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Sulforaphane-induced Cell Death in Human Prostate Cancer Cells Is Initiated by Reactive Oxygen Species

2005; Elsevier BV; Volume: 280; Issue: 20 Linguagem: Inglês

10.1074/jbc.m412443200

ISSN

1083-351X

Autores

Shivendra V. Singh, Sanjay Srivastava, Sunga Choi, Karen L. Lew, Jędrzej Antosiewicz, Dong Xiao, Yan Zeng, Simon C. Watkins, Candace S. Johnson, Donald L. Trump, Yong J. Lee, Hui Xiao, Anna Herman-Antosiewicz,

Tópico(s)

Synthesis and biological activity

Resumo

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent. We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent. Epidemiological data continue to support the premise that dietary intake of cruciferous vegetables may reduce the risk of different types of malignancies, including cancer of the prostate (1Verhoeven D.T. Goldbohm R.A. van Poppel G. Verhagen H. van den Brandt P.A. Cancer Epidemiol. Biomarkers Prev. 1996; 5: 733-748PubMed Google Scholar, 2Cohen J.H. Kristal A.R. Stanford J.L. J. Natl. Cancer Inst. 2000; 92: 61-68Crossref PubMed Scopus (614) Google Scholar, 3Zhang S.M. Hunter D.J. Rosner B.A. Giovannucci E.L. 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The anticarcinogenic effect of cruciferous vegetables is attributed to isothiocyanates (ITCs) 1The abbreviations used are: ITCs, isothiocyanates; SFN, sulforaphane; NAC, N-acetylcysteine; ROS, reactive oxygen species; DAPI, 4′,6-diamidino-2-phenylindole; HE, hydroethidine; H2DCFDA, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; TMRM, tetramethylrhodaminemethyl ester; DPI, diphenyleneiodonium chloride; SOD, superoxide dismutase; MEFs, mouse embryonic fibroblasts; PARP, poly(ADP-ribose) polymerase; DCF, 2′,7′-dichlorofluorescein; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; Cox IV, cytochrome c oxidase (complex IV); EGFP, enhanced green fluorescence protein; BSA, bovine serum albumin; ANOVA, analysis of variance; DMEM, Dulbecco's modified Eagle's medium; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Ad, adenoviral; FADD, Fas-associated death domain; WT, wild type. that occur naturally as thioglucoside conjugates (glucosinolates) in a variety of edible plants including watercress, broccoli, cabbage, and so forth (reviewed in Refs. 5Hecht S.S. Drug. Metab. Rev. 2000; 32: 395-411Crossref PubMed Scopus (389) Google Scholar, 6Fahey J.W. Zalcmann A.T. Talalay P. Phytochemistry. 2001; 56: 5-51Crossref PubMed Scopus (2210) Google Scholar, 7Conaway C.C. Yang Y.M. Chung F.L. Curr. Drug Metab. 2002; 3: 233-255Crossref PubMed Scopus (357) Google Scholar). Organic ITCs are generated due to hydrolysis of corresponding glucosinolates through catalytic mediation of myrosinase (6Fahey J.W. Zalcmann A.T. Talalay P. Phytochemistry. 2001; 56: 5-51Crossref PubMed Scopus (2210) Google Scholar). Naturally occurring ITCs, including phenethyl-ITC and benzyl-ITC, have been shown to offer significant protection against cancer in animal models induced by a variety of chemicals including tobacco smoke-derived carcinogens (reviewed in Refs. 5Hecht S.S. Drug. Metab. Rev. 2000; 32: 395-411Crossref PubMed Scopus (389) Google Scholar and 7Conaway C.C. Yang Y.M. Chung F.L. Curr. Drug Metab. 2002; 3: 233-255Crossref PubMed Scopus (357) Google Scholar). Sulforaphane (SFN; 1-isothiocyanato-4-(methylsulfinyl)-butane), a naturally occurring member of the ITC family of chemopreventive agents, has received particular attention because of its anticancer effects (8Zhang Y. Talalay P. Cho C.G. Posner G.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2399-2403Crossref PubMed Scopus (1513) Google Scholar, 9Zhang Y. Kensler T.W. Cho C.G. Posner G.H. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3147-3150Crossref PubMed Scopus (693) Google Scholar, 10Fahey J.W. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10367-10372Crossref PubMed Scopus (1120) Google Scholar, 11Chung F.L. Conaway C.C. Rao C.V. Reddy B.S. Carcinogenesis. 