A Polyphenolic Fraction from Grape Seeds Causes IrreversibleGrowth Inhibition of Breast Carcinoma MDA-MB468 Cellsby Inhibiting Mitogen-activated Protein Kinases Activationand Inducing G1 Arrest and Differentiation 1

Chapla Agarwal, Yogesh Sharma, Jifu Zhao, andRajesh Agarwal2

Center for Cancer Causation and Prevention, AMC Cancer ResearchCenter, Denver, Colorado 80214 [C. A., Y. S., J. Z., R. A.], andUniversity of Colorado Cancer Center, University of Colorado HealthSciences Center, Denver, Colorado 80262 [R. A.]

ABSTRACTIn recent years, significant emphasis is being placed on

identifying naturally occurring cancer preventive and inter-ventive agents. In this regard, a polyphenolic fraction iso-lated from grape seeds (hereafter referred as GSP) hasrecently been shown by us and others to prevent tumorigen-esis in mouse skin models. Chemical analysis of GSP hasshown that it is largely constituted with procyanidins thatare strong antioxidants. Breast cancer is the most commoninvasive malignancy and the second leading cause of cancer-related deaths in United States women. Accordingly, here weinvestigated the effect of GSP on mitogenic signaling andregulators of cell cycle and apoptosis as molecular targetsfor the growth arrest, apoptotic death, and/or differentiationof estrogen-independent MDA-MB468 human breast carci-noma cells. Treatment of cells with GSP (at 25-, 50-, and75-mg/ml doses for 1–3 days) resulted in a highly significantinhibition (90% to complete, P < 0.001) of constitutiveactivation of mitogen-activated protein kinase (MAPK)/ex-tracellular signal-regulated protein kinase1/2 in a dose-dependent manner after 72 h of treatment. Whereas GSPtreatment of cells did not show a conclusive effect on MAPK/JNK1 activation, a moderate to highly significant inhibition(15–70%, P < 0.1–0.001) of constitutive activation ofMAPK/p38 was also observed in a dose-dependent manneras early as 24 h of GSP treatment. GSP-treated cells alsoshowed a strong induction (1.7–2.7 fold,P < 0.001) of cyclin-dependent kinase inhibitor Cip1/p21 and a decrease (10–

50%, P < 0.1–0.001) in cyclin-dependent kinase 4. Consist-ent with these findings, GSP-treated cells resulted in theiraccumulation in G1 phase of the cell cycle in a dose-depen-dent manner. An irreversible growth inhibition (44–88%,P < 0.001) was also observed in 50 and 75mg/ml GSP-treated cells in a time-dependent manner. Additional studiesassessing the biological fate of GSP-treated cells showed thatthey do not undergo apoptotic death, as evidenced by a lackof DNA fragmentation, poly (ADP ribose) polymerase cleav-age, and apoptotic morphology of the cells. A morphologicalchange suggestive of differentiation was observed in GSP-treated cells that was further confirmed by a significantinduction (1.7–2.6 fold, P < 0.001), in both a dose- andtime-dependent manner, in cytokeratin 8 protein level, amarker of differentiation. An irreversible growth-inhibitoryeffect of GSP possibly via terminal differentiation of humanbreast carcinoma cells suggests that GSP and the procyani-dins present therein should be studied more extensively to bedeveloped as preventive and/or interventive agents againstbreast cancer in humans.

INTRODUCTIONBreast cancer is the most commonly diagnosed invasive

malignancy and a leading cause (after lung cancer) of cancerdeaths in American women (1). One approach to control thismalignancy is its prevention. In most of the cancer preventionclinical trials including breast cancer, the term “prevention orintervention” is also frequently used to chemopreventive sup-pression or reversal of premalignant lesions although the lesionis not permanently eliminated (2, 3). Several studies in animaltumor models including rat mammary tumors, followed byepidemiological studies orvice versa, have convincingly estab-lished that consumption of yellow-green vegetables and fruits isassociated with a decreased risk of several malignancies, includ-ing breast cancer (4–12). Accordingly, there has been an in-creased effort to define the mechanisms by which fruits andvegetables afford protection against cancer and to identify thenutritive and nonnutritive components of the fruits and vegeta-bles that exert such effects. One group of cancer preventivephytochemicals from fruits and vegetables that is receivingincreasing attention in recent years is polyphenolic antioxidants(13–18).

Grapes (Vitis vinifera) are one of the most widely con-sumed fruits in the world. Grapes are rich in polyphenols, andabout 60–70% of grape polyphenols exist in grape seeds asdimers, trimers and other oligomers of flavan-3-ols commonlyknown as procyanidins (or proanthocyanidins; Refs. 19–21).Commercial preparations of grape seed polyphenols are mar-

Received 11/23/99; revised 3/10/00; accepted 3/17/00.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 Supported in part by USPHS Grants CA64514 and CA83741, U. S.Army Medical Research and Materiel Command Grant DAMD17-98-1-8588, and AMC Cancer Research Center Institutional Funds.2 To whom requests for reprints should be addressed, at Center forCancer Causation and Prevention, AMC Cancer Research Center, 1600Pierce Street, Denver, CO 80214. Phone: (303) 239-3580; Fax: (303)239-3534; E-mail: agarwalr@amc.org.

