Alpna Tyagi 1, Rana P.Singh 1, Chapla Agarwal 1,2 and Rajesh Agarwal 1,2,

Carcinogenesis vol.27 no.11 pp.2269 2280, 2006 doi:10.1093/carcin/bgl098 Advance Access publication June 15, 2006 Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2 Alpna Tyagi 1, Rana P.Singh 1, Chapla Agarwal 1,2 and Rajesh Agarwal 1,2, 1 Department of Pharmaceutical Sciences, School of Pharmacy and 2 University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO, USA To whom correspondence should be addressed: R. Agarwal, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box C238, Denver, CO 80262, USA. Tel: +1 303 315 1381; Fax: +1 303 315 6281; Email: Rajesh.Agarwal@uchsc.edu Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-a, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 sirna. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspasemediated cleavage of Cip1/p21. Introduction Apoptosis or programmed cell death is a major mechanism to eliminate cancer cells. The understanding of apoptosis has provided the basis for novel therapies that can induce death in cancer cells or sensitize them to cytotoxic agents and radiation therapy (1). Tumor suppressor p53 gene product (p53) promotes apoptosis in response to death stimuli by transactivation of target genes or interaction with other proteins (2). p53 can activate genes in the extrinsic and intrinsic pathways through transcription-dependent mechanism or induce apoptosis through transcription-independent mechanism (3). Many p53 targets are part of basic apoptotic Abbreviations: ATM, ataxia telangiectasia-mutated; ATR, ATM-related Rad-3 kinase; Chk2, checkpoint kinase 2, PI, propidium iodide; JNK, c-jun NH 2 -terminal kinase; z-vdvad, caspase 2 specific inhibitor; z-letd, caspase 8 specific inhibitor; z-vad, pan-caspase inhibitor. machinery; however, they can also be activated independent of p53 (4). In apoptotic pathway, p53 protein accumulation and stabilization via phosphorylation are linked to the increased permeability of mitochondrial membrane to release cytochrome c for caspase activation (5). It has been observed that in all major types of tumors, p53 becomes non-functional by two common mechanisms, either via germline mutation or via frequent protein inactivation by interaction with oncoproteins or defective upstream signaling (6). Activation of p53 in tumors harboring the functional p53 gene has been proved to be an effective mechanism in rapid killing of the tumor cells (7). Human bladder transitional-cell papilloma RT4 cells have the functional p53 gene; therefore, we used this model to study the signaling regulating p53 activation as well as to define its role in apoptosis induction by silibinin, an anticancer agent, which is shown to induce apoptosis in this model (8). The ataxia telangiectasia-mutated (ATM) gene, which is homologous to the yeast checkpoint gene Mec1, plays a critical role in sensing DNA double-strand breaks (DSBs) in mammalian DNA (9). Upon activation, ATM phosphorylates a variant histone H2A.X in response to DNA DSBs (10). There are reports that ATM, ATR (ATM-related Rad-3 kinase) as well as cell cycle checkpoint kinase 2 (Chk2), which are activated by DNA damaging stimuli, can phosphorylate p53 for cell cycle arrest or apoptosis induction as an inherent response for cellular defense (11 13). However, neoplastic cells overcome to this mechanism and keep proliferating without any regulation and therefore present an excellent target for cancer control. In the classical apoptotic pathway, the loss of mitochondrial membrane potential releases cytochrome c into the cytosol, which subsequently forms a complex with apaf-1 and pro-caspase 9 for the activation of caspase 9 (14). Activated caspase 9 cleaves and activates downstream caspase 7, 6 and 3 for the apoptotic response (15). Some studies show that in response to DNA damage, activation of caspase 2 is required before mitochondrial permeabilization and cytochrome c release for apoptosis (16 18). Caspase 2 cleaves proapoptotic protein Bid in cytoplasm, which translocates to mitochondria and facilitates cytochrome c release (19). In the present study, we investigated these mechanisms in detail in response to silibinin for both extrinsic (cytoplasmic) and intrinsic (mitochondrial) pathways in RT4 cells. Recently, it has been observed that cyclin-dependent kinase inhibitor Cip1/p21 is an effecter molecule for caspases in apoptosis induction (20). For example, butyrate is shown to induce Cip1/p21 expression as well as its cleavage by caspase underlying the apoptotic response of this agent in colorectal cancer cells (21). Therefore, we also investigated whether silibinininduced Cip1/p21 protein expression is targeted by caspase activity for the apoptotic death of bladder cancer cells. The significance of the study lies in the fact that cancer causes more than 7 million deaths each year worldwide and # The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 2269

A.Tyagi et al. bladder cancer commonly ranks fourth in men and eight in women in the USA (22). USA alone has 63 210 new bladder cancer cases each year with 16 280 associated deaths (22). Therefore, chemoprevention/chemointervention by anticancer natural agents could be a realistic approach to control bladder cancer. This approach is further supported by the fact that high dietary intake of fruits and vegetables rich in flavonoids is consistently associated with a reduced risk of common human cancers, including bladder cancer (23). Our earlier studies show that silibinin downregulates survivin, activates caspase and induces apoptosis in RT4 cells (8). We have also observed the apoptotic effect of silibinin in two different human bladder transitional-cell carcinoma cell lines, TCC-SUP and T24 (24). In the present study we provide evidence for the silibinin-induced apoptotic mechanisms in RT4 cells. Materials and methods Cell lines and reagents Human bladder transitional-cell papilloma RT4 cell line was purchased from American Type Culture Collection (Manassas, VA) and cells were cultured in Dulbecco s modified Eagle s medium (DMEM; Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (FBS) under standard culture conditions (37 C, 95% humidified air and 5% CO 2 ). Silibinin was from Sigma-Aldrich chemical company (St Louis, MO) and its purity was confirmed by high performance liquid chromatography as 100% pure. The primary antibody for Anti-ATM (ps1981) was from Rockland Immunochemicals (Gilbertsville, PA) and for ATR was from Novus (Littleton, CO). Primary antibodies against phosphorylated and/or total c-jun NH 2 -terminal kinase 1/2 (JNK1/2); Chk2 (Thr68); Bid; cleaved caspases 2, 3, 8 and 9 and the p53 antibody kit; cleaved poly(adp-ribose) polymerase (PARP); peroxidase-conjugated secondary anti-rabbit antibody; and the JNK kinase assay kit were from Cell Signaling Technology (Beverly, MA). Antibodies for total Chk2 and p53 (DO1) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-gH2A.X (Ser139) and Cip1/p21 antibodies were from Upstate Biotechnologies (Lake Placid, NY). Cytochrome c antibody was from BD Pharmingen (San Diego, CA). Caffeine and antibody for b-actin were from Sigma. Secondary antimouse antibody and ECL detection system were from Amersham (Arlington Heights, IL). The Annexin V/propidium iodide (PI) kit and JC-1 dye were from Molecular Probes (Eugene, OR). Caspase inhibitors, Z-VAD.fmk, Z-Leu-Glu(OMe)-Thr-Asp(OMe)-fmk (Z-LETD-fmk) and Z-Val-Asp(OMe)- Val-Ala-Asp(OMe)-fmk (Z-VDVAD-fmk) were purchased from Enzyme Systems Products (Livermore, CA). p53 inhibitor (pifithrin-a) was from Alexis Biochemicals (San Diego, CA) and JNK specific inhibitor (SP600125) was from BioMol (Plymouth Meeting, PA). Cell culture and treatments RT4 cells were cultured in DMEM containing 10% FBS and 1% penicillin streptomycin under standard culture conditions. At 60% confluency, cultures were treated with desired doses of silibinin (100, 150 and 200 mm, final concentrations in medium) dissolved in dimethysulfoxide (DMSO) or DMSO alone for different time-points (24 48 h) or for early time kinetics (3 24 h) and for other studies we used 150 mm dose of silibinin. For lysate preparation, cells were treated with silibinin for desired time in serum containing medium. In caffeine and caspase inhibitor studies, as desired, cells were treated with caffeine (5 mm), z-vad.fmk (pan-caspase inhibitor), z-letd.fmk (caspase 8 inhibitor) and z-vdvad (caspase 2 inhibitor) (50 mm) 2 h prior to silibinin treatment (12 h). RT4 cells were pretreated with pifithrin-a (p53 inhibitor) or SP600125 (JNK inhibitor), each 50 mm for 24 h and the medium was replaced with fresh medium containing inhibitor with or without silibinin for 12 h. Cell lysates were prepared in nondenaturing lysis buffer [10 mm Tris HCl (ph 7.4), 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 0.3 mm phenyl methyl sulfonyl fluoride (PMSF), 0.2 mm sodium orthovanadate, 0.5% NP-40 and 5 U/ml aprotinin]. For total cell lysate preparation, the medium was aspirated and cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated in lysis buffer for 10 min on ice. Then cells were scraped off and kept on ice for 30 min, and finally cell lysates were cleared by centrifugation at 4 C for 30 min at 14 000 r.p.m. Protein concentration was determined by the Lowry method using the Bio-Rad DC protein assay kit (Bio-Rad laboratories, Hercules, CA). 2270 Immunoblot analysis Total cell lysates were denatured with 2 sample buffer (0.5 M Tris, ph 6.8, 10% SDS, 2.74 M glycerol, 1.28 M 2-mercaptoethanol and 0.4 mg/ml bromophenol blue). Samples were subjected to SDS PAGE on 6, 12 or 16% Tris glycine gels and separated proteins were transferred onto nitrocellulose membrane by western blotting. Membranes were blocked with blocking buffer for 1 h at room temperature and, as desired, probed with primary antibody for desired molecule over night at 4 C followed by peroxidaseconjugated appropriate secondary antibody for 1 h at room temperature. Finally, proteins were visualized by ECL detection and exposure to X-ray film. JNK kinase activity assay The JNK kinase activity was determined by using the non-radioactive assay kit following manufacturer s protocol. Briefly, 200 250 mg protein/cell lysate was incubated with N-terminal c-jun (1 89) fusion protein (contains high affinity binding site for JNK) bound to glutathione sepharose beads to selectively pull down JNK phosphorylated at Ser63 and Ser73. The beads were washed twice with lysis buffer and then with kinase buffer, and kinase reaction was carried out in the presence of cold ATP for 30 min at 37 C. The reaction was stopped by adding sample buffer and boiling the sample. Phosphorylation of c-jun is measured by immunoblotting using a phospho-cjun specific antibody and ECL detection. Quantitative apoptotic cell death assay To quantify silibinin-induced apoptotic death of RT4 cells, Annexin V and PI staining was performed, followed by flow cytometry. After desired treatments with different caspase inhibitors, pifithrin-a, JNK inhibitor or caffeine in combination with silibinin or silibinin alone as indicated in the figures, cells were collected by trypsinization, and cell pellets were washed twice with icecold PBS. The cells were then stained with Annexin V PI by using the Vybrant Apoptosis Assay Kit2 following the step-by-step protocol provided by the manufacture and then analyzed by flow cytometry. JC-1 staining for mitochondrial membrane potential Mitochondrial membrane potential was assessed by JC-1 staining (25). Briefly, cells were treated with caspase 2 inhibitor or pifithrin-a or silibinin or with the combination of inhibitor and silibinin. Cells were trypsinized and washed with PBS and then incubated with JC-1 (10 mg/ml in PBS) at 37 C for 10 min. Stained cells were washed twice with PBS, pelleted down in 0.5 ml of PBS followed by fluorescence-activated cell sorter (FACS) analysis, and mitochondrial function was assessed as JC-1 green (uncoupled mitochondria, detector FL-1) or red (intact mitochondria, detector FL-2) fluorescence. Valinomycin-treated cells (10 mg/ml) were used as positive control. Analysis of cytochrome c release Cytochrome c release from mitochondria into cytosol was measured in RT4 cells following desired treatments. Briefly, cells were washed thrice with icecold PBS and then incubated in permeabilization buffer (20 mm HEPES KOH, ph 7.4, 50 mm KCl, 210 mm mannitol, 70 mm sucrose, 5 mm MgCl 2, 1 mm DTT and 0.1 mm PMSF) containing complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) for 20 min on ice. Cells were homogenized using a glass Dounce and pestle for 40 45 strokes. After a short centrifugation at 1000 g for 5 min, the supernatants were again centrifuged at 14 000 g for 30 min, and the cytosolic supernatants and mitochondrial pellets were collected. The pellets were washed once with extraction buffer and then finally suspended in mitochondrial lysis buffer (150 mm NaCl, 50 mm Tris HCl, ph 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mm EGTA and protease inhibitor). Cytosolic and mitochondrial fractions were analyzed by immunoblotting. Cip1/p21 sirna transfection For transfection, cells were plated in a 6-well plate with complete medium. When cells were 50% confluent the old medium was replaced with fresh medium. To knock-down the expression of Cip1/p21, commercially available sirna for Cip1/p21 from Cell Signaling was used according to the manufacturer s instructions. Briefly, lipofectamine and serum-free medium was incubated for 30 min and sirna was then added in that mixture and incubated for 20 min at room temperature. The cells in each well were then transfected with the equal concentration (10 nm) of the sirna complex. After 24 h of transfection, the medium was replaced with fresh medium and cells were treated with 150 mm dose of silibinin for 24 h, and cells were harvested for immunoblot or apoptosis analysis as detailed above. Statistical analysis Statistical significance of differences between the control and treated samples was calculated by the Student s t-test (SigmaStat 2.03). P-values of <0.05

