Silibinin i activates p53-caspase-2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis

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1 Silibinin i 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 Authors : Alpna Tyagi, Rana P. Singh, Chapla Agarwal and Rajesh Agarwal From : Carcinogenesis, 2006

2 Introduction

3 Apoptosis or programmed cell death p p (. f g ) p Apoptosis *Apoptosis : (Gr. "falling") a process seen in multicellular organisms, by which specific cells are killed and removed for the benefit of the organism. Kerr, J.F.R., Wyllie, A.H. and Currie, A.R., 1972 *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. CA Cancer J Clin.,2005

4 The history of p53 *The p53 protein was first described in as a transformation-related protein and as a protein associating with the SV40 DNA tumour virus large T antigen. Lane and Crawford, 1979; Linzer and Levine, p53 Proto- Tumor suppressor Oncogene? gene? *Almost 10 years later, it was discovered that the oncogenic properties of p53 were in fact due to mutations in p53, and subsequent research with wild-type p53 clearly demonstrated that p53 was in fact a tumour suppressor gene. Baker et al., 1989; Vogelstein et al., 2000 *Following more than 20 years of extensive studies, we now know that p53 is a member of a family of proteins that has three members: p53, p63 and p73. Dobbelstein and Roth, 1998;

5 To die or not to die: how does p53 decide? DNA damage p53 Cell cycle arrest Cell cycle arrest The p53 modulates cellular functions such as gene transcription, DNA synthesis, DNA repair, cell cycle arrest, senescence and apoptosis. DNA repair Irreparable able damage survival Apoptosis elimination of damage and potentially cancerous cells Oncogene, 2004

6 How does p53 induce apoptosis? *Tumor suppressor p53gene product (p53) promotes apoptosis pt in response to death stimuli by transactivation of target genes or interaction with other proteins. Oncogene, 2004 *p53 can activate genes in the extrinsic and intrinsic pathways through transcription-dependent mechanism or induce apoptosis through transcription-independent mechanism. Biochem. Biophys. Res. Commun.,2005 Transactivate Pathway by which p53 induces apoptosis DNA binding Transrepress Mitochondria bind *In apoptotic pathway, p53 protein accumulation and stabilization via phosphorylation is linked to the increased permeability of mitochondrial membrane to release cytochrome C for caspase activation. J. Biol. Chem., 2005 Bax PUMA Noxa.. Bcl-2 MAP4 Bcl-2 Bcl-XL Oncogene, 2004

7 p53 and Cancer cell *However, neoplastic cells overcome to this mechanism and keep proliferating without any regulation, and therefore, present an excellent target for cancer control. *It has been observed that in all major types of tumors, p53 becomes nonfunctional by two common mechanisms, either via germline mutation or via frequent protein inactivation by interaction with oncoproteins or defective upstream signaling. EMBO J, 2005 *Activation of p53 in tumors harboring functional p53 gene has been proved to be an effective mechanism in rapid killing of the tumor cells. J. Biol. Chem., 2000

8 Activation i of caspases NH 2 Pro-domaine larger domain small domain Initiator caspases -caspase 2, 8, 9 and 10 NH 2 Asp X Asp X Low High Sequence homology among caspases COOH Effector caspases -caspase 3, 6 and 7 Asp X Asp X COOH Asp X Homodimer Activate form D'après Amarante-Mendes et Green, 1999

9 Apoptosis & caspases *In the classical apoptotic pathway, the loss of mitochondrial membrane potential releases cytochrome C in to the cytosol, which subsequently forms a complex with apaf-1 and procaspase- 9 for the activation of caspase-9. Oncogene, 2005 *Activated caspase-9 cleaves and activates downstream caspase-7, 6 and 3 for the apoptotic response. Cell,, 1997 *Some studies show that in response to DNA damage, activation of caspase- 2 is required before mitochondrial permeabilization and cytochrome C release for apoptosis. J. Biol. Chem., 2002; Science, 2002

10 Structure of caspase-2 *Caspase-2 is one of the best conserved caspases across species. 416 Caspase-9 *This enzyme is unique among caspases in that it has features of both initiator and effector caspases. Caspase Molecular Cell Biology, 2004 *Like other initiator caspases, pro-caspase-2 (51 kda) contains a long pro-domain domain, containing a caspase-recruitment domain (CARD). In addition, pro-caspase-2 contains two subunits, p19 and p12, which are important for processing and activation of this enzyme. *However the cleavage specificity of caspase-2 (VDVAD) is more closely related to the *However, the cleavage specificity of caspase 2 (VDVAD) is more closely related to the effector caspases, caspase-3 and -7 (DEVD). Biochem. Biophys. Res. Commun.,2005

11 Where is Caspase-2? *Subcellular fractionation studies have revealed that procaspase-2 resides in the Golgi complex, mitochondria, nucleus, and soluble cytoplasm. Biochem. Biophys. Res. Commun.,2005 Where is caspase-2? *However, using a set of antibodies raised to different epitopes on caspase-2 a recent investigation failed to provide evidence for its presence in mitochondria. Cell Death Differ., 2002 *Importantly, pro-caspase-2 is the only pro-caspase present constitutively in the nucleus. u J. Cell Biol., 2000