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keted in the United States as GSE3, with 95% standardizedprocyanidins as dietary supplement due to its health benefits,particularly a very strong antioxidant activity of procyanidins. Inaddition to grape seed, procyanidins are a diverse group ofpolyphenolics that are widely distributed in fruits and vegetables(22).

Several studies in recent years have shown the healthbenefits of procyanidins as well as wine consumption, which isalso a source of procyanidins (23–26). In terms of its anticarci-nogenic potential, oral feeding of 1% GSE in diet is shown toinhibit adenomatous polyposis coli mutation-associated intesti-nal adenoma formation in MIN mice (27). Furthermore, recentstudies by us and others (28, 29) have shown that topicalapplication of a polyphenolic fraction isolated from grape seeds(hereafter referred as GSP) or commercial GSE results in ahighly significant protection against phorbol ester-induced tu-mor promotion in chemical carcinogen-initiated mouse skin.With regard to epidemiology, a case-control study showed thatincreased consumption of grapes is associated with reducedcancer risk (30). Together, these studies suggested that GSP andthe procyanidins present therein could also be effective in breastcancer prevention and/or intervention.

In an effort to develop GSP and procyanidins as interven-tive agents against breast cancer, in this study we focused ourattention on the effect of GSP on MAPK activation and cellcycle and apoptosis regulators. The selection of these moleculartargets was based on the fact that they are the major, possiblycausative, mitogenic and antiapoptotic signaling contributors tothe multifactorial mechanisms of uncontrolled breast cancergrowth. For example, enhanced expression of EGF receptorfamily members [erbB1 (or EGFR) and Her-2/neu/erbB2] andassociated ligands (e.g.,transforming growth factora/EGF) hasbeen shown with high frequency in both estrogen-dependent and-independent breast carcinomas and derived cells (31–36). Thishigh expression of growth factors and receptors leads to anautocrine loop for both mitogenic and antiapoptotic signaling,leading to autonomous growth and metastasis of breast cancer(31–36). The activation of these and other signaling cascadesultimately activate MAPKs that, following their translocation tonucleus, activate transcription factors and command cell cycleregulatory molecules for cell growth, proliferation, and differ-entiation (37–47). Several studies have shown that MAPKs areconstitutively active in human breast carcinomas and derivedcell lines as well as in rat mammary tumors (48–52). Together,it can be appreciated that targeting the MAPK signaling path-way should be a useful strategy for the prevention and/or inter-vention of breast cancer. Using estrogen receptor-negativebreast carcinoma MDA-MB468 cells, in this study we demon-strate the irreversible inhibitory effect of GSP on cell growth

and suggest that this effect is possibly via inhibition of MAPKactivation and modulation of cell cycle regulators involved inG1 phase. The observed molecular effects of GSP on MDA-MB468 cells result in a G1 arrest in cell cycle, followed byterminal differentiation, not the apoptotic cell death.

MATERIALS AND METHODSCell Line and Other Reagents. The MDA-MB468 cell

line was obtained from the American Type Culture Collection(Manassas, VA). Cells were grown in DMEM with 10% fetalbovine serum, 100 units/ml penicillin, and 100mg/ml strepto-mycin at 37°C in a 5% CO2 atmosphere. Anti-Cip1/p21 anti-body was from Calbiochem (Cambridge, MA). Anti-cytokeratin8 and anti-CDK4 antibodies were from Neomarkers, Inc. (Fre-mont, CA). Antibodies to CDK2, cyclin D1, and cyclin E andrabbit antimouse immunoglobulin- and goat antirabbit immuno-globulin-horseradish peroxidase-conjugated secondary antibod-ies were purchased from Santa Cruz Biotechnology, Inc. (SantaCruz, CA). Phospho- (and regular) MAPK/ERK1/2, JNK1, andp38 antibodies were from New England Biolabs (Beverly, MA).PARP antibody was from PharMingen (San Diego, CA). TheECL detection system was from Amersham (Arlington Heights,IL). The GSP preparation used was that described in detailrecently (29). Approximately 60% (w/w) of this total GSPpreparation has been identified and defined by us to containpolyphenols, namely procyanidin B3 (3%), procyanidin B1(0.7%), catechin (5%), procyanidin B4 (2%), procyanidin B2(3.5%), epicatechin (6%), procyanidin C1 (6%), procyanidinB5–39-gallate (15%), and procyanidin B5 (19%), each w/w oftotal GSP (29). For all of the studies, GSP was dissolved inDMSO as a 10-mg/ml stock solution and diluted as desireddirectly in the medium. Unless specified otherwise, the finalconcentration of DMSO in culture medium during GSP treat-ment did not exceed 0.75% (v/v), and, therefore, the sameconcentration of DMSO was present in control dishes.

