European Journal of Pharmacology 591 (2008) 96–101

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European Journal of Pharmacology

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Catechin and proanthocyanidin B4 from grape seeds prevent doxorubicin-inducedtoxicity in cardiomyocytes

Yu Du, Hongxiang Lou ⁎Department of Natural Products, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, PR China

⁎ Corresponding author. 44 Wenhua Xilu, Jinan, Shand531 88382018; fax: +86 531 88382019.

E-mail address: louhongxiang@sdu.edu.cn (H. Lou).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.06.068

A B S T R A C T

A R T I C L E I N F O

Article history:

The clinical use of doxorubic Received 29 March 2008Received in revised form 4 June 2008Accepted 12 June 2008Available online 24 June 2008

Keywords:CatechinProanthocyanidinDoxorubicinApoptosisReactive oxygen speciesCardiomyocyte

in, a highly active anticancer drug, is limited by its severe cardiotoxic side effects.Grape seed extract has been reported to exert protective effects on doxorubicin-induced cardiotoxicity. Thecardiovascular protective effects of grape seed extract are believed to be ascribed to its antioxidativeproperties. A series of studies have has demonstrated that polyphenols are instrumental for the antioxidativeproperties of grape seed extract. This study was designed to investigate whether two major polyphenolsisolated from grape seed extract, catechin and proanthocyanidin B4 (Pc B4), had protective effects againstdoxorubicin-induced toxicity in cardiomyocytes and their underlying mechanisms. The results showed thatgrape seed polyphenols catechin and Pc B4 pretreatment would protect cardiomyocytes against doxorubicin-induced toxicity by decreasing reactive oxygen species generation as well as the number of apoptotic cells,preventing DNA fragmentation, regulating the expression levels of the pro-apoptotic protein Bax-α and theanti-apoptotic protein Bcl-2, and inhibiting apoptotic signaling pathways.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Doxorubicin is a powerful anthracycline antibiotic used to treat amultitude of human neoplasms, it is among the most effective andwidely used anticancer drugs in the clinic, however, cardiotoxicitycompromises the clinical usefulness of the drug (Wouters et al., 2005).The chronic side effects of doxorubicin are irreversible and devastat-ing for the patient, including the development of cardiomyopathy andultimately congestive heart failure (Mott, 1997; Shan et al., 1996). Thecause of doxorubicin cardiotoxicity is multifactorial, but mostdoxorubicin-induced cardiotoxicity can be attributed to the formationof reactive oxygen species, which ultimately results in myocytesapoptosis (Horenstein et al., 2000). An alternative approach is the useof cardioprotective drugs as adjuvants to doxorubicin therapy. Agentswhich prevent cardiotoxicity would allow us to exploit the fulltherapeutic potential of doxorubicin, making a tremendous impact oncancer therapy.

Grape seeds are waste products of the winery and grape juiceindustry. These seeds contain lipid, protein, carbohydrates, and 5–8%polyphenols depending on the variety. Polyphenols in grape seeds aremainly flavonoids, including gallic acid, the monomeric flavan-3-olscatechin, epicatechin, gallocatechin, epigallocatechin, epicatechin 3-O-gallate, dimeric-, trimeric- and even more polymeric proanthocya-nidins(Shi et al., 2003). A large number of studies have been con-ducted on grape seed extract and have demonstrated excellent free

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radical scavenging and cardioprotective properties(Bagchi et al.,2000). Grape seed extract provided a unique protection against myo-cardial ischemia–reperfusion injury andmyocardial infarction in rats(Sato et al., 1999). Grape seed extract was also shown to confercardioprotection against exogenous H2O2- or antimycin A-inducedoxidant injury (Shao et al., 2003). Grape seed extract inhibitedplatelet function and release of reactive oxygen intermediates(Vitseva et al., 2005). Grape seed extract also provided protectionagainst doxorubicin-induced cardiotoxicity in mice (Ray et al., 2000).However, the protective mechanisms underlying it remain elusive. Inmost cases, the activity of grape seed extract are related to itsantioxidative properties and are mainly attributed to the phenoliccompounds (Yilmaz and Toledo, 2004). In prior studies, we purifiedeleven phenolic compounds from grape seeds by gel chromatogra-phy and high performance liquid chromatography (HPLC). Amongthose compounds, catechin and Pc B4 (Fig. 1) exhibited betterantioxidative activity against an oxidative damage to DNA in micespleen cells than other compounds (Fan and Lou, 2004). Recently, weincubated cardiac H9C2 cells with micromolar catechin or Pc B4, anddemonstrated that there was a significant induction of cellularantioxidant enzymes in a concentration-dependent fashion. Further-more, catechine or Pc B4 pretreatment led to a marked reduction inxanthine oxidase (XO)/xanthine-induced intracellular reactive oxy-gen species accumulation and cardiac cell apoptosis (Du et al., 2007).Because polyphenols are believed to be the major bioactive com-pounds in grape seed extract, in this study, we examined the effectsof grape seed polyphenols catechin and Pc B4 on doxorubicindoxorubicin-induced toxicity in cardiomyocytes. Our goal was toelucidate the potential mechanisms underlying the protective effects