Silibinin activates p53-caspase 2 pathway for apoptosis were considered significant. The immunoblot and cell cycle analysis data are representative of at least 2 4 independent studies with reproducible results. Results Silibinin induces p53 and caspase activation First, we assessed the dose- and time-dependent effect of silibinin (100 200 mm for 24 and 48 h) on p53 serine 15 phosphorylation as well as total p53 protein levels in RT4 cells. Silibinin caused a prominent increase in both phosphorylation of p53(ser15) and its total protein level in a dose-dependent manner (Figure 1). In this experiment, silibinin also increased the cleaved levels of caspase 9 (37 kda), caspase 3 (19 and 17 kda) and PARP (89 kda) mostly in a dose-dependent manner (Figure 1). These results suggested the possible role of p53 and caspase cascade in silibinin-induced apoptosis of RT4 cells. Next, we selected 150 mm dose of silibinin to first define the role of caspase activation in silibinin-induced apoptosis. Silibinin-induced apoptosis is mediated via caspase activation Effect of silibinin on caspase activation was investigated on early treatment time-points (3 24 h), which showed the activation of caspase 9 and 3 as early as 6 h of treatment and reached to the optimum at 12 h of treatment (Figure 2A). Further, we assessed the activation status of caspases 2 and 8, which are activated via the extrinsic pathway. Silibinin showed a slight increase in cleaved levels of caspase 2 (13 kda) and 8 (18 kda) at 6 h, which became prominent at 12 h of silibinin treatment (Figure 2A). Since this extrinsic pathway is known to cleave proapoptotic protein Bid, we also probed the membrane for the truncated Bid protein (tbid, 15 kda) level, and observed an increasing trend in tbid level similar to as cleaved caspases 2 and 8 (Figure 2A). To confirm the role of caspase-cascade activation in silibinininduced apoptosis, we used 50 and 100 mm doses of a broad spectrum pan-caspase inhibitor (z-vad.fmk), the pretreatment of which (100 mm) almost completely reversed silibinin-caused cleavage of Bid, caspases 9 and 3, and PARP (Figure 2B). In similar treatment, we analyzed the apoptotic cell population by annexin V PI staining that clearly showed a significant (P < 0.001) increase in apoptotic cell death by silibinin, which was completely (P < 0.001) reversed by pretreatment with z-vad.fmk (Figure 2C). These results suggested that silibinin-induced apoptosis in RT4 cells involves caspase-dependent mechanisms. Role of silibinin-induced activation of caspases 2 and 8 in apoptosis In some studies, it has been shown that caspase 2 is an initiator caspase for some types of DNA damaging agents, while in others caspase 8 has been shown to be the initiator caspase (26). However, their role in silibinin-induced apoptosis has not been investigated. To examine the regulatory relationship between these initiator caspases, we verified the deficiency of caspases 2 and 8 activation by Fig. 1. Silibinin induces p53 and caspase activation in RT4 cells. Cells were treated with either DMSO alone (control) or varying concentrations of silibinin as labeled in the figure for 24 and 48 h. At the end of the treatments, total cell lysates were prepared and western blotting was carried out for Ser15 phosphorylated and total levels of p53, cleaved caspases 9, 3 and PARP using specific antibodies. Protein loading was checked by stripping and re-probing the membranes for b-actin. Fig. 2. Silibinin-induced apoptosis is mediated via caspase activation. RT4 cells were treated with either DMSO alone (control) or 150 mm silibinin for 3 24 h as labeled in the figure. (A) At the end of the treatments, total cell lysates were analyzed by western blotting for cleaved caspases 2, 8, 9, 3 and tbid and protein loading was checked by stripping and re-probing the same membranes for b-actin. (B) Cells were pretreated with the indicated doses of z-vad for 2 h and then treated with or without silibinin (150 mm) for 12 h. At the end of the treatment, cell lysates were analyzed by western blotting for cleaved levels of Bid and caspases 9 and 3, and protein loading was checked by stripping and re-probing the same membranes for b-actin; or (C) in similar treatments with 100 mm z-vad, cells were harvested and processed for flow cytometric analysis of annexin V PI-stained apoptotic cells as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Sb, 150 mm silibinin; z-vad, pan-caspase inhibitor. 2271

A.Tyagi et al. Fig. 3. Role of silibinin-induced activation of caspases 2 and 8 in apoptosis. RT4 cells were pretreated with z-vdvad (A) or z-letd (B) for 2 h and then treated with or without silibinin for 12 h. At the end of the treatments, total cell lysates were prepared and western blotting was carried out for (A) cleaved caspases 2, 8, 9, 3 and Bid; (B) cleaved caspases 8, 2, 9, 3 and Bid, protein loading was checked by stripping and re-probing the same membranes for b-actin in each case. (C and D) Cells were harvested after similar treatments and processed for flow cytometric analysis of annexin V PI-stained apoptotic cells as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Sb, 150 mm silibinin; z-vdvad, 50 mm caspase 2 specific inhibitor; z-letd, 50 mm caspase 8 specific inhibitor. caspase inhibitor approach. Caspases 2 and 8 were inactivated by pretreatment of RT4 cells with z-vdvad.fmk and z-letd.fmk, respectively. Results showed that z-vdvad. fmk inhibited silibinin-induced activation of caspase 8 and the level of tbid (Figure 3A). As expected, it also inhibited silibinin-induced caspases 9 and 3 activation (Figure 3A). In other study, z-letd.fmk inhibited silibinin-induced caspase 8 activation as well as caspase 2 but did not show any effect on the tbid level (Figure 