GSP Treatment of Cells and Western Immunoblotting.MDA-MB468 cells were grown in 100-mm dishes, as detailedabove, and at 70% confluency were treated with either DMSOalone or varying concentrations of GSP. After 24, 48, and 72 hof treatments, medium was aspirated, cells were washed twotimes with cold PBS, and cell lysates were prepared as describedin detail recently (53). For Western immunoblotting, 40–100mgof protein lysate per sample was denatured with 23 samplebuffer, samples were subjected to SDS-PAGE on 12% gels, andseparated proteins were transferred onto membrane. The levelsof phospho- and regular ERK1/2, JNK1, p38, Cip1/p21, CDK4,CDK2, cyclin D1, cyclin E, PARP, and cytokeratin 8 weredetermined using specific primary antibodies, followed by per-oxidase-conjugated appropriate secondary antibody and visual-ization by the ECL detection system, as described in detailrecently (53).

Cell Cycle Analysis. MDA-MB468 cells at 70% conflu-ency were treated with either DMSO alone or varying concen-trations of GSP. After 24, 48, and 72 h of treatments, mediumwas aspirated, cells were quickly washed two times with coldPBS and trypsinized, and cell pellets were collected. Approxi-mately 0.53 106 cells in 0.5 ml of saponin/PI solution [0.3%saponin (w/v), 25mg/ml PI (w/v), 0.1 mM EDTA, and 10mg/ml

3 The abbreviations used are: GSE, grape seed extract; CDK, cyclin-dependent kinase; CDKI, CDK inhibitor; ERK1/2, extracellular signal-regulated protein kinase 1/2; GSP, a polyphenolic fraction isolated fromgrape seeds; IGF, insulin-like growth factor; IGF-1R, IGF-1 receptor;MAPK, mitogen-activated protein kinase; PARP, poly(ADP ribose)polymerase; ECL, enhanced chemiluminescence; EGF, epidermalgrowth factor; JNK, c-jun-NH2-kinase; FACS, fluorescence-activatedcell-sorting; PI, propidium iodide.

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RNase (w/v) in PBS] were left at 4°C for 24 h in the dark. Cellcycle distribution was then analyzed by flow cytometry usingthe FACS Analysis Core Services of the University of ColoradoCancer Center (Denver, CO).

Cell Growth Assay. MDA-MB468 cells were plated at5000 cells/cm2 density in 35-mm dishes under the culture con-ditions detailed above. After 24 h, cells were fed with freshmedium and treated with either DMSO alone or varying con-centrations of GSP. The cultures were fed with fresh mediumwith or without the same concentrations of GSP every alternateday up to the end of the experiment; each treatment and timepoint had four plates. After 2, 4, and 6 days of treatments, cellswere trypsinized, collected, and counted using a hemocytome-ter. Trypan blue dye exclusion was used to determine cellviability.

In another study, MDA-MB468 cells were plated at 5000cells/cm2 density in 35-mm dishes and, after 24 h, treated withDMSO alone or varying doses of GSP. After 72 h of treatments,total cell growth was determined by cell counting. At this point,in separate dishes, after 72 h of treatments with GSP, cultureswere washed several times with medium to remove GSP andgrown in fresh medium without GSP for another 24, 48, or 72 h.The cell number was determined at these time periods.

DNA Ladder Assay. MDA-MB468 cells were grown in100-mm dishes, as detailed above, and at 70% confluency weretreated with either DMSO alone or varying concentrations ofGSP. After 24, 48, and 72 h of treatments, trypsinized cells(together with any floating cells) were collected and the DNAladder analysis was done as described in detail recently (54).

Morphological Analysis. MDA-MB468 cells weregrown in 100-mm dishes, as detailed above, and at 50% con-fluency were treated with either DMSO alone or 50- and 75-mg/ml concentrations of GSP. After 24, 48, 72, and 96 h oftreatments, pictures were taken using phase-contrast microscopeat 3200 magnification. The cell pellets were then collected forcytokeratin 8 protein expression by Western immunoblotting, asdetailed above.

Densitometric and Statistical Analysis. Autoradio-grams of the Western immunoblots were scanned using AdobePhotoshop. The blots were adjusted for brightness and contrastfor minimum background, and the mean density for each bandwas analyzed using Scanimage Program. As needed, a two-tailed Student’st test was used to assess statistical significanceof difference between vehicle- and GSP-treated samples. Untiland unless specified otherwise, the results shown in each caseare representative of three independent experiments with similarfindings.

