and lysed using an equal volume of 4%SDS. Protein concentration was then measured using proteinassay reagents (Pierce, Rockford, IL). Cell extracts were boiledfor 10 min and chilled on ice, subjected to 14 or 10% SDS-PAGE, and electrophoretically transferred to a nitrocellulosemembrane. Each membrane was incubated with monoclonalBcl-2, c-erbB-2 (1:500, Oncogene, Cambridge, MA), Bax (1:5000; Biomol, Plymouth Meeting, PA), and rabbit polyclonalb-actin (1:5000, Sigma, MO) antibodies, washed with Tween 20in Tris-buffered saline, and incubated with secondary antibodyconjugated with peroxidase. The signal was then detected usingthe chemiluminescent detection system (Pierce).

Densitometric Analysis for c-erbB-2 Expression. Au-toradiograms of the Western blots forc-erbB-2 and actin protein

expression were scanned with the Gel Doc 1000 image scanner(Bio-Rad, Hercules, CA). The bidimensional optical densities ofc-erbB-2 and actin proteins on the films were quantified andanalyzed with Molecular Analyst software (Bio-Rad). The Ra-tios of c-erbB-2/actin were calculated by standardizing theratios of each control to the unit value.

Zymography of MMP Activity. Gelatin zymographywas performed according to the methods published previously(14, 19), with a slight modification. The breast cancer cells(MDA-MB-435, 435.neo, and 435.eB) were plated and culturedin complete medium and allowed to attach overnight. The cellswere washed three times with DMEM/F12 and incubated inconditioned medium for 24, 48, and 72 h. The conditionedmedia were DMEM/F12 without serum and phenol red and with150 or 300 nM flavopiridol or DMSO. The culture supernatantswere collected, and cell debris was spun off. The supernatantswere concentrated using spin columns (Amicon, Beverly, MA),and the protein concentration was measured using protein assayreagents (Pierce). The samples with equal proteins were thensubjected to 10% nonreducing SDS-PAGE (containing 0.1%gelatin). After electrophoresis, the gels were washed with 2.5%Triton X-100 for 30 min and with developing buffer (10 mM Trisbase, 40 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, and 0.02%Briji 35) for 1 h. After washing, the gels were incubated withfresh developing buffer overnight at 37°C. The gels were thenstained with Coomassie solution (0.5% Coomassie blue in 10%methanol, 5% acetic acid) for 1 h and de-stained with samesolution without Coomassie blue.

DNA Ladder Formation. Cellular cytoplasmic DNAfrom cells treated with 300 nM flavopiridol for 24, 48, 72 h orwith DMSO for 24 h (as control) was extracted using 10 mM

Tris (pH 8.0), 1 mM EDTA, and 0.2% Triton X-100. The lysatewas centrifuged for 15 min at 13,0003 g to separate thefragment DNA (soluble) from intact chromatin (nuclear pellet).The supernatant from the lysate was treated with RNase, fol-lowed by SDS-Proteinase K digestion, phenol chloroform ex-traction, and isopropanol precipitation. DNA was separatedthrough a 1.5% agarose gel. After electrophoresis, gels werestained with ethidium bromide, and the DNA was visualized byUV light.

Analysis of Cleavage of CPP32 and PARP. Cellstreated with 100 nM flavopiridol or with DMSO (as control) for24 and 48 h were lysed in lysis buffer [10 mM Tris-HCl (pH 7.1),50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM

sodium fluoride, 100mM sodium orthovanadate, 2 mM iodoace-tic acid, 5mM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, and0.5% Triton X100]. The lysates were kept on ice for 30 min andvigorously vortexed before centrifugation at 12,5003 g for 20min. Fifty micrograms of total proteins were resolved on 14% or10% SDS-PAGE and then transferred to nitrocellulose mem-brane. The membrane was incubated with primary monoclonalantihuman CPP32 antibody (1:250, Santa Cruz, CA) or PARPantibody (1:5000; Biomol), washed with TTBS, and incubatedwith secondary antibody conjugated with peroxidase. The signalwas then detected using the chemiluminescent detection system(Pierce).

Cell Invasion Assay. The invasion assay was carried outfollowing standard methods using the matrigel invasion assayprocedure as described below. Matrigel was purchased from3 The abbreviation used is: MMP, matrix metalloproteinase.