12 How is Caspase-2 Activation i? UV irradiation DNA damage TNF, TRAIL Cytokine Pathogens, virus Casp pase-2 p53, p73 RAIDD PACAP ARC. TRAF2 RIP1 Apoptosis NF- B activation p38 MAPK activation Consequences of caspase-2 interaction with different proteins. RAIDD, RIP-associated Ich-1/Ced-3-homologue protein with a death domain; DECAP, death effector filament- forming Ced-4 like apoptosis proteins; PACAP, pro-apoptotic caspase adaptor protein; ARC, apoptosis repressor with caspase recruitment domain, CARD; GMEB1, glucocorticoid modulatory element-binding protein 1; RAF2, TNFR-associated factor-2; RIP1, the serine-threonine kinase containing death domain; and NF- B, nuclear factor kappa B. Biochem. Biophys. Res. Commun.,2005

13 Apoptosis & caspase-2 *Caspase-2 cleaves proapoptotic protein Bid in cytoplasm, which translocates to mitochondria and facilitates cytochrome C. release. J. Biol. Chem., 2004 *In several experimental systems, a link has been shown between the p53 family proteins and caspase-2 activation leading to cell death. Biochem. Biophys. Res. Commun.,2005

14 Bladder cancer In USA *The significance of this study lies in the fact that cancer causes more than seven million deaths each year worldwide and bladder cancer commonly ranks fourth in men and eight in women in the USA. year worldwide and bladder cancer commonly ranks Tobacco Sex *USA alone has 63,210 new bladder cancer cases each year with 16,280 associated deaths. CA. Cancer J. Clin., 2005 Occupation Age Bladder cancer Race *Therefore, chemo-prevention/-intervention by anticancer natural agents could be a realistic approach to control bladder cancer. Infections

15 Silibinin i *Our earlier studies show that silibinin down-regulates survivin, activates caspase, and induces apoptosis in RT4 cells. Biochem. Biophys. Res. Commun., 2003 *We have also observed the apoptotic effect of silibinin in two different human bladder transitional-cell carcinoma cell lines, TCC-SUP and T24. Carcinogenesis,.,2004 H bl dd t iti l ll ill RT4 ll h f ti l 53 th f *Human bladder transitional-cell papilloma RT4 cells have 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.

16 Materials & Methods

17 Immunoblot analysis JNK1/2, Chk2 (Thr68), Bid, cleaved caspase 2, 3, 8 and 9 and p53 etc. JNK kinase activity assay pull down :JNK phosphorylated p at Ser63 and Ser73. substrate: c-jun RT4 cell Human bladder transitional-cell papilloma cell Quantitative apoptotic cell death assay Annexin V/PI Mitochondrial membrane potential JC-1 staining Analysis of cytochrome C release Cip1/p21 sirna transfection

18 Results & Discussions

19 Silibinin induces p53 and caspase activation *The phosphorylation of p53(ser15) promotes both the accumulation and functional activation of p53 in response to apoptotic stimuli or DNA damage. These results suggested the possible role of p53 and caspase cascade in silibinin-induced apoptosis of RT4 cells. 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 p and total levels of p53, cleaved-caspase 9, -3 and PARP using specific antibodies. Protein loading was checked by stripping and re-probing the membranes for β-actin.

20 Silibinin-induced induced apoptosis is mediated via caspase activation Fig. 2. Silibinin-induced apoptosis is mediated via caspase activation. RT4 cells were treated with either DMSO alone (control) or 150 µm 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 caspase-2, -8, -9, -3 and protein loading was checked by stripping and re-probing the same membranes for β-actin.

21 Silibinin-induced induced apoptosis is mediated via caspase activation These results suggested that silibinin-induced apoptosis in RT4 cells involves caspase-dependent mechanisms. Fig. 2. Silibinin-induced apoptosis is mediated via caspase activation. RT4 cells were treated with either DMSO alone (control) or 150 µm silibinin for 3-24 h as labeled in the figure. (B) Cells were pre-treated with the indicated doses of z-vad for 2 h and then treated with or without silibinin (150 µm) for 12 h. (C) in similar treatments with 100 µm z-vad, cells were harvested and processed for flow cytometric analysis of annexin V/PI-stained apoptotic p cells as described under methods.

22 Silibinin? pp53 Caspase-2 Caspase-8 *In some studies, it has been shown that casapse-2 is an initiator caspase for some types of DNA damaging agents, while in others caspase-8. (FEBS J., 2005) To examine the regulatory relationship between these initiator caspases, we verified the deficiency of caspase-2 and -8 activation by caspase inhibitor approach.

23 Role of silibinin-induced induced activation of caspase-2 and 8 in apoptosis caspase-2 inhibitor caspase-8 inhibitor Fig. 3. Role of silibinin-induced activation of caspase-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 fo r12 h.