3B). However, downstream, it showed considerable reversal in caspases 9 and 3 activation (Figure 3B). These results suggested that (i) caspases 2 and 8 can activate each other in response to silibinin to initiate the activation of caspase cascade and (ii) caspase 2 can also cleave Bid independent of caspase 8 activation in RT4 cells. To further establish the role of caspases 2 and 8 in silibinin-induced apoptosis in RT4 cells, in similar treatments as in Figure 3A and B, cells were analyzed for apoptotic cells by flow cytometry. Treatment of cells with the inhibitor alone did not show any significant change in the number of apoptotic cells (Figure 3C and D). Caspase 2 inhibitor, z-vavad.fmk, showed 80% (P < 0.001) reversal in silibinin-induced apoptotic cells (Figure 3C). Similarly, caspase 8 inhibitor, z-letd.fmk, showed 75% (P < 0.001) reversal in silibinin-induced apoptosis (Figure 3D). A slightly more reversal in apoptosis by caspase 2 inhibitor as compared with caspase 8 inhibitor suggests that caspase 2-mediated apoptosis may also involve caspase 8 independent pathway. 2272 Silibinin activates p53 via ATM-Chk2 pathway Since we observed p53 activation by silibinin, to explore its upstream molecular events first we assessed a time-dependent (3 24 h) effect of silibinin on ATM activation. Silibinin induced ATM(Ser1981) phosphorylation as early as 3 h of treatment, which sustained till 18 h of the treatment (Figure 4A), without showing any change in total protein levels of ATM and ATR (data not shown). Three controls for increasing time-points were included for the fact that stress signaling including ATM activation may occur as cells grow more densely and begin to undergo the oxidative stress of nutrient deprivation. Next we analyzed the phosphorylation of Chk2 and H2A.X, which are known to be phosphorylated by ATM (10,27). The increase in Chk2(Thr68) phosphorylation by silibinin was also observed at 3 h, but prominently at 6 h of the treatment, which remained sustained till 18 h of the treatment and did not show any change in total Chk2 protein level (Figure 4A). H2A.X(Ser139) phosphorylation by silibinin was detectable at 6 h of the treatment; however, it remained sustained even at 24 h of the treatment (Figure 4A). In the case of p53, silibinin resulted in an increase in phosphorylation (Ser15) and total protein levels as early as 3 h of the treatment (Figure 4A). Silibinin did not show any observable effect on other p53 phosphorylation sites (data not shown). These results suggested a possible sequential activation of ATM-Chk2-p53 by silibinin in the induction of early apoptosis as marked by H2A.X(Ser139) phosphorylation.

Silibinin activates p53-caspase 2 pathway for apoptosis Fig. 4. Silibinin activates p53 and caspase cascade via ATM-Chk-2 pathway. RT4 cells were treated with either DMSO alone (control) or 150 mm silibinin for 3 24 h (A) as labeled in the figure, and total cell lysates were prepared and western blotting was carried out for patm(ser1981), pchk2(thr68), total Chk2, pp53(ser15), total p53 and H2A.X(Ser139). Dash sign in lanes 1, 4 and 7 represents control for 3, 12 and 24 h, respectively. (B) RT4 cells were pretreated with the caffeine for 2 h and then treated with or without silibinin (150 mm) for 12 h. At the end of the treatment, cell lysates were analyzed by western blotting for pp53(ser15); total p53; pchk2(thr68); total Chk2; cleaved caspases 2, 9, 3 and Bid; and H2A.X (Ser139). Protein loading was checked by stripping and re-probing the same membranes for b-actin in each case. (C) In similar treatment as in B, cells were analyzed for annexin V PI-stained apoptotic cells as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Caffeine abrogates the effects of silibinin on Chk2, p53, caspase and H2A.X Caffeine is a known inhibitor of the protein kinase activity of ATM (28). To define the role of silibinin-induced ATM activation, RT4 cells were treated with caffeine and/or silibinin as shown in Figure 4B. Caffeine almost completely reversed silibinin-induced phosphorylation of Chk2(Thr68) and p53(ser15) (Figure 4B). On the other hand, it also inhibited silibinin-caused activation of caspases 2, 9 and 3 as well as the cleavage of Bid (Figure 4B). As expected, caffeine also reversed silibinin-caused phosphorylation of H2A.X(Ser139) (Figure 4B). Interestingly, ATM(Ser1981) phosphorylation was not inhibited when caffeine was used in combination with silibinin (data not shown). There are some evidences where caffeine has been shown to directly inhibit Chk2 activation without having any effect on ATM and ATR activation (29). We did the annexin PI staining in the presence of caffeine and/or silibinin to confirm the role of this mechanism in apoptotic cell death. Results showed a strong reversal of silibinin-caused apoptosis by caffeine pretreatment (Figure 4C). Overall, these results confirmed the role of ATM-Chk2 in p53 activation by silibinin as well as in causing caspase-mediated apoptosis in RT4 cells. Role of p53 and caspase 2 activation in silibinin-induced apoptosis First, we further established the role of silibinin-induced p53 activation in the cleavage of caspases 8, 9 and 3, Bid and PARP by using the p53 specific inhibitor, pifithrin-a, in RT4 cells. Pifithrin-a inhibited silibinin-caused p53(ser15) phosphorylation and reduced its total protein level (Figure 5A). Subsequently, it also inhibited silibinin-caused activation of caspases 8, 9 and 3 as well as the cleavage of Bid and PARP (Figure 5A). Further, pifithrin-a reversed silibinin-induced activation of the initiator caspase 2, suggesting the upstream role of p53 in caspase-mediated apoptosis (Figure 5B). However, in another experiment we observed that caspase 2 inhibitor reverses silibinin-induced p53(ser15) phosphorylation without having any observable effect on ATM(Ser1981) phosphorylation (Figure 5C). These results suggest that (i) caspase 2 activation is downstream of ATM activation and (ii) p53 and caspase 2 can activate each other and therefore, for the first time, indicated the presence of a bidirectional regulatory mechanism/s for their activation. Silibinin induces translocation of p53 and Bid protein into mitochondria Silibinin-induced Bid cleavage and activation of caspase 9 suggested the possible role of mitochondrial apoptosis. It has also been reported that induced activation and expression of p53 targeted to mitochondria results in apoptosis (7). Mitochondrial p53 localization is shown to be specific for p53-dependent apoptosis and it does not occur during p53- independent apoptosis or during p53-mediated cell cycle arrest (7). Bid