224 Effects of Flavopiridol in Breast Cancer

Becton Dickinson Labware (Bedford, MA), and Costar tran-swell culture inserts were purchased from Fisher scientific(Itasca, IL). The transwell was coated with 100ml of matrigel,which were diluted in ice cold DMEM at a final concentrationof 400mg/ml and incubated at 37°C for 3 h to allow the matrigelto polymerize. DMEM was allowed to evaporate under sterileconditions under the culture hood. The matrigel-coated insertswere rehydrated by adding 100ml of serum-free medium andincubating for 2 h at 37°C. The breast cancer cells were culturedas described earlier, trypsinized, collected by centrifugation, andresuspended in serum-free medium, and the cell numbers werecalculated by using the trypan blue exclusion method using thehemocytometer. A different number of cells in 0.5 ml wasdispensed into each culture inserts, and the cells were incubatedin a tissue culture incubator for 18 h without and with 150 and300 nM flavopiridol. After the incubation, the medium from theculture inserts was discarded, and the inner surface was wipedwith cotton wipes to remove any noninvading cells through thematrigel. The inserts were fixed for 3 min in Hema 3 fixative,then consecutively stained with Hema 3/Eosin and Hema3/methylene blue obtained from Fisher Scientific (Itasca, IL).The inserts were washed in distilled water, allowed to air dry,and then examined under a light microscope counting fourrandom fields from each insert. The percent inhibition of cellinvasion relative to controls was plotted using graph pad prismsoftware.

RESULTSEffects of Flavopiridol on Cell Growth. The effect of

flavopiridol on the cell growth of MDA-MB-435 and 435 trans-fectants is depicted in Fig. 1. The treatment of MDA-MB-435,435.neo, 435.eB1, and 435.eB4 breast cancer cells for 1–3 dayswith 70 nM, 150 nM, and 300 nM of flavopiridol resulted in inhibi-tion of cell proliferation, which was dose-dependent. However, thedose-dependent growth inhibition was not influenced by the statusof c-erbB-2 overexpression (IC50s for all three cell lines was about70 nM). The data on the inhibition of cell growth in parental and435.neo was found to be identical; hence, Fig. 1 represents parentalcells as the control. Furthermore, in all our subsequent experiments,we could not find any difference between the parental cell line and435.neo; therefore, all subsequent data have been presented with435 cells as the control, except Fig. 6, in which 435.neo has beenpresented. The overall inhibition of cell proliferation could be dueto the induction of apoptosis elicited by flavopiridol. We, therefore,investigated whether flavopiridol could induce apoptosis in thesebreast cancer cells.

Induction of Apoptosis. Apoptosis was demonstrated inall of the cell lines treated with 300 nM flavopiridol by the DNAladder shown in Fig. 2A. The induction of apoptosis was pro-nounced at 24 h of treatment, and it is directly correlated withthe inhibition of cell growth. Western blot analysis revealed thatinactive CPP32 (32 kDa) was cleaved to yield an active frag-ment (;17 kDa) after flavopiridol treatment for 24 h (Fig. 2B).Furthermore, PARP cleavage analysis showed that the full-sizePARP (116 kDa) protein was also cleaved to yield an 85-kDafragment after treatment with flavopiridol for 24 h (Fig. 2B) inall cell lines tested, corresponding with the activation of CPP32.These three independent methods of measuring apoptosis pro-

vided strong evidence that apoptosis was induced in all cell linestreated with flavopiridol and that the apoptosis inducing activityof flavopiridol was found to be independent of c-erbB-2 over-expression. To explore the mechanisms by which flavopiridol

Fig. 1 Cell growth inhibition by flavopiridol.

225Clinical Cancer Research

induces apoptosis, we investigated the alterations in the expres-sion of genes, which are known to be involved in the apoptoticpathway.