2000; 21: 2287-2291Crossref PubMed Scopus (325) Google Scholar, 12Fahey J.W. Haristoy X. Dolan P.M. Kensler T.W. Scholtus I. Stephenson K.K. Talalay P. Lozniewski A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7610-7615Crossref PubMed Scopus (680) Google Scholar). This phytochemical is a potent inducer of the phase 2 enzymes implicated in carcinogen detoxification (13Brooks J.D. Paton V.G. Vidanes G. Cancer Epidemiol. Biomarkers Prev. 2001; 10: 949-954PubMed Google Scholar) and a competitive inhibitor of CYP2E1, which is involved in the activation of carcinogenic chemicals (14Barcelo S. Gardiner J.M. Gescher A. Chipman J.K. Carcinogenesis. 1996; 17: 277-282Crossref PubMed Scopus (196) Google Scholar). Cancer chemoprevention by SFN or its N-acetylcysteine (NAC) conjugate has been observed against 9,10-dimethyl-1,2-benz[a]anthracene-induced mammary tumorigenesis in rats (9Zhang Y. Kensler T.W. Cho C.G. Posner G.H. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3147-3150Crossref PubMed Scopus (693) Google Scholar), azoxymethane-induced colonic aberrant crypt foci formation in rats (11Chung F.L. Conaway C.C. Rao C.V. Reddy B.S. Carcinogenesis. 2000; 21: 2287-2291Crossref PubMed Scopus (325) Google Scholar), and benzo[a]pyrene-induced forestomach cancer in mice (12Fahey J.W. Haristoy X. Dolan P.M. Kensler T.W. Scholtus I. Stephenson K.K. Talalay P. Lozniewski A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7610-7615Crossref PubMed Scopus (680) Google Scholar). Evidence is accumulating to indicate that SFN can suppress proliferation of cancer cells in culture by causing cell cycle arrest and apoptosis induction (15Gamet-Payrastre L. Li P. Lumeau S. Cassar G. Dupont M.A. Chevolleau S. Gasc N. Tulliez J. Terce F. Cancer Res. 2000; 60: 1426-1433PubMed Google Scholar, 16Fimognari C. Nüsse M. Cesari R. Iori R. Cantelli-Forti G. Hrelia P. Carcinogenesis. 2002; 23: 581-586Crossref PubMed Scopus (214) Google Scholar, 17Misiewicz I. Skupinska K. Kasprzycka-Guttman T. Oncol. Rep. 2003; 10: 2045-2050PubMed Google Scholar, 18Wang L. Liu D. Ahmed T. Chung F.L. Conaway C. Chiao J.W. Int. J. Oncol. 2004; 24: 187-192PubMed Google Scholar, 19Jackson S.J.T. Singletary K.W. Carcinogenesis. 2004; 25: 219-227Crossref PubMed Scopus (176) Google Scholar, 20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar, 21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Growth inhibition, cell cycle arrest, and/or apoptosis induction by SFN has been observed in human colon, leukemia, and prostate cancer cells (15Gamet-Payrastre L. Li P. Lumeau S. Cassar G. Dupont M.A. Chevolleau S. Gasc N. Tulliez J. Terce F. Cancer Res. 2000; 60: 1426-1433PubMed Google Scholar, 16Fimognari C. Nüsse M. Cesari R. Iori R. Cantelli-Forti G. Hrelia P. Carcinogenesis. 2002; 23: 581-586Crossref PubMed Scopus (214) Google Scholar, 17Misiewicz I. Skupinska K. Kasprzycka-Guttman T. Oncol. Rep. 2003; 10: 2045-2050PubMed Google Scholar, 18Wang L. Liu D. Ahmed T. Chung F.L. Conaway C. Chiao J.W. Int. J. Oncol. 2004; 24: 187-192PubMed Google Scholar, 19Jackson S.J.T. Singletary K.W. Carcinogenesis. 2004; 25: 219-227Crossref PubMed Scopus (176) Google Scholar, 20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar, 21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Most interestingly, the NAC conjugate of SFN was shown recently to inhibit histone deacetylase activity (22Myzak M.C. Karplus P.A. Chung F.L. Dashwood R.H. Cancer Res. 2004; 64: 5767-5774Crossref PubMed Scopus (440) Google Scholar). Our own work has revealed that orally administered SFN at a dietary achievable dose significantly retards growth of PC-3 human prostate cancer xenografts in athymic mice without causing weight loss or any other side effects (20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar). Recent studies have offered novel insights into the mechanism by which SFN inhibits cell cycle progression (15Gamet-Payrastre L. Li P. Lumeau S. Cassar G. Dupont M.A. Chevolleau S. Gasc N. Tulliez J. Terce F. Cancer Res. 2000; 60: 1426-1433PubMed Google Scholar, 16Fimognari C. Nüsse M. Cesari R. Iori R. Cantelli-Forti G. Hrelia P. Carcinogenesis. 