Fig. 1. Structures of catechin and Pc B4.

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of grape seed extract grape seed extract which have been observed indoxorubicin cardiotoxicity.

2. Materials and methods

2.1. Reagents

Catechin and Pc B4 were extracted from grape seeds in ourlaboratory (Fan and Lou, 2004); doxorubicin, 2′, 7′-dichlorodihydro-fluorescein diacetate (DCFH), Hoechst 33258, and bovine serum albu-min (BSA) were from Sigma Chemical (St. Louis, MO, USA). Dulbecco’sDulbecco's modified Eagle's medium (DMEM) was from Gibco-Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was from HangzhouSijiqingBiotechnologyCo. (Hangzhou, China). Trizolwas from InvitrogenLife Technologies (Grand Island, NY, USA). M-MLV reverse transcriptasewas from Promega (Madison, WI, USA). Enhanced chemiluminescencereagent was from Amersham Life Sciences (Arlington Heights, IL, USA).Kodak X-omat film was from Eastman Kodak (Rochester, NY, USA).Antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,USA). Non-itemized chemicals were all of the highest possible quality.Only deionized, glass-distilled water was used in this study.

2.2. Cell culture

Rat heart cell line H9C2(2-1) (ATCC, Rockville, MD) were culturedin DMEM supplemented with 10% FBS, 100 U/ml of penicillin, and80 μg/ml of streptomycin in tissue culture flasks at 37 °C in ahumidified atmosphere of 5% CO2. The cells were fed every 2–3 days,and subcultured once they reached 70–80% confluence.

2.3. Flow-cytometric assay of 2′, 7′-dichlorodihydrofluorescein (DCFH)

DCFH was used to detect intracellular reactive oxygen specieslevels in H9C2 cells. To detect doxorubicin-induced intracellularreactive oxygen species accumulation, H9C2 cells grown on 6-welltissue culture plates were rinsed once with PBS and then the cellswere treated with 20 μM doxorubicin at 37 °C for 24 h. After thisincubation, DCFH was added at a final concentration of 20 μM andincubated for another 30 min at 37 °C. After three washes, cells wereobserved under a fluorescence microscope (IX-71, Olympus, Japan)and then maintained in 500 μl PBS. Cellular fluorescence wasdetermined by a flow cytometry apparatus (FACS-SCAN Becton-Dickinson. Franklin Lakes, NJ, USA). Measurements were taken at

510 to 540 nm after excitation of cells at 488 nm with an argon ionlaser.

2.4. Flow Flow-cytometric detection of apoptosis

FITC-Annexin V/PI double staining. The cells were labeled withFITC-Annexin V and propidium iodide according to kit’s kit's instruc-tions (Jinmei Biotechnology Co. Ltd, Beijing, China).

2.5. Fluorescent staining of nuclei

The nuclei of H9C2 cells were stainedwith chromatin dye (Hoechst33258). Briefly, cells were fixed with 3.7% paraformaldehyde for10 min at room temperature, washed twice with PBS, and incubatedwith 10 μM Hoechst 33258 in PBS at room temperature for 30 min.After three washes, cells were observed under a fluorescencemicroscope (IX-71, Olympus, Japan).

2.6. RT-PCR

Total RNA was isolated from H9C2 cells using Trizol reagentsand quantified spectrophotometrically by its absorbance at 260 nm.First-strand cDNA was synthesized by reverse transcription of 2 μgtotal RNA employing random primers using M-MLV reverse tran-scriptase. The quantity of mRNA was normalized for glyceraldehydephosphate dehydrogenase (GAPDH).