exists in the cytosolic fraction of living cells as an inactive precursor that becomes activated upon cleavage. After cleavage, the COOH fragment of Bid (tbid) translocates into mitochondria, an event that is independent of its BH3 domain. The tbid by itself is sufficient to induce release of cytochrome c from mitochondria. To further investigate the role of these mechanisms in the apoptotic activity of silibinin, first we studied the time-dependent effect of silibinin on mitochondrial and cytosolic levels of phospho-p53(ser15), p53 and tbid. We observed an increased fraction of phosphoprotein as well as total p53 protein localization into mitochondria at 3 6 h of silibinin treatment as compared with the control (Figure 6A). Similar to p53 translocation to mitochondria, silibinin also caused tbid translocation from cytosol to mitochondria, which was prominently observable as early as 12 h of the treatment (Figure 6A), further suggesting an upstream role of p53-related events in the initiation of apoptosis. In mitochondrial apoptosis, cytochrome c release from mitochondria into cytosol is a critical event to activate caspase 9 and downstream caspases (30). Since silibinin 2273

A.Tyagi et al. Fig. 5. Role of p53 and caspase 2 activation in silibinin-induced apoptosis. RT4 cells were pretreated with the pifithrin-a for 24 h and then added with fresh media containing pifithrin-a with or without silibinin for 12 h. At the end of the treatments, total cell lysates were prepared and western blotting was carried out for (A) pp53(ser15); total p53; cleaved caspases 8, 9, 3 and Bid; and PARP; (B) cleaved caspase 2 and protein loading was checked by stripping and re-probing the same membranes for b-actin in each case. (C) RT4 cells were pretreated with the z-vdvad for 2 h and then with or without silibinin for 12 h. At the end of the treatments, total cell lysates were prepared and western blotting was carried out for pp53(ser 15), total p53 and patm(ser1981) and protein loading was checked by stripping and re-probing the same membranes for b-actin. z-vdvad, caspase 2 specific inhibitor; pifithrin-a, p53 specific inhibitor. induced the translocation of p53 as well as tbid into mitochondria, we also investigated its effect on cytochrome c levels. Results showed an increased level of cytosolic cytochrome c after silibinin treatment showing a slight effect at 6 h, which became optimum at 12 h of the treatment (Figure 6A). These results suggest that silibinin-caused increased p53 and tbid translocation into mitochondria could have led to the enhanced cytochrome c release from mitochondria into the cytosol. p53 and caspase 2 activation mediates silibinin-induced mitochondrial membrane depolarization and apoptosis p53 and caspase 2 activation are required before mitochondrial permeabilization and cytochrome c release (7,16,31,32). 2274 Fig. 6. Silibinin induces translocation of p53 and Bid protein into mitochondria. RT4 cells were treated with either DMSO alone (control) or 150 mm silibinin for 3 24 h (A) as labeled in the figure. At the end of the treatments, mitochondrial and cytosolic lysates were prepared as described in Materials and methods and analyzed by western blotting for pp53(ser15), total p53, cleaved Bid and cytochrome c, and protein loading as well as purity of both the extracts were checked by stripping and re-probing the same membranes for tubulin. (B) RT4 cells were pretreated with pifithrin-a (24 h) or z-vdvad for 2 h and then treated with or without silibinin (150 mm) for 12 h. At the end of the treatments, mitochondrial and cytosolic lysates were prepared and western blotting was carried out for cytochrome c and protein loading was checked by stripping and re-probing the same membranes for tubulin; and (C) cells were harvested and stained with JC-1 dye, and the percentage of cells positive for JC-1 monomers was analyzed by FACS analysis as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Sb, silibinin; z-vdvad, caspase 2 specific inhibitor; pifithrin-a, p53 specific inhibitor. Since we observed that p53 and caspase 2 have roles in silibinin-induced apoptosis, our next question was whether caspase 2 and p53 are required for silibinin-induced apoptosis before mitochondrial damage, which were measured by cytochrome c release from mitochondria into cytosol and mitochondrial membrane depolarization using pifithrin-a and z-vdvad.fmk, the irreversible p53 and caspase 2 inhibitors, respectively. Results showed that both pifithrin-a and z-vdvad.fmk inhibited silibinin-induced cytochrome c release into cytosol from mitochondria (Figure 6B). The inhibitory effect of pifithrin-a was more than that of z-vdvad.fmk, suggesting a possible caspase 2-independent effect of p53 on mitochondrial apoptosis. This could be, partly, due to the increased p53 translocation into

Silibinin activates p53-caspase 2 pathway for apoptosis mitochondria and thereby regulation of mitochondrial proteins modulating apoptosis. Further, by using a mitochondrial membrane potential sensitive JC-1 dye and flow cytometry, we observed a strong increase in mitochondrial membrane permeabilization by silibinin, which was reversed by pretreatment with p53 or caspase 2 inhibitor (Figure 6C). Moreover, again we observed that p53 inhibitor showed complete reversal while caspase 2 inhibitor showed partial reversal of silibinin-caused mitochondrial membrane permeabilization, and therefore further suggested the caspase 2- independent effect of p53 on mitochondrial apoptosis. However, to further confirm this assumption, different doses of inhibitors and treatment times need to be employed in future studies. Overall, these results suggested the role of p53 and caspase 2 in causing mitochondrial membrane disruption and cytochrome c release into cytosol by silibinin. This also, in part, involved a caspase 2-independent effect of p53 on mitochondrial apoptosis. Silibinin activates JNK, which mediates caspase 2 activation, cytochrome c release and apoptosis Since we observed a partial caspase 2-independent effect of p53 on mitochondrial apoptosis, we also assessed whether JNK1/2 activation has any role in silibinin-induced mitochondrial apoptosis. In time kinetics study, silibinin caused a strong JNK1/2 phosphorylation from 6 up to 24 h of the treatment, which was correlated with the temporal activation of p53, except for the 3 h of the treatment where only p53 activation was observed (Figure 7A). To investigate the functional significance of this event, we pretreated cells with SP600125, a specific inhibitor of JNK, followed by the addition of silibinin for 12 h, and analyzed