RESULTSGSP Inhibits Constitutive Activation of ERK1/2 and

p38, But Causes Moderate Effect on JNK1 in MDA-MB468Cells. First, we focused our attention on the effect of GSP onconstitutive activation of MAPKs. Because the MAPK familyincludes ERKs, JNK (stress-activated protein kinase), and p38(HOG), which are functional units involved in three distinctsignaling pathways (37–47), we assessed the effect of GSP onall of these MAPKs. As shown in Fig. 1A, treatment of MDA-MB468 cells with GSP resulted in a moderate to complete

Fig. 1 Effect of GSP on the constitutive activation of MAPK sig-naling in human breast carcinoma MDA-MB468 cells. Cells werecultured in DMEM with 10% serum and at 70% confluency weretreated either with DMSO alone or 25-, 50-, and 75mg/ml concen-trations of GSP for 24, 48, and 72 h. Cell lysates were prepared andsubjected to SDS-PAGE, followed by Western blotting, as detailed in“Materials and Methods.” The membranes were probed with an-tiphospho-ERK1/2, antiphospho-JNK1, and antiphospho-p38 MAPKantibodies and then peroxidase-conjugated appropriate secondaryantibody. Visualization of proteins was done using ECL detectionsystem.A, phosphorylation of ERK1/2.B, phosphorylation of JNK1.C, phosphorylation of p38. Treatments are as labeled in the figure.IB, Western immunoblot. In eachpanel, the densitometric analysisbars represent the mean6 SE values (arbitrary units) of threeindependent experiments. At each time point of the study, thevehicle-treated control value is presented as 100%.

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inhibition of constitutive activation of ERK1/2 in both a dose-and time-dependent manner. The densitometric analysis of threedifferent blots showed that compared with DMSO control, after24 h of GSP treatment, 25- and 50-mg/ml doses were noteffective, but a 75-mg/ml dose showed moderate (althoughstatistically not significant) inhibition (10%,P , 0.1) of con-stitutive ERK1/2 activation. However, a higher inhibition (20–25%,P , 0.05) was observed at both 50- and 75-mg/ml dosesafter 48 h of GSP treatment, and after 72 h these doses showedcomplete inhibition (P, 0.001) of constitutive ERK1/2 activa-tion; even a 25-mg/ml dose resulted in 90% inhibition (P ,0.001) after this treatment time (Fig. 1A). The observed de-creases in constitutive ERK1/2 activation by GSP were not dueto a change in its protein levels following these treatments (datanot shown), suggesting that GSP impairs mitogenic signaling ofERK1/2. These results are specifically important because of adose and time response where it could be suggested that a longertreatment time with a lower dose(s) produce comparable effi-cacy to that with higher dose and shorter treatment time.

Unlike its effect on ERK1/2 activation, GSP did not showa conclusive effect on JNK1 activation (Fig. 1B). For example,24 h of treatment with 25-, 50-, and 75-mg/ml doses did notresult in any change in constitutive activation of JNK1, whereasafter 48 h of treatment a 75-mg/ml dose showed a strongstimulation (Fig. 2B). Conversely, 50- and 75-mg/ml doses ofGSP showed;35% inhibition (P, 0.05) after 72 h of treatment(Fig. 2B). Similar to ERK1/2, GSP did not show any change inJNK1 protein expression following these treatments (data notshown).

With regard to p38, as shown in Fig. 1C, compared withDMSO control, GSP treatment of MDA-MB468 cells resulted ina highly significant inhibition of p38 activation in both a dose-and time-dependent manner. The observed effect of GSP on p38was much stronger than ERK1/2 activation at both 24 and 48 hat all three doses examined, but was less effective than ERK1/2after 72 h of treatment (Fig. 1,C versus A). Quantitative analysisof the blots showed that 24 h of GSP treatment at a 25-mg/mldose was not effective, however, 50- and 75-mg/ml doses re-sulted in 15% (P, 0.1) and 70% (P, 0.001) decrease,respectively, in constitutive p38 activation. After 48 h of GSPtreatment of cells, as much as a 15% (P, 0.1), 68% (P,0.001), and 70% (P, 0.001) decrease was evident in p38activation by these doses, respectively. No further effect, how-ever, was observed following additional treatment time up to72 h. An interesting observation was a strong increase in con-stitutive activation of p38 in control samples after 48 h, ascompared with those at 24 and 72 h (Fig. 1C). The reason forthis increase remains to be established. Taken together, thesefindings suggest that GSP treatment of MDA-MB468 humanbreast carcinoma cells results in a significant decrease inERK1/2 and p38 activation, the two MAPK pathways associatedwith cell growth and differentiation (55), but has moderate, ifany, effect on JNK1, the MAPK pathway associated largelywith cell death (55–57).

GSP Induces Cip1/p21 Levels and Decreases G1 PhaseRegulators CDK4 and Cyclin D1 in MDA-MB468 Cells. Acontrolled cell growth, proliferation, differentiation, and/or ap-optosis are mediated via cell cycle progression that is governedby cellular signaling (Refs. 58–62 and references therein).