Amplification of the cDNA by polymerase chain reaction (PCR) wasperformed using PCR Supermix (Bio-Rad, Hercules, California, USA)with the following primers:

1. for GAPDH:5′-GAGGGGCCATCCACAGTCTT-3′ and 5′-TTCATTGACCTCAACTACAT-3′;

2. for Bax-α: 5′-CCAAGAAGCTGAGCGAGTGTCTC-3′ and 5′-AGTTGCCGTCTGCAAACATGTCA-3′;

3. for Bcl-2: 5′-AGCTGCACCTGACGCCCTT-3′ and 5′-CAGCCAGGAGAAATCAAACAGAGG-3′.

The amplified products were 506 bases for GAPDH, 146 bases forBax-α, and 293 bases for Bcl-2, respectively.

The amplified transcripts were analyzed on 2.0% agarose gel usingethidium bromide. The signal intensities of the bands were quantifiedby densitometry using AlphaEaseFC Image Analysis Software (AlphaInnotech, San Leandro, CA, USA) and compared with the internalstandard GAPDH.

2.7. Western blotting analysis

The H9c2 cells were lysed on ice in a sample buffer (50 mM Tris–HCl [pH 6.8], 2% SDS, 10% Glycerol, 100 mM dithiothreitol, 0.1%bromophenol blue), and the protein concentration of each samplewasmeasured by the BCA method using the bovine serum albumin as astandard. Samples containing 20 μg total protein were loaded onto a12% SDS-PAGE and then electrophoretically transferred to a poly-vinylidene difluoride membrane. Membranes were incubated with 5%non-fat dried milk at room temperature for 1 h, then incubated withspecific primary antibody at room temperature for 2 h. Antibodyrecognitionwas detected using the specific secondary antibody, eitheranti-mouse or anti-rabbit at room temperature for 1 h. The signalswere visualized by an enhanced chemiluminescence reagent andexposed to Kodak X-omat films. Relative intensities of protein bandswere quantified by densitometry using AlphaEaseFC Image AnalysisSoftware and compared with the internal standard β-actin.

2.8. Caspase-3 and caspase-9 activity assay

The activities of caspase-3 and caspase-9 were determined by thedetection of chromophore p-nitroanilide after cleavage from the

Fig. 3. Flow Flow-cytometric analysis of apoptosis in H9C2 cells that were pretreatedwith catechin and Pc B4. The upper panels are results of one experiment: (A) Untreatedcontrol cells; (B) cells that were treated with 20 μM doxorubicin for 24 h; (C) cells thatwere pretreated with 100 μM catechin for 24 h and then incubated with doxorubicin for24 h; (D) cells that were pretreated with 100 μM Pc B4 for 24 h and then incubated withdoxorubicin for 24 h. The lower panel (E) shows a column bar graph analysis. Valuesrepresent means±S.D. from three independent experiments. ⁎Significantly differentfrom doxorubicin, Pb0.001; ⁎⁎Significantly different from doxorubicin, Pb0.01;⁎⁎⁎Significantly different from doxorubicin, Pb0.05.

Fig. 2. Effects of catechin and Pc B4 treatment on intracellular reactive oxygen speciesaccumulation induced by doxorubicin in H9C2 cells. Following pretreatment with100 μM catechin, or Pc B4 for 24 h, the medium was changed, followed by incubationwith 20 μM doxorubicin for another 24 h. The intracellular reactive oxygen speciesaccumulation was determined by measuring the DCF-derived fluorescence afterincubation of cells with DCF-DA for 30 min. (A) Cells were visualized under fluorescentmicroscope, size-bars represent 5 μm. (B) Column bar graph mean cell fluorescence forDCF. The fluorescence intensities in untreated control cells are expressed as 100%.Results are means±S.D. of three independent experiments. ⁎Pb0.05 vs. doxorubicin.

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labeled substrate DEVD-p-nitroanilide using assay kits (NanjingKeyGen Biotech. Inc., Nanjing, China). In brief, 3×106 cells weresolubilized, then equal amounts of protein lysates were reacted withDEVD-p-nitroanilide reaction buffer and caspase-3 or caspase-9substance at 37 °C for 4 h. The activity was read in a microplatespectrophotometer at 405 nM. The protein content was determined bythe BCA method using the bovine serum albumin as a standard.

2.9. Statistical analysis

All data are expressed as means±S.D. from at least three inde-pendent experiments. Differences between mean values of multiplegroups were analyzed by one-way analysis of variance (ANOVA).Statistical significance was considered at Pb0.05.

3. Results

3.1. Catechin and Pc B4 regulate intracellular reactive oxygen species

Treatment with 20 μM doxorubicin for 24 h caused a 1.5-foldincrease in DCF fluorescence in H9C2 cells, suggesting intracellular

production of reactive oxygen species. Pretreatment with 100 μMcatechin or 100 μM Pc B4 for 24 h dramatically lowered doxorubicin-induced free radical release (Fig. 2).