2002; 23: 581-586Crossref PubMed Scopus (214) Google Scholar, 18Wang L. Liu D. Ahmed T. Chung F.L. Conaway C. Chiao J.W. Int. J. Oncol. 2004; 24: 187-192PubMed Google Scholar, 19Jackson S.J.T. Singletary K.W. Carcinogenesis. 2004; 25: 219-227Crossref PubMed Scopus (176) Google Scholar, 21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Our own work revealed that the SFN-mediated G2-M phase cell cycle arrest in human prostate cancer cells was associated with activation of checkpoint kinase 2, which promoted Ser216 phosphorylation of Cdc25C leading to its translocation from the nucleus to the cytosol (21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). The net result of these effects was accumulation of Tyr15-phosphorylated (inactive) cyclin-dependent kinase 1 (21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), which together with the B-type cyclins plays an important role in the regulation of G2-M transition. Thus, significant progress has been made toward our understanding of the mechanism of cell cycle arrest by SFN (15Gamet-Payrastre L. Li P. Lumeau S. Cassar G. Dupont M.A. Chevolleau S. Gasc N. Tulliez J. Terce F. Cancer Res. 2000; 60: 1426-1433PubMed Google Scholar, 16Fimognari C. Nüsse M. Cesari R. Iori R. Cantelli-Forti G. Hrelia P. Carcinogenesis. 2002; 23: 581-586Crossref PubMed Scopus (214) Google Scholar, 18Wang L. Liu D. Ahmed T. Chung F.L. Conaway C. Chiao J.W. Int. J. Oncol. 2004; 24: 187-192PubMed Google Scholar, 19Jackson S.J.T. Singletary K.W. Carcinogenesis. 2004; 25: 219-227Crossref PubMed Scopus (176) Google Scholar, 21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, the signaling pathway by which SFN initiates the cell death process is poorly defined. An understanding of the mechanism of SFN-induced apoptosis is critical for its clinical development because this knowledge could lead to identification of mechanism-based biomarkers potentially useful in future clinical trials. Previous studies have documented down-regulation of Bcl-2 in SFN-treated cells (16Fimognari C. Nüsse M. Cesari R. Iori R. Cantelli-Forti G. Hrelia P. Carcinogenesis. 2002; 23: 581-586Crossref PubMed Scopus (214) Google Scholar, 18Wang L. Liu D. Ahmed T. Chung F.L. Conaway C. Chiao J.W. Int. J. Oncol. 2004; 24: 187-192PubMed Google Scholar, 19Jackson S.J.T. Singletary K.W. Carcinogenesis. 2004; 25: 219-227Crossref PubMed Scopus (176) Google Scholar, 20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar), yet studies are lacking that could directly test the role of this anti-apoptotic protein in cell death caused by SFN. Similarly, activation of caspases on treatment with SFN has been reported (20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar), but the mechanism of caspase activation remains elusive. Using PC-3 and DU145 human prostate cancer cells as a model, we demonstrate the following: (a) cell death caused by SFN is initiated by reactive oxygen species (ROS); (b) the SFN-induced apoptosis is significantly attenuated by ectopic expression of Bcl-xL but not Bcl-2; and (c) both intrinsic and extrinsic caspase cascades contribute to the cell death caused by SFN. Reagents—SFN (purity >99%) was purchased from LKT Laboratories (St. Paul, MN). Reagents for cell culture including F-12K Nutrient Mixture, DMEM, penicillin, streptomycin antibiotic mixture, and serum were purchased from Invitrogen. The kits for determination of catalase (catalog number 707002) and superoxide dismutase (catalog number 706002) activities and glutathione (GSH) levels (catalog number 703002) were purchased from Cayman Chemical (Ann Arbor, MI). Propidium iodide, NAC, RNaseA, 4′,6-diamidino-2-phenylindole (DAPI), and rotenone were from Sigma; hydroethidine (HE), 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and tetramethylrhodamine methyl ester (TMRM) were from Molecular Probes (Eugene, OR); and diphenyleneiodonium chloride (DPI) was from Calbiochem. The combined SOD/catalase mimetic EUK-134 was a generous gift from Eukarion, Inc. (Bedford, MA). The antibodies against Cu,Zn-superoxide dismutase (Cu,Zn-SOD; catalog number 