Fig. 2 GSP induces Cip1/p21 levels and decreases G1 phase regulatorsCDK4 and cyclin D1 in human breast carcinoma MDA-MB468 cells.Cells were cultured in DMEM with 10% serum and at 70% confluencywere treated with either DMSO alone or 25-, 50-, and 75-mg/ml con-centrations of GSP for 24, 48 and 72 h. Cell lysates were prepared andsubjected to SDS-PAGE, followed by Western blotting, as detailed in“Materials and Methods.” The membranes were probed with anti-Cip1/p21, anti-CDK4, and anti-cyclin D1 antibodies and then peroxidase-conjugated appropriate secondary antibody. Visualization of proteinswas done using an ECL detection system.A, protein expression ofCip1/p21. B, protein expression of CDK4.C, protein expression ofcyclin D1. Treatments are as labeled in the figure.IB, Western immu-noblot. In eachpanel, the densitometric analysisbars represent themean6 SE values (arbitrary units) of three independent experiments. Ateach time point of the study, the vehicle-treated control value is pre-sented as 100%.

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However, cancer cells often display defects in the genes thatgovern the cellular responses to a growth factor(s)-growth factorreceptor(s) interaction (61). Perturbations in cell cycle regula-tion have been demonstrated to be one of the most commonfeatures of transformed cells (61), which could be associatedwith gain of function for uncontrolled growth due to enhancedexpression of growth factor-receptor autocrine loop (31–36), alack of CDKI or loss in its function (58–63), and so on.Accordingly, next we assessed the effect of GSP on cell cycleregulators involved in G1 phase. As shown by data in Fig. 2A,treatment of cells with GSP resulted in a significant induction ofCip1/p21 in a dose-dependent manner. The densitometric anal-ysis of blots showed that 24 h of GSP treatment caused a 1.7-,2.3-, and 2.7-fold increase (P, 0.001) in Cip1/p21 at doses of25, 50, and 75mg/ml, respectively. Although 48 and 72 h ofGSP treatment also showed significant Cip1/p21 induction, itwas less profound than that at 24 h and amounted for 1.2–1.6-fold increases (P, 0.1 to 0.001). Similar to its affect onCip1/p21, GSP treatment of cells also resulted in a significantdecrease in CDK4 protein levels (Fig. 2B). In this case, 24 h ofGSP treatment caused a 10% (P, 0.1), 30% (P, 0.05), and50% (P, 0.001) decrease in CDK4 expression at 25-, 50-, and75-mg/ml doses, respectively. Similar effects of GSP were alsoobserved following its treatment for 48 and 72 h at identicaldoses. Unlike its effect on Cip1/p21 and CDK4, GSP treatmentof cells for 24 h resulted in a marginal increase in cyclin D1protein levels, however, 48 and 72 of treatment caused marginaldecrease (Fig. 2C); GSP treatment did not show any effect onCDK2 and cyclin E protein expression (data not shown).

GSP Induces G1 Arrest in Cell Cycle Progression ofHuman Breast Carcinoma MDA-MB468 Cells. On the ba-sis of the results showing a strong induction in Cip1/p21 and adecrease in CDK4 by GSP, we next examined its effect on cellcycle progression. As shown in Fig. 3, FACS analysis ofDMSO-treated cells showed a cell cycle distribution that fol-lowed a proliferation pattern. However, a moderate to strongdifference in cell cycle progression was observed followingGSP treatment where all of the doses and time points examinedshowed a G1 arrest in the cell cycle progression of MDA-MB468 cells largely at the expense of S phase population (Fig.3). Within 24 h after GSP treatment, compared with DMSO-treated control, 25- and 50-mg/ml doses showed marginal accu-mulation of cells in G1 phase (55% and 58% of cells in G1,respectively, compared with 53% in control; Fig. 3A). However,as much as 71% of cells accumulated in G1 phase following a75-mg/ml dose of GSP treatment for 24 h (Fig. 3A). The increasein G1 population by GSP at all of the doses was accompanied bya significant decrease of the cells in S phase (Fig. 3B). Anaccountable increase in G2-M population of the cells was alsoobserved following a 24-h treatment with GSP at these doses(Fig. 3C). When MDA-MB468 cells were treated for a longertime (i.e.,48 and 72 h) with GSP, even a lower dose (25mg/ml)resulted in a strong accumulation of the cells in G1 phase that ismoderately affected by increasing the doses to 50 and 75mg/ml(Fig. 3A). Once again, this increase was mainly due to a de-crease in S phase population (Fig. 3B) with moderate alterationsin G2-M phase (Fig. 3C). Together, these results were consistentwith other findings, showing that (a) maximum effect of GSP on

Cip1/p21 was at 24 h (Fig. 2A) and (b) GSP modulates cell cycleregulators associated with G1 phase (Fig. 2).