the cells for both JNK1/2 activation and annexin V-positive apoptotic cells. JNK inhibitor completely blocked silibinin-induced JNK1/2 phosphorylation (Figure 7B) and also significantly (P < 0.001) reversed silibinin-induced apoptosis (Figure 7C). In order to determine the relationship between JNK1/2 activation and p53 in this cell system, we next pretreated cells with pifithrin-a, which completely reversed silibinininduced phosphorylation of JNK1/2 (Figure 7D) as well as its kinase activity for GST-cJun (Figure 7E). These results provided the evidence that JNK1/2 is activated by p53 in response to silibinin treatment. Next, we defined the role of JNK1/2 activation in cytochrome c release from mitochondria into cytosol. Results showed that JNK inhibitor partially reversed silibinin-induced cytosolic translocation of cytochrome c from mitochondria (Figure 7F). Further, we observed that JNK inhibitor as well as p53 inhibitor reverses silibinin-induced caspase 3 activation; however, the reversal effect was more prominent with p53 inhibitor as compared with JNK inhibitor (Figure 7G). These data lend further support to the notion that p53 and JNK1/2 are active upstream of cytochrome c; however, p53 can also partially activate caspases, independent of JNK1/2 pathway. In order to further analyze whether p53 is the critical molecule in mediating silibinin-caused apoptosis, and also whether caspases are the sole mediator of the p53-induced apoptotic mechanisms, we performed the apoptosis assay using p53 and pan-caspase inhibitors. Results showed a strong reversal of silibinin-caused apoptosis by p53 inhibitor, while with pan-caspase inhibitor almost complete reversal in apoptosis was observed (Figure 7H). Combining these two inhibitors preceding silibinin treatment showed a complete reversal of silibinin-induced apoptosis (Figure 7H). Overall, these results suggested the major role of p53 in caspase-mediated apoptosis and JNK1/2 activation as a connecting link for p53-mediated activation of caspase 2 and subsequent mitochondrial apoptosis. Silibinin causes caspase-dependent cleavage of Cip1/p21 Recently, it has been observed that caspase-mediated cleavage of Cip1/p21 is an important event in apoptosis (20). Consistent with this report, in time kinetics study, silibinin treatment caused cleavage of Cip1/p21 protein showing a 15 kda band for cleaved Cip1/p21 as early as 6 h of treatment, which became optimum at 12 h of treatment (Figure 8A). Caspase 2 inhibitor almost completely suppressed silibinin-induced cleavage of Cip1/p21 (Figure 8B), while caspase 8 inhibitor could show only partial reversal (Figure 8C). Pan-caspase inhibitor completely blocked silibinin-caused cleavage of Cip1/p21 (Figure 8D). These results suggest that caspase 2 activated both extrinsic and intrinsic caspase pathways to cleave Cip1/p21 in response to silibinin. Further, to investigate the role of Cip1/p21 cleavage in silibinin-induced apoptosis, we used sirna strategy to knock-down Cip1/p21 expression. We transfected the cells with Cip1/p21 sirna (10 nm) to achieve a complete gene silencing of Cip1/p21 expression, which subsequently also eliminated the silibinin-induced cleavage of Cip1/p21 (Figure 8E). In similar treatments, apoptotic cells were measured by annexin V PI staining. Results showed that knocking down Cip1/p21 by sirna, and thereby its cleavage, almost completely (P < 0.001) reversed silibinininduced apoptosis (Figure 8F). These results suggest that caspase-mediated cleavage of Cip1/p21 plays a critical role in driving cells for apoptotic death by silibinin. Discussion The central findings in the present study are that silibinin activates p53 via ATM-Chk2 pathway and causes caspasedependent apoptosis in human bladder transitional-cell papilloma RT4 cells. p53-mediated apoptosis involved caspase 2 activated mechanisms, which was also regulated, in part, by p53-mediated activation of JNK1/2, leading to mitochondrial apoptosis. p53 and caspase 2 both activated each other, suggesting a regulatory loop for their activation. Activated caspase 2 cleaved caspase 8 and Bid, while activated caspase 8 could cleave only caspase 2 and had only partial effect on Bid cleavage, suggesting another bidirectional regulatory mechanism between caspases 2 and 8 and their different role in Bid activation. Further, we observed that caspase-cascade activation cleaved Cip1/p21 for the apoptotic response of silibinin. Overall, these findings elucidate many novel biochemical mechanisms for apoptosis induction by silibinin in RT4 cell line as depicted in Figure 9. The cellular responses to induce apoptosis are very complex involving many cellular factors that form an extensive signal transduction network. Several protein kinases including ATM in humans are increasingly recognized for their potential roles in the sensing of DNA damage (9) and initiating the activation of subsequent protein kinase cascade for cell cycle arrest, DNA repair and apoptosis. Furthermore, it has also been shown that in response to DNA damage, activation of caspase 2 is indeed required before mitochondrial permeabilization (31) and for apoptosis to take place 2275

A.Tyagi et al. Fig. 7. Silibinin causes p53-mediated activation of JNK for apoptosis. RT4 cells were treated with either DMSO alone (control) or 150 mm silibinin for 3 24 h (A) as labeled in the figure. At the end of the treatments, total cell lysates were prepared and analyzed by western blotting for pjnk1/2 and total JNK1/2. (B and C) Cells were pretreated with the SP600125 for 24 h and then added with fresh media containing SP600125 with or without silibinin for 12 h. At the end of the treatments, (B) total cell lysates were analyzed for pjnk1/2, total JNK1/2, and cleaved caspase 2 protein levels; or (C) cells were harvested and analyzed for annexin V PI-stained apoptotic cells as described under Materials and methods. (D and E) Cells were pretreated with the pifithrin-a for 24 h and then added with fresh media containing pifithrin a with or without silibinin for 12 h. At the end of the treatments, total cell lysates were analyzed (D) by western blotting for pjnk1/2 and total JNK1/2; or (E) for the kinase activity of the JNK1/2 by substrate phosphorylation of cjun as described in Materials and methods. (F) In the similar treatments as in B, mitochondrial and cytosolic lysates were prepared and western blotting was carried out for cytochrome c, and membrane was stripped and re-probed for tubulin. (G) Cell lysates were prepared after pretreatments with pifithrin-a and SP600125 followed by silibinin for 12 h and western blotting was carried