3.2. Catechin and Pc B4-induced cell resistance to doxorubicin-inducedapoptosis in H9C2 cells

Exposure of cardiac H9C2 cells to 20 μM doxorubicin for 24 hinduced apoptosis, as demonstrated by the increase from 1 (controlcells) to 16% (doxorubicin -treated cells) in the number of AnnexinV-FITC positive fluorescent cells. Pretreatment with 100 μM catechinor 100 μM Pc B4 for 24 h both led to significant decreases in thenumber of Annexin V-FITC positive fluorescent cells (Fig. 3E). Doxo-rubicin induced the rapid changes in the nuclear morphology ofH9C2 cells with heterogeneous intensity and chromatin condensa-tion while control cells were shown as round-shaped nuclei withhomogeneous fluorescence intensity. Pretreatment of 100 μM cat-echin or 100 μM Pc B4 significantly protected cells from morphol-ogical changes by doxorubicin (Fig. 4).

Fig. 6. Bax-a and Bcl-2 protein expression in H9C2. Following pretreatment with 100 μMcatechin, or Pc B4 for 24 h, themediumwas changed, followed by incubationwith 20 μMdoxorubicin for another 24 h. Then cells were treated as described in Materials andmethods. (A) shows the results of one experiment. (B) shows a column bar graphanalysis. Normalization relative to β-actin was performed. The mean protein levels inuntreated control cells are expressed as 100%. Results presented in bar graph are themeans±S.D. of three independent experiments., ⁎Significantly different from doxor-ubicin, Pb0.05; ⁎⁎Significantly different from doxorubicin, Pb0.01; ⁎⁎⁎Significantlydifferent from doxorubicin, Pb0.001.

Fig. 5. Bax-α and Bcl-2 mRNA expression in H9C2 cells. Following pretreatment with100 μM catechin, or Pc B4 for 24 h, the medium was changed, followed by incubationwith 20 μM doxorubicin for another 24 h. Then cells were treated as described inMaterials and methods. (A) shows the results of one experiment. (B) shows a columnbar graph analysis. Normalization relative to GAPDH was performed. The mean mRNAlevels in untreated control cells are expressed as 100%. Results presented in bar graphare the means±S.D. of three independent experiments. ⁎Significantly different fromdoxorubicin, Pb0.05; ⁎⁎Significantly different from doxorubicin, Pb0.01; ⁎⁎⁎Signifi-cantly different from doxorubicin, Pb0.001.

Fig. 4. Fluorescent staining of nuclei of H9C2 cells. Cells were treated as described inMaterials and methods. Then, cells were visualized under fluorescent microscope.(A) Untreated control cells; (B) cells that were treated with 20 μM doxorubicin for 24 h;(C) cells that were pretreated with 100 μM catechin for 24 h and then incubated with20 μM doxorubicin for 24 h; (D) cells that were pretreated with 100 μM Pc B4 for 24 hand then incubated with 20 μM doxorubicin for 24 h. Size-bars represent 10 μm.

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3.3. Catechin and Pc B4 regulate the expression of Bax-α and Bcl-2 indoxorubicin-treated H9C2 cells

To explore effects of Catechin and Pc B4 on the apoptotic signalingchanges in H9C2 cells, we investigated the mRNA and proteinexpressions of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax-α(Figs. 5 and 6). Treatment with 20 μM doxorubicin for 24 h induced adrop in the expression of Bcl-2 and an increase in the expression ofBax-α in H9C2 cells. Catechin and Pc B4 pretreatment prevented theexpression changes of Bcl-2 and Bax-α caused by doxorubicin.

3.4. Catechin and Pc B4 inhibit doxorubicin-induced caspase-3, caspase-9 activation in H9C2 cells

Activation of caspase cascades is critical in the initiation of apo-ptosis in diverse biological systems (Budihardjo et al., 1999). Invarious cell apoptosis, caspase-3 constitutes the major pool ofexecutor caspases and it is also a downstream target of caspase-9. Inorder to investigate the apoptotic signaling downstream from Bcl-2and Bax, and determine effects of catechin and Pc B4 on caspasecascades, the catalytic activities of caspase-3 and caspase-9 weretested. As shown in Fig. 7, significant 8.5-fold and 8.9-fold increasesin cellular caspase-3 and caspase-9 activities were observed after20 μM doxorubicin treatment. In keeping with their effects onAnnexin V-FITC/propidium iodide staining and Bax/Bcl-2 expression,

Fig. 7. The activities of caspase-3 and caspase-9 were measured by the detection ofchromophorep-nitroanilide and expressed aspercentof control. Results aremeans±S.D. ofthree independent experiments. ⁎Pb0.05 vs. doxorubicin. ⁎Significantly different fromdoxorubicin, Pb0.05; ⁎⁎Significantly different from doxorubicin, Pb0.01; ⁎⁎⁎Significantlydifferent from doxorubicin, Pb0.001.