GSP Irreversibly Inhibits the Growth of MDA-MB468Cells. The studies assessing the biological significance of theobserved effects of GSP showed that it causes the inhibition ofhuman breast carcinoma MDA-MB468 cells and that this inhi-bition was irreversible at higher doses examined. As shown bydata in Fig. 4A, GSP treatment of growing cells resulted in asignificant to almost complete inhibition of cell growth in botha dose- and time-dependent manner. Compared with DMSO-treated controls, whereas a 10-mg/ml dose was not effective, a25-mg/ml dose resulted in 20–50% (P, 0.001) inhibition after2–6 days of treatment (Fig. 4A). A much higher inhibitory effectwas observed at 50- and 75-mg/ml doses of GSP, accounting for

Fig. 3 GSP induces G1 arrest in cell cycle progression of human breastcarcinoma MDA-MB468 cells. Cells were cultured in DMEM with 10%serum and at 70% confluency were treated either with DMSO alone or25-, 50-, and 75-mg/ml concentrations of GSP for 24, 48 and 72 h. Afterthese treatments, cells were trypsinized and cell pellets were collected.Approximately 0.53 106 cells were labeled with PI using 0.5 ml ofsaponin/PI solution, at 4°C for 24 h in dark, as detailed in “Materials andMethods.” Cell cycle distribution was then analyzed by flow cytometryusing Becton Dickinson FACS System.A, percentage of total cells in G1phase.B, percentage of total cells in S phase.C, percentage of total cellsin G2-M phases. The data shown are representative of two independentexperiments (each done in duplicate) with,5% variation.

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40–65% and 63–84% (P, 0.001) inhibition, respectively,during 2–6 days of treatment (Fig. 4A). A dose of 100mg/mlGSP resulted in 63%, 90%, and 92% (P , 0.001) inhibition ofanchorage-dependent cell growth after 2, 4, and 6 days oftreatment, respectively (Fig. 4A).

On the basis of the data shown in Fig. 4A, we next exam-ined whether the observed inhibitory effect of GSP on cellgrowth is due to its cytostatic effect, or is an irreversibleactivity. For these studies, cells were treated with 25-, 50-, and75-mg/ml doses for 72 h and then washed several times withmedium to remove GSP from the culture. The cells were thenleft in normal medium without GSP for their possible growth.

As shown in Fig. 4B, compared with DMSO-treated controls,72 h of GSP treatment at a 25-mg/ml dose, followed by awashout study for 1–3 days, did not show any change in cellgrowth. However, treatment of cells with 50mg/ml GSP for 72 hand then following their growth after 24, 48 and 72 h of washoutshowed that there was 44%, 46%, and 52% inhibition (P ,0.001) in cell growth, respectively (Fig. 4B). A much strongerinhibition was evident in this washout study where 75mg/mlGSP led to a 77%, 86%, and 88% inhibition in cell growth after24, 48 and 72 h of GSP washout, respectively (Fig. 4B). Becauseanother interpretation of the data shown in Fig. 4Bleads to theargument that the observed irreversible inhibition is due to thefact that 72 h of GSP treatment at different doses alreadyreduced the total number of cells to begin with in washoutstudies, we also followed cell growth pattern during 72 h afterGSP washout. As shown in Fig. 4B, in case of DMSO-treatedand 25 mg/ml GSP-treated cultures, after washout, the cellgrowth pattern was comparable and was 205%, 172%, and125% (compared with the previous 24 h) after 24, 48, and 72 hof washout, respectively. However, under similar treatment con-ditions, these patterns were 140%, 164%, and 110% in case of50 mg/ml GSP-treated cultures, and 140%, 104%, and 100% incase of 75mg/ml GSP-treated cultures (Fig. 4B). Together, theseresults provide convincing evidence that the observed cellgrowth inhibitory effects of GSP are not just cytostatic, but areirreversible.

Irreversible Growth Inhibitory Effect of GSP Leads toDifferentiation of MDA-MB468 Cells. The observed irre-versible growth inhibitory effect of GSP on MDA-MB468 cellsled to the question that what was the ultimate fate of the growtharrested cells. Both apoptotic cell death and differentiation pos-sibilities, therefore, were explored to answer this question. Forthe apoptotic cell death studies, cultures were treated with 25-,50-, and 75-mg/ml doses of GSP for 24, 48 and 72 h, and bothfloating and attached cells were subjected to several apoptosis-associated assays. In these studies, none of the GSP doses andtime points examined showed: (a) DNA ladder; (b) PARPcleavage; (c) morphological changes suggestive of apoptosis;(d) TUNEL staining; and (e) sub-G1 population in cell cycleassay, as compared with positive findings with paclitaxel usedas a positive control (data not shown).

On the contrary, GSP-treated MDA-MB468 cells showedunique morphological changes. As shown in Fig. 5, comparedwith DMSO-treated control cultures, cells treated with 50mg/mlGSP for 4 days were much larger in their size with a change inmorphology to spindle shape (Fig. 5,A versus B). A moreprofound effect of GSP was evident at a 75-mg/ml dose for 4days where cells became much larger in size with significantchanges in morphology because they were both elongated inlength and had spindle shape (Fig. 5,C versus A). Although lessprofound, 48- and 72-h treatment time points at these doses ofGSP also showed morphological changes (data not shown).These morphological changes were similar to that of epithelialcell differentiation, suggesting that GSP induces differentiationof MDA-MB468 cells. Following these morphological changes,the cells started detaching from the culture dishes presumablydue to their death, suggesting that the observed morphologicalchanges with GSP are associated with terminal differentiationthat ultimately causes cell death. In other studies, as shown in