out for cleaved caspase 3. As needed, protein loading was checked by stripping and re-probing the same membranes for b-actin in each case of western blot analysis. (H) Cells were pretreated with pifithrin-a and z-vad followed by silibinin and processed for flow cytometric analysis of annexin V PI-stained apoptotic cells as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Sb, 150 mm silibinin; z-vad, 100 mm pan-caspase inhibitor; pifithrin-a, 50 mm p53 specific inhibitor; SP600125, JNK1/2 specific inhibitor. (16 18,31). Accumulating evidence suggests that ATM functions upstream of the Chk2 and p53 tumor suppressor protein (33,34). Functional ATM is required for optimal Chk2 and p53 induction and activation following cellular exposure to agents that induce DNA DSBs (12). Although ATM may regulate p53(ser15) activity by multiple mechanisms (35), recent findings suggest a role of ATM in the DNA damage-induced phosphorylation of Chk2(Thr68) and p53(ser15) (11,12). Consistent with these reports, in the present study, strong DNA damaging stress of silibinin on bladder cancer cells would have caused phosphorylation of ATM, which in turn followed phosphorylation of 2276 Chk2(Thr68) and p53(ser15). gh2a.x(ser139) phosphorylation by ATM is regarded as an early marker for DNA damage and apoptosis induction. Consistent with its effect on ATM activation, silibinin also caused increased phosphorylation of gh2a.x(ser139). Additional studies employing caffeine (ATM/ATR inhibitor) with silibinin showed marked reversal of silibinin-induced phosphorylation of Chk2(Thr68), p53(ser15) and gh2a.x(ser139), which clearly suggest that activation of Chk2 and p53 happens in an ATM-dependent manner. The activation of ATM-Chk2 followed by H2A.X phosphorylation by silibinin as early as 3 h after the treatment indicates that silibinin might be

Silibinin activates p53-caspase 2 pathway for apoptosis Apoptotic/DNA Damaging Stimuli patm(ser1981) Enhancing effect of Silibinin ph2a.x(ser139) pchk2(thr68) pp53(ser15) pjnk1/2 Cleaved Caspase-2 tbid Cleaved Caspase-8 Cytochrome C Release Fig. 8. Silibinin causes caspase-dependent cleavage of Cip1/p21. RT4 cells were treated with either DMSO alone (control) or 150 mm silibinin for 3 24 h (A) as labeled in the figure. At the end of the treatments, total cell lysates were analyzed by western blotting for Cip1/p21. Cells were pretreated with (B) z-vdvad, (C) z-letd and (D) z-vad for 2 h and then treated with or without silibinin for 12 h. At the end of the treatments, total cell lysates were analyzed by western blotting for Cip1/p21. (E) Cells were transfected with Cip1/p21siRNA and then treated with silibinin as described in Materials and methods. At the end of the treatments, total cell lysates were analyzed by western blotting for Cip1/p21. (A E) In each case, protein loading was checked by stripping and re-probing the same membranes for b-actin. (F) Cells were harvested after similar treatments as in E and processed for flow cytometric analysis of annexin V PI-stained apoptotic cells as described in Materials and methods. Quantitative data are presented as mean ± SE of triplicate samples. Sb, 150 mm silibinin; z-vdvad, caspase 2 specific inhibitor; z-letd, caspase 8 specific inhibitor; z-vad, pan-caspase inhibitor. directly causing DNA damage to induce the signaling events as observed with many direct DNA damaging agents. On the other hand, it could also be possible that silibinin interaction with receptor itself would lead to the activation of ATM Cleaved Caspase-9 Cleaved Caspase-3 Cleaved Cip1/p21 Apoptosis Fig. 9. Schematic diagram showing silibinin-induced mechanisms of apoptosis. Silibinin induces p53 phosphorylation via ATM-Chk2 pathway. p53 activates caspase 2 via JNK1/2 activation leading to the activation of caspase cascade involving Bid cleavage and cytochrome c release from mitochondria. Caspase 2 also regulates the activation of p53 for apoptosis induction. Caspases 2 and 8, both function as initiator caspase with a relatively more involvement of caspase 2. Downstream caspase-mediated cleavage of Cip1/p21 is necessary for the silibinin-induced apoptosis in human bladder transitional-cell papilloma RT4 cells. signaling, driving the cell toward apoptosis. In this regard, more studies are needed to single out the initial molecular event targeted by silibinin. The phosphorylation of p53(ser15) promotes both the accumulation and functional activation of p53 in response to apoptotic stimuli or DNA damage. It has been shown that phospho-p53(ser15) can translocate to the mitochondria (intrinsic pathway) to induce apoptosis by mitochondrial pathway (7). Consistent with this report, in the present study, silibinin induced p53(ser15) translocation from cytosol into the mitochondria followed by permeabilization of mitochondrial membrane leading to cytochrome c release and caspase activation for apoptotic cell death. In these studies, by using p53 inhibitor (pifithrin-a), we also confirmed that mitochondrial membrane depolarization and cytochrome c release in cytosol accompanied by caspase activation were p53- dependent. Studies were also performed to define the regulatory role of p53 for caspase activation. Caspases are a group of cysteine proteases that cleave protein substrates after aspartic acids and play a central role in the regulation and execution of apoptosis (36). Caspase 2 is one of the best conserved caspase across species and is unique among caspases in that it has features of both initiator and effecter caspases (37). Caspase 2 appears to be necessary for the onset of apoptosis triggered by several stimuli, including DNA damage, tumor necrosis factor (TNF), and different pathogens and viruses (37). Several recent findings have suggested that caspase 2 triggers apoptosis via activation of the mitochondrial-mediated pathway (31). 2277