574597), Mn-SOD (catalog number 574596) and catalase (catalog number 219010) were from Calbiochem; antibodies against cytochrome c (catalog number 556432), caspase-8 (catalog number 551242), caspase-9 (catalog number 552036), and Bcl-xL (catalog number 556361) were from Pharmingen (Palo Alto, CA); antibodies against Bid (catalog number sc-6538), Bok (catalog number sc-11424), Bim EL (catalog number sc-11425), and FADD (catalog number sc-5559) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Bcl-2 antibody (catalog number M0887) was from DAKO (Carpinteria, CA); anti-Fas antibody (catalog number AAP-221) was from Stressgen Biotechnologies (Victoria, Canada); anti-cytochrome c oxidase (complex IV) antibody (Cox IV; catalog numberA21348) was from Molecular Probes (Eugene, OR); antibody against poly(ADP-ribose) polymerase (PARP; catalog number SA-250) was from Biomol (Plymouth Meeting, PA); and anti-actin antibody was from Oncogene Research Products (San Diego, CA). Cell Lines and Cell Culture—Monolayer cultures of PC-3 cells were maintained in F-12K Nutrient Mixture (Kaighn's modification) supplemented with 7% nonheat-inactivated fetal bovine serum and antibiotics. PC-3 cells stably transfected with Bcl-2 (PC-3/Bcl-2) and vector-transfected control cells (PC-3/neo) were generously provided by Dr. Natasha Kyprianou (University of Maryland School of Medicine, Baltimore, MD) and cultured similarly except in the presence of 500 μg of G418/ml (Invitrogen). The DU145 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, and antibiotics. Primary mouse embryonic fibroblasts (MEFs) derived from wild-type and Bid knock-out (Bid–/–) mice and immortalized by transfection with a plasmid containing SV40 genomic DNA were generously provided by Dr. Stanley J. Korsmeyer (Dana-Farber Cancer Institute, Boston) (23Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3354) Google Scholar). The MEFs were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mm nonessential amino acids, 0.1 μm 2-mercaptoethanol, and antibiotics. Each cell line was maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The effect of SFN on cell survival was determined by trypan blue dye exclusion assay as described by us previously (20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar, 24Xiao D. Choi S. Johnson D.E. Vogel V.G. Johnson C.S. Trump D.L. Lee Y.J. Singh S.V. Oncogene. 2004; 23: 5594-5606Crossref PubMed Scopus (238) Google Scholar). Measurement of ROS—Intracellular ROS generation was measured by flow cytometry following staining with HE and H2DCFDA, which have been shown to be somewhat specific for detection of O2−˙ and H2O2, respectively (25Rothe G. Valet G.J. J. Leukocyte Biol. 1990; 47: 440-448Crossref PubMed Scopus (781) Google Scholar, 26Narayanan P.K. Goodwin E.H. Lehnert B.E. Cancer Res. 1997; 57: 3963-3971PubMed Google Scholar). The HE is oxidized to ethidium bromide, whereas H2DCFDA is cleaved by nonspecific cellular esterases and oxidized in the presence of H2O2 and peroxidases to yield fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, 5 × 105 cells were plated in 60-mm dishes, allowed to attach overnight, and exposed to different concentrations of SFN for specified time intervals. Stock solution of SFN was prepared in dimethyl sulfoxide (Me2SO), and an equal volume of Me2SO was added to the controls. The cells were counterstained with 2 μm HE and 5 μm H2DCFDA for 30 min at 37 °C. The cells were collected, and the fluorescence was analyzed using a Coulter Epics XL Flow Cytometer. In some experiments, cells were pretreated with NAC, DPI, or rotenone prior to SFN exposure and analysis of ROS generation. Immunoblotting—The cells were treated with SFN as described above, and both floating and attached cells were collected and lysed as described by us previously (20Singh A.V. Xiao D. Lew K.L. Dhir R. Singh S.V. Carcinogenesis. 