Expression of Bcl-2 and Bax. The effect of flavopiridolon Bcl-2 and Bax expression in MDA-MB-435, 435.neo, and435.eB cells were studied by Western blot analysis. The levelsof Bcl-2 expression in all 435 cell lines were found to bemodestly down-regulated with the addition of flavopiridol whenexposed for 24–72 h (Fig. 3A). Furthermore, the expression ofBax was also found to be modestly up-regulated after 24 h of150 nM or 300 nM flavopiridol treatment (Fig. 3B). We could not

find any substantial difference in Bcl-2 and Bax expressionamong these 435 cell lines, suggesting that the modulation inBax and Bcl-2 by flavopiridol is not dependent onc-erbB-2expression.

Expression of c-erbB-2. To explore the effect of fla-vopiridol on c-erbB-2 that has been correlated with metastaticpotential, the expression ofc-erbB-2 in MDA-MB-435 and 435transfectant cells with and without flavopiridol treatment wastested by Western blot analysis. As Tanet al. (14) reported,there were higher expressions ofc-erbB-2 in 435.eB1 cells and435.eB4 cells than in parental MDA-MB-435 and 435.neo cells.The expression ofc-erbB-2 protein in MDA-MB-435 and435.eB cells treated with 150 nM or 300 nM flavopiridol wassignificantly down-regulated at various degrees (Fig. 4A); how-ever, the down-regulation was much more pronounced in pa-rental cells compared to those overexpressing c-erbB-2. It isimportant to note that to detect erbB-2 expression, the blots withparental cells were exposed for a longer period of time as

Fig. 2 A, DNA ladder formation in MDA-MB-435 cells and 435.eBtransfectants treated with flavopiridol.Control: cells treated withDMSO for 24 h; Day 1, Day 2, Day 3:cells treated with 300 nMflavopiridol for 24, 48, and 72 h, respectively.B, Western blot analysisof CPP32 and PARP cleavage in MDA-MB-435 cells and 435.eBtransfectants treated with flavopiridol.Control: cells treated withDMSO; Day 1, Day 2:cells treated with 100 nM flavopiridol for 24 and48 h, respectively.

Fig. 3 A, Western blot analysis of Bcl-2 in MDA-MB-435 cells and435.eB transfectants treated with flavopiridol.C: cells treated withDMSO; 150, 300 nM: Cells treated with 150, 300 nM flavopiridol,respectively.B, Western blot analysis of Bax in MDA-MB-435 cells and435.eB transfectants treated with flavopiridol.C: cells treated withDMSO; 150, 300 nM: cells treated with 150 and 300 nM flavopiridol,respectively.

226 Effects of Flavopiridol in Breast Cancer

compared to those with erbB-2 overexpression. To obtain aquantitative value for the protein expression ofc-erbB-2, ab-sorbance measurement was conducted as described under “Ma-terials and Methods.” The ratios ofc-erbB-2 to actin proteinexpression revealed that cells treated with flavopiridol showed a55–90% decrease inc-erbB-2 compared to the untreated control(Fig. 5).

Enzyme Activities of MMPs. The activities of the base-ment membrane degrading MMPs in MDA-MB-435 and 435transfectants with or without flavopiridol treatment were measuredby zymographic analysis. Significantly higher levels of MMP-2 (72kDa) and MMP-9 (92 kDa) were detected in the conditionedmedium of the 435.eB1 and 435.eB4 transfectants than in parentalMDA-MB-435 and 435.neo as reported previously (14), whereasdecreasing levels of MMPs were observed in the conditionedmedium of all 435 cells treated with flavopiridol (Fig. 4B). Thesedata suggested that increasedc-erbB-2 expression in 435.eB cellscan lead to increased secretion of MMPs as reported earlier (14)and that flavopiridol can inhibit the expression ofc-erbB-2 and,therefore, decrease the secretion of MMPs, which may be impor-tant for the decrease in invasion and metastatic ability of breastcancer cells exposed to flavopiridol. However, the decline inMMPs 2 and 9 could be a direct effect on MMPs and not neces-sarily dependent on the down-regulation of c-erbB-2, which re-

quires further investigation. Whether flavopiridol could have anyeffect on their ability to invade through matrigel was subsequentlyinvestigated as described below.