Fig. 4 GSP irreversibly inhibits anchorage-dependent growth of hu-man breast carcinoma MDA-MB468 cells.A, cell growth inhibitoryeffect of GSP. Cells were plated at 5000 cells/cm2 in 35-mm dishes.After 24 h, cultures were treated with DMSO or GSP at the a concen-tration of 10–100mg/ml medium, and the total number of cells werecounted after 2, 4, and 6 days of these treatments. The cell growth datashown are mean6 SE of four independent plates; each sample wascounted in duplicate.B, washout study showing that the cell growthinhibitory effect of GSP is irreversible. Cells were plated at 5000cells/cm2 in 35-mm dishes and after 24 h were treated with indicateddoses of GSP, and total cell growth was determined after 72 h oftreatments. At this point, in separate dishes, 72 h after indicated treat-ments of GSP, cultures were washed several times with medium toremove GSP and grown in fresh medium without GSP for the indicatedtime. The cell number was determined by cell counting at the indicatedtime after GSP washout. The cell growth data shown are mean6 SE offour independent plates; each sample was counted in duplicate. Thebarsnot clear in some data bars are due to their lower values.

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Fig. 5D, we found that GSP treatment of cells also results in asignificant induction of cytokeratin 8 protein expression, amarker of epithelial cell differentiation (64). The densitometricanalysis of the blot showed that compared with DMSO, both 50-and 75-mg/ml doses of GSP resulted in a 1.7-fold increase (P,0.001) in cytokeratin 8 expression following a 48-h treatment.Whereas no further increase was observed at these doses after72 h of treatment, 96 h of GSP treatment at these doses showed2.4- and 2.6-fold increases (P, 0.001) in cytokeratin 8 levels,respectively.

DISCUSSIONThe central finding in the present study is that a polyphe-

nolic fraction isolated from grape seeds that is rich in procy-anidins, inhibits constitutive activation of MAPK/ERK1/2 andMAPK/p38, and causes an induction of CDKI Cip1/p21 and adecrease in CDK4. These effects of GSP result in a G1 arrest incell cycle, followed by an irreversible inhibition of cell growth.The GSP-treated cells unable to grow do not die by apoptosisbut undergo terminal differentiation, followed by cell death. Ourfindings that GSP inhibits constitutive activation of ERK1/2 arespecifically significant because constitutive activation of thispathway has been shown to be associated with human breastcarcinomas and derived cell lines for uncontrolled growth(48–52).

The MAPK family includes ERKs, JNK (stress-activatedprotein kinase), and p38 (HOG), which are functional unitsinvolved in three distinct signaling pathways (37–47). ActivatedMAPKs transport to the nucleus where they phosphorylate andactivate a series of transcription factors such as Elk-1, cMyc,ATF-2, cJun, CREB, and so on (37–47, 65–67). The differentialresponses of ERKs, JNKs, and p38 in terms of activation ofdifferent transcription factors suggest that these signaling cas-cades are distinct (37–47). Furthermore, several studies havelinked ERKs and p38 signaling to proliferation and differenti-ation, whereas the JNK pathway is largely associated withapoptosis (55–57). Consistent with these reports, in the presentstudy we found that GSP inhibits constitutive activation ofERK1/2 and p38, but had little (if any) effect on JNK activation.These results were in accord with the biological effects of GSPwhere it showed growth inhibitory and differentiation inducingpotential but did not cause apoptotic death. The observed inhib-itory effect of GSP on ERK1/2 activation is similar to thosereported with other polyphenolic antioxidants, such as silyma-rin, genistein, quercetin, and others (54), however, to the best ofour knowledge, this is the first study showing the inhibitoryeffect of a polyphenolic antioxidant fraction on p38 activation.

Earlier, it has been shown that erbB1 and other members of

Fig. 5 GSP induces both morphological changes and expression ofcytokeratin 8, suggesting differentiation of human breast carcinomaMDA-MB468 cells. Cells were cultured in DMEM with 10% serum andat 50% confluency were treated with either DMSO alone or 50- and75-mg/ml concentrations of GSP in DMSO. After 96 h of these treat-ments, pictures were taken using phase-contrast microscope at3200magnification.A, DMSO alone.B, 50 mg/ml GSP in DMSO.C, 75mg/ml GSP in DMSO.D, stimulatory effect of GSP on cytokeratin 8

protein levels. Cells at 50% confluency were treated either with DMSOalone or 50- and 75-mg/ml concentrations of GSP for 48, 72, and 96 h.Cell lysates were prepared and subjected to SDS-PAGE, followed byWestern blotting, as detailed in “Materials and Methods.” The mem-branes were probed with anti-cytokeratin 8 antibody, followed by per-oxidase-conjugated appropriate secondary antibody. Visualization ofproteins was done using ECL detection system. Treatments are aslabeled in the figure.IB, Western immunoblot.