2004; 25: 83-90Crossref PubMed Scopus (308) Google Scholar, 21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Cytosolic and mitochondrial fractions from control and SFN-treated cells were prepared using a kit from BioVision, Inc., Mountain View, CA (catalog number K256-100). The lysate proteins were resolved by 6–12.5% SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membrane was incubated with a solution containing Tris-buffered saline, 0.05% Tween 20, and 5–10% (w/v) nonfat dry milk and then exposed to the desired primary antibody for 1 h at room temperature. Following treatment with the appropriate secondary antibody, the bands were visualized using enhanced chemiluminescence method. The blots were stripped and reprobed with anti-actin antibody to correct for differences in protein loading. The change in protein level was determined by densitometric scanning of the immunoreactive bands followed by correction for actin loading control. The immunoblotting for each protein was performed at least twice using independently prepared lysates, and the results were similar. SOD and Catalase Activity Determination—PC-3 cells (5 × 105) were plated in T25 flasks, allowed to attach overnight, and exposed to Me2SO (control) or SFN (20 or 40 μm) for 6 h at 37 °C. The cells were collected, washed with PBS, and pelleted by centrifugation at 500 × g for 6 min. The cell pellet was suspended in ice-cold 50 mm potassium phosphate (pH 7) containing 1 mm EDTA, sonicated, and centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant fraction was used for determination of catalase or SOD activity using kits from Cayman Chemical according to the manufacturer's instructions. Glutathione Assay—The effect of SFN treatment on the intracellular level of GSH was determined by using a kit from Cayman Chemical. Briefly, PC-3 cells (1 × 106) were plated in T25 flasks, and allowed to attach overnight. The cells were exposed to Me2SO (control) or 40 μm SFN for the specified time period at 37 °C. The cells were collected by scraping, washed with PBS, resuspended in 0.5 ml of PBS, and counted. An equal number of cells from each treatment group was used for the determination of GSH according to the manufacturer's instructions. Measurement of Mitochondrial Membrane Potential—Mitochondrial membrane potential was measured using fluorescent lipophilic cationic dye TMRM, which accumulates within mitochondria in a potential-dependent manner. Briefly, cells (5 × 105) were plated in 60-mm culture dishes, allowed to attach overnight, exposed to desired concentrations of SFN for specified time period, and collected by trypsinization. The cells were then resuspended in growth medium and stained with 0.2 μm TMRM for 15 min at 37 °C in the dark. The cells were washed twice with ice-cold PBS, and the fluorescence was measured using a Coulter Epics XL flow cytometer. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 25 μm), an uncoupler of mitochondrial oxidative phosphorylation, was used as a positive control. Electron Microscopy—Transmission electron microscopy to determine the effect of SFN treatment on mitochondrial morphology was performed essentially as described previously (27Watkins S.C. Cullen M.J. J. Neurol. Sci. 1987; 82: 181-192Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, PC-3 cells (2 × 105) were plated in 6-well plates and allowed to attach overnight. The cells were then treated with either Me2SO (control) or 40 μm SFN for 6 h at 37 °C. The cells were fixed in ice-cold 2.5% electron microscopy grade glutaraldehyde (in 0.1 m PBS (pH 7.3)). The specimens were rinsed with PBS, post-fixed in 1% osmium tetroxide with 0.1% potassium ferricyanide, dehydrated through a graded series of ethanol (30–90%,) and embedded in Epon (dodecenyl succinic anhydride, nadic methyl anhydride, scipoxy 812 resin, and dimethylaminomethyl; Energy Beam Sciences). Semi-thin (300 nm) sections were cut using a Reichart Ultracut, stained with 0.5% toluidine blue, and examined under a light microscope. Ultrathin sections (65 nm) were stained with 2% uranyl acetate and Reynold's lead citrate and examined on a JEOL 1210 transmission electron microscope at ×50,000 magnification. Apoptosis Assays—Apoptosis induction by SFN was assessed