Inhibition of Cell Invasion by Flavopiridol. It has beenreported earlier that c-erbB-2 transfected MDA-MB-435(435.eB1 and 435.eB4) cells secret MMPs, and these cellsacquire invasion and metastatic potential compared to parentalcells (435) or cells transfected with control plasmid (435.neo;Ref. 14). We have performed cell invasion assays using thesebreast cancer cells treated with flavopiridol, which showedinhibition of cell invasion through matrigel within 18 h (Fig. 6),suggesting that the down-regulation of c-erbB-2 followed by thedown-regulation of MMPs may play a direct role in the invasioncharacteristic of these cells when exposed to flavopiridol. Thepercent inhibition in cell invasion was found to be higher inparental cells, which secret less MMPs compared to 435.eB1,which secrets higher amounts of MMPs and, thus, the residualamount of MMPs observed in flavopiridol treated 435.eB1remains relatively higher compared to parental cells, whichcould explain the differential effect on cellular invasion amongthese cell lines. Collectively, these results provide strong evi-dence for the antitumor as well as anti-invasive properties ofbreast cancer cells when exposed to flavopiridol irrespective ofc-erbB-2 overexpression, suggesting broader application of fla-vopiridol in breast cancer.

Fig. 4 A,Western blot analysis ofc-erbB-2 in MDA-MB-435 cells and435.eB transfectants treated with flavopiridol.C: cells treated withDMSO; 150, 300 nM: cells treated with 150 and 300 nM flavopiridol,respectively.B, zymographic analysis of MMPs in MDA-MB-435 cellsand 435.eB transfectants treated with flavopiridol.C: cells treated withDMSO; 150, 300 nM: Cells treated with 150 and 300 nM flavopiridol,respectively.

Fig. 5 Densitometric analysis ofc-erbB-2 in MDA-MB-435 cells and435.eB transfectants treated with flavopiridol.C: cells treated withDMSO; 150, 300 nM: cells treated with 150 and 300 nM flavopiridol,respectively.

227Clinical Cancer Research

DISCUSSIONFlavopiridol is known to potently inhibit the activity of mul-

tiple cyclin-dependent kinases. It has been reported to inhibit cellgrowth of several cancer cell lines (1, 5, 20, 21). A previous reportshowed that whereas flavopiridol caused cell cycle arrest in G1 andG2 in PC3 cells, apoptosis of SUDHL4 cells occurred withoutevidence of cell cycle arrest, suggesting that antiproliferative ac-tivity of flavopiridol may be different in different cell types fromcell cycle arrest to apoptosis (22). In our study, we found thatflavopiridol can inhibit the growth of MDA-MB-435, 435.neo, and435.eB transfectants. To explore if the inhibition of cell growthobserved in flavopiridol-treated cells may be due to apoptotic celldeath, we used several methods to detect apoptosis in our system.The nucleosomal DNA ladder has been widely used as biochemicalmarkers of apoptosis for several years (23). In addition, the cleav-age of Caspase-3 (CPP32) and poly (ADP-ribose) polymerase(PARP) has also been used as early markers of apoptosis (24, 25).Using these various techniques, we found induced apoptosis in theMDA-MB-435 and 435.eB transfectants treated with flavopiridol.DNA ladder formation, CPP32 activation, and PARP cleavagewere observed in the breast cancer cells after treatment with fla-vopiridol for 24 h. Our results clearly suggest that flavopiridol caninhibit the growth of breast cancer cells and induce apoptosis.These results are also consistent with other reports documentingthat apoptosis was observed in flavopiridol-treated head and neckcancer cells, lymphoid cells, umbilical vein endothelial cells, leu-kemia cells, and lung cancer cells (5, 21, 26–28).

It has been demonstrated that Bcl-2 and Bax play a major rolein determining whether cells will undergo apoptosis under experi-mental conditions that promote cell death. Bcl-2 protects cells fromapoptosis, whereas increased expression of Bax can induce apo-ptosis (9, 10). It has also been found that the ratio of Bax:Bcl-2,rather than Bcl-2 alone, is important for the survival of drug-induced apoptosis in leukemia cell lines (10). In our study, adecrease in Bcl-2 expression and up-regulation of Bax expression