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catechin or Pc B4 dose dependently inhibited the activation ofcaspase-3 and caspase-9. Pretreatment with catechin (50 and 100 μM)for 24 h limited the increases in caspase-3 activity to 6.2-, 4.8-fold, andcaspase-9 activity to 6.5-, 4.6-fold, respectively, but 25 μM catechin didnot show significant effects. Pretreatment with Pc B4 (25, 50 and100 μM) for 24 h also dramatically limited the increases in caspase-3activity to 7.3-, 6.0-, 4.1-fold, and caspase-9 activity to 7.2-, 5.9-, 4.2-fold,respectively.

4. Discussion

Grape seed polyphenols mainly include proanthocyanidins. Gen-erally the dimer- and trimer-proanthocyanidins, referred to asextractable proanthocyanidins or bioflabonoids, have been shown tobe highly bioavailable and provide excellent health benefits (Bagchiet al., 2000). These low molecular proanthocyanidins are also knownas sustained release antioxidants, and can remain in the plasma andtissues for up to 7–10 days and exert antioxidant properties, which ismechanistically different from other soluble antioxidants (de Vrieset al., 1998). We used gel chromatography and semi-preparative HPLC,11 pure individual phenolic compounds were separated from grapeseed extract. Epicatechin lactone A, B and catechin lactone are beyondthe usual varieties of compounds from grape seed extract, which arepresumed oxidative derivatives of epicatechin and catechin afteranalyzing the homology of their structures. In previous studies, weobserved that catechin and Pc B4 had substantial antioxidantactivities, which protected mice spleen cells from the DNA damages

induced by reactive oxygen species (Fan and Lou, 2004). Our exper-iments also confirmed that a number of antioxidant enzymes incultured cardiomyocytes could be induced by low micromolar con-centrations catechin or Pc B4, this chemically mediated upregulationof cellular defenses was accompanied by a markedly increasedresistance to cardiac cell apoptosis elicited by reactive oxygen species(Du et al., 2007).

Reactive oxygen species are considered to be chemically reactivewith and damaging to biomolecules including DNA, protein and lipid,and excessive exposure to reactive oxygen species induces oxidativestress and causes genetic mutations. There are increasing evidencesthat reactive oxygen species play an important role in the develop-ment of vasculopathies, including those that define atherosclerosis,hypertension, and restenosis after angioplasty. It is now clear not onlythat diverse reactive oxygen species are produced in the vessel wall,but that they individually and in combination contribute to many ofthe abnormalities associated with vascular disease (Griendling andFitzGerald, 2003).

Doxorubicin is the mainstay treatment of a variety of hematolo-gical malignancies and solid tumors. Unfortunately, the clinical use ofthis drug is limited by cumulative dose-related cardiotoxicity whichmay ultimately lead to a severe and irreversible form of cardiomyo-pathy (Orhan, 1999; Silber and Barber, 1995; Wouters et al., 2005).Toxicity is believed to occur by doxorubicin-induced mitochondrialdysfunctions and subsequent oxidants production (De Beer et al.,2001). Doxorubicin also caused morphological alterations in mito-chondrial, nuclear, and fibrous protein structures in H9C2 cells (Sardaoet al., 2008).

A series of studies were was conducted using grape seed extract todemonstrate its cardioprotective ability in animals and humans. Grapeseed extract supplementation improved cardiac functional assess-ment including post-ischemic left ventricular function, reducedmyocardial infarct size, reduced VF and VT, decreased the amount ofreactive oxygen species and reduced malondialdehyde (MDA) forma-tion in the heart perfusate (Bagchi et al., 2003). Grape seed extract alsoprovided protection against doxorubicin-induced cardiotoxicity inmice (Ray et al., 2000). However, the protective mechanism under-lying it remained to be elusive. In this report, based on prior ex-periments (Du et al., 2007), we chose 100 μM catechin and 100 μM PcB4 to detect their effects on doxorubicin-induced toxicity incardiomyocytes. We demonstrated that incubation of cardiomyocyteswith catechin and Pc B4 significantly inhibited doxorubicin-inducedintracellular reactive oxygen species accumulation, decreased thenumber of apoptotic cells, prevented DNA fragmentation, and inhib-ited apoptotic signaling pathways.