as follows: (a) by fluorescence microscopic analysis of cells with condensed and fragmented DNA following staining with DAPI; (b) quantitation of cytoplasmic histone-associated DNA fragmentation; and (c) flow cytometric analysis of cells with sub-G0-G1 DNA content following staining with propidium iodide. For DAPI assay, cells (2 × 104) were plated on coverslips, allowed to attach overnight, and exposed to Me2SO or SFN. The cells were washed with PBS and fixed with 3% paraformaldehyde for 1 h at room temperature. The cells were washed three times with PBS, permeabilized with 1% Triton X-100 for 4 min, washed again, and stained by incubation with 1 μg/ml DAPI for 30 min. The cells with condensed and fragmented DNA (apoptotic cells) were scored using a Leica DC300F fluorescence microscope at ×40 magnification. Cytoplasmic histone-associated DNA fragmentation was determined using a kit from Roche Applied Science according to the manufacturer's recommendations. The sub-G0-G1 fraction was quantified by flow cytometry as described previously (21Singh S.V. Herman-Antosiewicz A. Singh A.V. Lew K.L. Srivastava S.K. Kamath R. Brown K.D. Zhang L. Baskaran R. J. Biol. Chem. 2004; 279: 25813-25822Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Catalase Overexpression—Adenoviral constructs for enhanced green fluorescence protein (Ad-EGFP) or catalase (Ad-catalase) have been described previously (28Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The DU145 cells (2 × 105) were plated in 6-well plates, allowed to attach, infected with Ad-EGFP (5 multiplicity of infections) or Ad-catalase (20 multiplicity of infections), and treated with Me2SO or 40 μm SFN for the specified time points. The cells were then processed for analysis of ROS generation, immunoblotting, or apoptosis as described above. Transfection of PC-3 Cells with Bcl-xL—PC-3 cells were transfected with pSFFV-Bcl-xL and pSFFV-neo plasmids (a generous gift from Dr. Stanley J. Korsmeyer) using Lipofectamine 2000 (Invitrogen). Transfected cells were grown in media containing 800 μg of G418/ml for 3 weeks. Several G418-resistant clones were expanded and screened for the Bcl-xL protein levels by immunoblotting. The clone with the highest expression (clone 18) was selected for functional studies and maintained in the presence of 500 μg of G418/ml. Determination of Caspase Activity—Caspase-8 and caspase-9 activities were determined by using a kit from Clontech and R & D Systems (Minneapolis, MN), respectively, according to the instructions provided by the manufacturer. Briefly, 2 × 106 cells were plated and allowed to attach overnight. The medium was replaced with fresh completed medium containing 40 μm SFN. After incubation for the specified time point at 37 °C, cells were trypsinized, washed with PBS, and lysed using 50 μl of manufacturer supplied lysis buffer. The reaction mixture contained 50 μl of cell lysate, 50 μl of reaction buffer, 5 μl of caspase-8- or caspase-9-specific peptide substrate conjugated to p-nitroanilide. The reaction mixture was incubated at 37 °C for 3 h. Absorbance at 405 nm, due to release of p-nitroanilide from the peptide substrate, was measured using a Labsystem Multiskan Plus plate reader. In some experiments, the cells were pretreated with specified concentration of NAC or EUK-134 prior to addition of SFN and determination of caspase activity. Immunohistochemistry for Cytochrome c Localization—The MEFs derived from wild-type and Bid–/– mice were cultured on coverslips and treated with 30 μm SFN or Me2SO (control) for 8 h. The MEFs were then washed with PBS and stained for 1 h at 37 °C with 100 nm of the mitochondria-specific dye MitoTracker Red (Molecular Probes, Eugene, OR; catalog number M7513). After washing with PBS, MEFs were fixed with 2% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS and BSA buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.15% glycine), the MEFs were incubated with normal goat serum (1:20 dilution with BSA buffer, Sigma; catalog

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