A.Tyagi et al. Caspase 2 activates caspase 8 and cleaves the Bcl-2 family member Bid, which generate a truncated Bid fragment that collaborate with Bax, another Bcl-2 relative, to promote the release of mitochondrial factors necessary for activation of executioner caspases and apoptosis (19). Ectopically expressed caspase 2 is shown to trigger Bid translocation to mitochondria and release cytochrome c (18). Recently, p53 is shown to activate caspase 2 for apoptosis (37); however, there is no report of p53 regulation by caspase 2. Consistent with the published report, silibinin showed p53-dependent caspase 2 activation accompanied by Bid cleavage and caspase activation. However, for the first time we also observed that p53 phosphorylation, and subsequent apoptosis, was regulated by caspase 2. How does caspase 2 regulate p53 phosphorylation and p53-mediated apoptosis remained to be studied. In case of caspase 2, using caffeine and pifithrin-a, we observed that silibinin-caused activation of p53 via ATM is necessary for the activation of caspase 2, and therefore indicated their upstream role in caspase 2 activation. Our studies also showed that caspase 2 inhibitor causes more reversal of silibinin-induced apoptotic cells as compared with caspase 8 inhibitor. Caspase 2 induced-apoptosis could be via caspase 8, Bid and caspase 3 (intrinsic pathway) or, in part, direct activation of caspase 3 (extrinsic pathway). All these molecules activated by silibinin were almost completely inhibited by caspase 2 inhibitor; however, caspase 8 inhibitor could show only partial reversal in Bid and caspase 3 cleavage, suggesting the predominant role of caspase 2 activation by silibinin. Further, caspase 8 inhibitor only partially reversed silibinin-induced caspase 2 activation suggesting that silibinin can activate caspase 2, in part, independent of caspase 8 activation. These results also indicated the presence of a bidirectional regulatory activation mechanism between caspase 2 and caspase 8 in RT4 cells. Interestingly, we also observed that caspase 2 inhibitor blocks silibinin-induced phosphorylation of H2A.X(Ser139) (data not shown), which is considered as an early event linked to apoptosis induction (10). Therefore, a possible regulatory effect of caspase 2 on phosphoinositide-3kinase (PI3K)-related kinase/s is also suspected. However, more studies are needed to explore such mechanism/s. The JNK pathway is activated rapidly by distinct extra cellular stimuli, such as ultraviolet irradiation, oxidative stress, DNA damaging agents, inflammatory cytokines and growth factors (38,39). JNK is activated more slowly by the initiation of the apoptotic cell death response by events such as ligation of the Fas protein (38,39). Activation of JNK has been shown to phosphorylate N-terminal domain of the transcription factor c-jun thereby inducing its transactivation potency and it has shown that c-jun activation leads to apoptosis (40). JNK is known to be involved in UV-triggered mitochondrial death pathways such as cytochrome c release and caspase activation (40,41). In this regard, we found that silibinin strongly increases JNK1/2 phosphorylation as well as the kinase activity against c-jun. In time kinetics study, JNK1/2 activation followed p53 phosphorylation. Accordingly, we anticipated that p53 activation would be feeding to JNK1/2 activation. To investigate such anticipation, we used p53 inhibitor and analyzed JNK1/2 activation in silibinin-treated cells. The p53 inhibitor pifithrin-a, a suppressor of p53 transactivation (42), completely blocked silibinin-induced JNK1/2 activation as well as mitochondrial apoptotic response. Further, we observed that JNK1/2 2278 inhibitor almost completely blocked caspase 2 activation and apoptotic responses. Comparing the silibinin-induced death in the presence or absence of JNK1/2 inhibitor revealed the involvement of JNK1/2-dependent pathway/s in silibinininduced apoptosis. Together, these results suggest that silibinin sequentially activates p53, JNK1/2 and caspase 2, and therefore provide an evidence for JNK1/2 as a connecting link between p53 and caspase 2 for apoptosis induction. Recently, the cleavage of Cip1/p21 during apoptosis has been reported in response to various stimuli ( 21). For example, TNF-a-induced apoptosis of human cervical carcinoma cells (43), growth factor-deprived human endothelial cells (44), butyrate-induced apoptosis in colorectal cancer cells (21), and DNA damage by g-irradiation as well as other DNA damaging agents (20,45) are shown to involve Cip1/p21 cleavage. However, it is still not clear how Cip1/p21 induction causes both G 1 arrest and apoptosis, but then the latter as a function of its caspase-dependent cleavage. It is more likely that the cells have to release from the cell cycle arrest in order to turn on the apoptotic mechanisms. It has been observed that the increase in the protein level of Cip1/p21 precedes the level of caspase activation, and later the appearance of its cleaved product correspond to apoptosis (21). Therefore, Cip1/p21 cleavage could possibly be a critical event in driving arrested cells for their apoptotic death. In the present study, silibinin caused a cleavage of Cip1/p21 producing a 15 kda fragment. Since Cip1/p21 has also been suggested as an apoptotic substrate, we investigated the role of caspase activation in silibinin-induced proteolytic cleavage of Cip1/p21. Our findings suggested that caspase 2 activation plays a major role as compared with caspase 8 activation to carry out this event. However, none of the cell permeable inhibitors of these caspases could completely block silibinin-induced Cip1/p21 cleavage. Then, we used pan-caspase inhibitor, z-vad.fmk, which completely blocked the silibinin-caused Cip1/p21 cleavage, suggesting indeed this event is mediated by the activation of caspase cascade by silibinin. To further confirm the role of silibinininduced Cip1/p21 cleavage in apoptosis, we used sirna knock-down strategy for Cip1/p21, which completely eliminated the 15 kda cleaved fragment in response to silibinin and also almost completely reversed silibinin-induced apoptosis. More likely, preventing the induction of Cip1/p21 by its proteolytic cleavage, the major mediator of p53-dependent cell cycle arrest, could be one of the mechanisms necessary to drive the cells for caspase-mediated apoptosis. Together, these data suggest the critical role of caspase-mediated Cip1/ p21 cleavage in silibinin-induced apoptosis of RT4 cells. Further, we would like to mention that for the relevance of these findings achieving pharmacological levels of silibinin in similar dose ranges as used in the present study are desirable without any toxicity. In this regard, our earlier studies with silibinin/silymarin feeding to mice show that silibinin is physiologically available without any apparent sign of toxicity. For example, our recent study with silibinin (2 g/kg dose) feeding for 16 days to SENCAR mice has resulted in up to 165 mm of silibinin in plasma without any toxicity, indicating the possibility of achieving even higher physiological levels of silibinin (46). Also, in an ongoing clinical trial with escalating doses of silibinin in prostate cancer patients, a peak level of >100 mm silibinin in plasma has been observed (47). In this trial, oral consumption of 13 g/day silibinin has shown even 150 mm plasma level of