were observed in breast cancer cells after treatment with flavopiri-dol for 24 h, corresponding with the induction of apoptosis afterflavopiridol treatment. These changes, however, were not relatedwith the status ofc-erbB-2 expression. Our results suggest thatup-regulation of Bax and down-regulation of Bcl-2 may be one ofthe molecular mechanisms through which flavopiridol induces ap-optosis. However, further in-depth studies are warranted given theimportance and potent biological activity of flavopiridol. It isimportant to note that Koniget al. (21) have found a significantdown-regulation of Bcl-2 only after long exposure and a higherconcentration of flavopirodol, but two other studies could not findany changes in the levels of Bcl-2 (20, 23). Our studies show somemodest down-regulation of Bcl-2, which could either be due to thedifferences in the cell lines used or this modest effect could be dueto the concentration of flavopiridol and the duration of treatment.Further studies with higher concentrations and longer exposuretime may provide results similar to the findings obtained by Koniget al.(21).The modulation of cdk as observed previously (1, 3, 26)and its association with the induction of apoptosis is still unknown,but the apparent modest down-regulation of Bcl-2 and up-regula-tion of Bax may be important for the induction of apoptosis elicitedby flavopiridol.

Overexpression ofc-erbB-2 has been found in breast cancers,and it has been correlated with lymph node metastasis in cancerpatients (29, 30). Tanet al. (14) introduced the humanc-erbB-2gene into the very lowc-erbB-2-expressing MDA-MB-435 humanbreast cancer cells and established 435.eB transfectants that expresshigh levels ofc-erbB-2. They compared the metastatic phenotypesof parental MDA-MB-435 cells and the 435.eB transfectants andfound that overexpression ofc-erbB-2 can enhance the MMPsecretion and metastatic potential of MDA-MB-435 human breastcancer cells. In our study, we found that flavopiridol significantlyinhibited c-erbB-2 expression (55% to 90%) in the 435.eB trans-fectants and parental MDA-MB-435 cells, including 435.neo. Be-cause MMPs were previously found to be up-regulated byc-erbB-2transfection and overexpression, we also compared the secretion ofMMPs in 435.eB transfectants and MDA-MB-435 with and with-out flavopiridol treatment. Higher levels of MMPs in 435.eB trans-fectants than in parental MDA-MB-435 were observed, corre-sponding with a higher metastatic potential of 435.eB transfectantsas previously reported (14). After treatment with flavopiridol, thelevels of MMPs decreased in 435.eB transfectants and MDA-MB-435 parental cells. The decrease in MMPs may be responsible forthe inhibition of cell invasion observed in this system (Fig. 6). It isimportant to note the we were able to observe inhibition of cellinvasion in all cell lines within 18 h in our assay system, suggestingthat the inhibition of cell invasion may be directly responsible forthe biological effect of flavopiridol. The inhibition of cell invasionappears not to be due to cell growth inhibition or apoptosis assuggested by the duration of the experiment and also based onparallel experiments where the initial plating cell density wascompensated for these variables. Our results suggest that flavopiri-dol can inhibit the expression ofc-erbB-2 and, in turn, decrease thesecretion of MMPs in breast cancer cells, which, in turn, inhibitscell invasion and further suggests that flavopiridol may also inhibitmetastatic properties of breast cancer cells. However, the down-regulation of MMPs could be independent of c-erbB-2 in flavopiri-dol-treated breast cancer cells; this requires further in depth inves-tigation.

Fig. 6 Matrigel cell invasion assay showing inhibition of cell invasionthrough matrigel induced by flavopiridol, where the data are presentedas percent inhibition of cell invasion relative to control untreated cells.

228 Effects of Flavopiridol in Breast Cancer

In conclusion, our results demonstrated that flavopiridolinhibited the growth of breast cancer cells, regulated the expres-sion of apoptosis-related genes, and induced apoptosis in MDA-MB-435 breast cancer cells. Furthermore, flavopiridol inhibitedthe expression ofc-erbB-2, the secretion of MMPs, and cellinvasion, suggesting that flavopiridol may also inhibit metasta-sis of breast cancer. Flavopiridol, thus, may be an effectivechemotherapeutic or preventive agent against breast cancer.However, much remains to be studied about the molecularmechanisms of flavopiridol as an anticancer agentin vivo.

ACKNOWLEDGMENTSWe sincerely thank Dr. Dihua Yu for providing 435.eB, 435.neo

transfectants, and MDA-MB-435 parental cells. We sincerely thankPatricia Arlauskas for her editorial assistance.

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