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the erbB family (Her-2/neu or erbB2) play an important role inhuman breast cancer (31–36). In addition, IGF-1R has also beenshown to play an important role in the growth, proliferation, andmetastasis of breast cancer, which is also associated with adecrease or loss of IGF-binding protein-3 that binds to IGF,leading to an inhibition of the binding of this ligand to IGF-1R(35). This impairs mitogenic and antiapoptotic signaling medi-ated by IGF-1/IGF-1R pathway causing a malignant cell growtharrest and apoptotic cell death (68). These and other signalingpathways ultimately activate MAPKs that then translocate to thenucleus and activate transcription factors for cell growth, dif-ferentiation, and proliferation (37–47, 68). Taken together,these reports raise the question whether the observed inhibitoryeffects of GSP on ERK1/2 and p38 activation are direct effect ordue to impairment of upstream signaling involving both receptortyrosine kinases and cytosolic signaling. This cause and effect isan important issue that is being studied in an ongoing program.

Several studies have shown that mitogenic signaling mustbe continually renewed, even if at quite low levels, throughoutmuch of the growth stimulating G1 phase of the mammalian cellcycle (58–62). For example, a cell arrest or growth signaldetermines whether the cells will be in a resting, nondividing“G0 ” phase or whether they will enter the G1 phase of cell cycleand thereby undergo cell replication and proliferation (58–62).Cyclin D1 and its associated CDK4 (or CDK6) normally controlcell cycle events in early G1 phase, and cyclin E coupled withCDK2 is involved in the transition from late G1 to S phase(58–62). Alternatively, impairment of a growth-stimulatory sig-naling pathway leads to the induction of CDKIs (such as Cip1/p21 and Kip1/p27) that inhibit the activity of cyclin-CDK com-plexes, leading to cell growth arrest (69). These studies suggestthat modulation of cell cycle regulatory molecules either di-rectly or via impairment of mitogenic signaling that regulatethem, could be a practical and translation approach for theprevention and/or intervention of human malignancies, includ-ing breast cancer. Consistent with these reports, in the presentstudy we found that GSP treatment of breast carcinoma MDA-MB468 cells results in a significant induction of Cip1/p21. Wealso found that GSP significantly inhibits CDK4 levels andmoderately alters cyclin D1 expression. Conversely, GSP wasnot effective in inhibiting cyclin E and CDK2 levels. Together,these data are consistent with the observed effect of GSP on G1

accumulation and suggest that GSP causes an early effect,leading to arrest of cells in early G1 phase. These effects of GSPon breast carcinoma cells are specifically important because anoverexpression of G1 phase cyclin D1 has been shown with highfrequency in human breast cancer (61).

An up-regulation of Cip1/p21 strongly correlates with cellgrowth inhibition that ultimately decides the fate of cell betweendifferentiation and death. For example, inhibition of Cip1/p21expression through transfection of Cip1/p21 antisense oligonu-cleotides has been shown to block growth factor-induced dif-ferentiation of SH-SY5Y neuroblastoma cells and resulted intheir death (70). Conversely, Cip1/p21 induction is shown in thedifferentiation of a variety of cells such as myogenic, keratino-cytic, promyelocytic (HL-60), and human melanoma cells (71–73). Consistent with these findings, we observed that GSP-caused induction of Cip1/p21 and resultant G1 arrest did notinduce apoptosis but caused terminal differentiation of cells,

followed by their death. An irreversible cell growth inhibitionby GSP further supports this observation.

An analysis of the time kinetics of the observed effects ofGSP on cell cycle regulators suggests that possibly GSP directlyaffects Cip1/p21 and CDK4 expression as an early response thatleads to cell growth inhibitory effect via G1 arrest. This growthinhibitory effect of GSP then presumably leads to an impairmentof autocrine loop, causing a decrease in constitutive activationof MAPK/ERK1/2 as a late response and a terminal differenti-ation of the cells. This presumption could be supported indi-rectly by the studies where at least in prostate carcinoma cells ithas been shown that an induction in MAPK/ERK1/2 activationis associated with the reversal of cellular differentiation (74).More studies, however, are needed to further establish this causeand effect relationship.

In summary, based on the results of the present study, weconclude that GSP and procyanidins present therein should bestudied in more detail to be developed as breast cancer preven-tive and/or interventive agents. In addition, studies are alsoneeded to establish whether the observed effective doses of GSPin cell culture are achievable physiologically in breast cancerpatients and that such doses are both nontoxic and effective forpreventive intervention of this deadly malignancy.

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2000;6:2921-2930. Clin Cancer Res   Chapla Agarwal, Yogesh Sharma, Jifu Zhao, et al.  

Arrest and Differentiation1Inducing GInhibiting Mitogen-activated Protein Kinases Activation andGrowth Inhibition of Breast Carcinoma MDA-MB468 Cells by A Polyphenolic Fraction from Grape Seeds Causes Irreversible

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