Several studies have indicated that apoptosis occurs in the humanheart during the end stage of cardiac failure or acute myocardialinfarction, which suggests that apoptosis is involved in cardiovasculardiseases (Formigli et al., 1998). Multiple lines of evidence support therole of reactive oxygen species in apoptotic cell death. A direct role ofoxygen free radicals has also been implicated in the pathogenesis ofapoptosis. Annexin V/PI staining (Fig. 3) showed that, at low micro-molar doxorubicin concentration, apoptosis was the dominant mech-anism for cardiac cell death, while pretreatment with 100 μM catechinor 100 μMPc B4 reduced the numbers of positive fluorescent cells. Ourexperiments also showed that pretreatment of 100 μM catechin or100 μMPc B4 significantly protected cells frommorphological changesinduced by doxorubicin (Fig. 4), but effect of Pc B4 was a little strongerthan catechin, presumably this may be related to its double number ofphenolic hydroxy groups.

Although the intracellular localization of reactive oxygen speciesformation elicited by doxorubicin is still under investigation, recentstudies suggest that doxorubicin accumulates over time in mitochon-dria, where it is enzymatically activated to form semi-quinone radicalsand superoxide anions (Green and Leeuwenburgh, 2002). It is increas-ingly apparent that mitochondrial dysfunction plays a key role in the

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regulation of apoptosis. It has been hypothesized that doxorubicin-induced oxidative cell signaling may alter the regulation of the Bcl-2family of proteins (Antonsson and Martinou, 2000). Bax and Bcl-2, thetwo main members of this family, influence the permeability of themitochondrial membrane. Bcl-2 is regarded as an important cellularcomponent that guards against apoptotic cell death and which is alsoinvolved in many other cellular events. It is associated with the outermitochondrial membrane where it stabilizes the membrane perme-ability, thus preserving mitochondrial integrity and suppressing therelease of cytochrome c (Yang et al., 1997). Bax is a pore-formingcytoplasmic protein, that in response to an enhanced oxidative stress,translocates to the outer mitochondrial membrane, influences itspermeability and induces cytochrome c loss from the intermembranespace of the mitochondria and subsequent release into the cytosol(Crompton, 2000). It has been reported that the prevention ofapoptosis is associated with the upregulation of Bcl-2 and thedownregulation of Bax (Shihab et al., 1999; Wang et al., 1999). Ourstudy showed that doxorubicin had profound effects on the Bcl-2family proteins. While it upregulated Bax expression, it also down-regulated the level of Bcl-2 expression. Pretreatment with 100 μMcatechin or 100 μM Pc B4 inhibited both the doxorubicin-induced Baxexpression and the reduction of Bcl-2.

Apoptosis is also generally associated with the activation ofcaspase cascades. Several studies have demonstrated that the releaseof cytochrome c from the mitochondria into the cytosol can result inthe activation of caspase-9, which is responsible for activating effectorcaspases, such as caspase-3, which leads to DNA fragmentation andcleavage of cytoskeletal and myofibrillar proteins, and ultimately tocell death (Kumar et al., 1999). In our experiments, pretreatment withcatechin or Pc B4 were also associated with the inhibition of thedownstream apoptotic signaling pathways, finally preventing activa-tion of caspase-3 and caspase-9 induced by doxorubicin.

In conclusion, our experiments confirmed that grape seed poly-phenols catechin and Pc B4 pretreatment would protect cardiomyo-cytes against doxorubicin-induced toxicity through decreasing ROSgeneration and the number of apoptotic cells, preventing DNA frag-mentation, regulating the levels of expression of the pro-apoptoticprotein Bax-α and the anti-apoptotic protein Bcl-2, and inhibitingapoptotic signaling pathways. Because grape seed polyphenols areinstrumental for the bioactive properties of grape seed extract, aboveresults may be important mechanisms underlying the protective ef-fects of grape seed extract observed in doxorubicin cardiotoxicity.Thereby, we provide the mechanistic basis of grape seed extract andgrape seed polyphenols in clinical application to protect patients fromdoxorubicin doxorubicin-induced cardiotoxicity.

Acknowledgements

This work was supported by a grant from the National NaturalFoundation of P.R. China (No. 30472072).

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