24 Silibinin 12 pp53 Caspase-2 Caspase-8 10 tbid 8 tbid 6 These results suggested that (a) caspase-2 and -8 can activate each other in response to silibinin to initiate the activation 2 of caspase cascade and (b) caspase-2 can also cleave Bid independent of caspase-8 activation in RT4 cells. 0 4 Silibinine Z-VDVAD-FMK Z-LETD-FMK

25 Role of silibinin-induced induced activation of caspase-2 and 8 in apoptosis ~80% ~75% A slightly more reversal in apoptosis by caspase-2 inhibitor as compared to caspase-8 inhibitor, suggest that t caspase-2-mediated d apoptosis may also involve caspase-8 independent pathway. Fig. 3. Role of silibinin-induced activation of caspase-2 and -8 in apoptosis. (C & D) Cells were harvested after similar treatments and processed for flow cytometric analysis of annexin V/PI-stained apoptotic cells as described under methods.

26 Silibinini??? pp53(ser15) Caspase-2 Caspase-8 tbid

27 DNA damage signaling: the ATM ATR p53 CHK connection DSB:DNA double-strand break NBS1:nibrin ATM:ataxia telangiectasia mutated ATR:ataxia telangiectasia and Rad3 related proteins CHK:checkpoint kinases H2AX:histone 2AX MDM2:E3 ubiquitin ligase TRENDS in Molecular Medicine, 2006

28 Silibinin activates p53 via ATM- Chk2 pathway These results suggested a possible sequential activation of ATM-Chk2-p53 by Silibinin silibinin did in not the show induction any observable of early apoptosis effect on as other marked p53 by phosphorylation H2A.X(Ser139) phosphorylation. sites (data not shown). Fig. 4. Silibinin activates p53 and caspase cascade via ATM-Chk-2 pathway. RT4 cells were treated with either DMSO alone (control) or 150 µm 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). - sign in lanes 1, 4 and 7 represents control for 3, 12 and d24h h, respectively.

29 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68)??? pp53(ser15) Caspase-2 Caspases Caspase-8 tbid??? Apoptosis

30 Caffeine abrogates the effects of silibinin i on Chk2, p53, caspase and H2A.X 1. Interestingly, ATM(Ser1981) phosphorylation p was not inhibited when caffeine was used in combination with silibinin (data not shown). 2. There are some evidences where caffeine has been shown to directly inhibit Chk2 activation without having any effect on ATM and ATR activation. J. Biol. Chem., 2003 Fig. 4. Silibinin activates p53 and caspase cascade via ATM-Chk-2 pathway. (B) RT4 cells were pretreated with the caffeine for 2 h and then treated with or without silibinin (150 µm) 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 caspase-2, 9, 3 and Bid, and H2A.X (Ser139).

31 Caffeine abrogates the effects of silibinin i on Chk2, p53, caspase and H2A.X Overall, these results confirmed the role of ATM-Chk2 in p53 activation by silibinin as well as in causing caspase-mediated apoptosis p in RT4 cells. Fig. 4. Silibinin activates p53 and caspase cascade via ATM-Chk-2 pathway. (C) In similar treatment as in B, cells were analyzed for annexin V/PI-stained apoptotic cells as described under methods. Quantitative data are presented as mean ±S.E. of triplicate samples.

32 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspases Caspase-8? tbid

33 Role of p53 and caspase-2 activation in silibinin-induced i i d apoptosis pifithrin-α Fig. 5. Role of p53 and caspase-2 activation in silibinin-induced apoptosis. RT4 cells were pretreated with the pifithrin-α for 24 h and then added with fresh media containing pifithrin-α 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 caspase-8, -9, -3, Bid and PARP.

34 Role of p53 and caspase-2 activation in silibinin-induced i i d apoptosis These results suggest that (a) caspase-2 activation is down-stream of ATM activation and (b) p53 and caspase-2 can activate each other and therefore, for the first time, indicated the presence of a bidirectional regulatory Fig. 5. Role of p53 and caspase-2 activation in silibinin-induced apoptosis. (B) cleaved caspase-2 and protein loading was checked by stripping and re-probing the same membranes for β-actin in each case. (C) RT4 cells were pretreated mechanism/s with the z-vdvad for for their 2 h and activation. 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, patm(ser1981).

35 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspase-8 tbid Cytochrome c release Caspase-9 Apoptosis Caspase-3

36 Silibinin induces translocation of p53 and Bid protein in to mitochondria These results suggest that silibinin caused increased p53 and tbid translocation into mitochondria could have led to the enhanced cytochrome C release from mitochondria in to the cytosol. Fig. 6. Silibinin induces translocation of p53 and Bid protein in to mitochondria. RT4 cells were treated with either DMSO alone (control) or 150 µm 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 under methods.

37 P53 and caspase-2 activation are required before mitochondrial permeabilization and cytochrome C release (7, 16, 31, 32). Since we observed that p53 and caspase-2 have role in silibinin-induced apoptosis (Fig 3 and 4), our next question was whether caspase-2 and p53 are required for silibinin-induced apoptosis before mitochondrial damage which h were measured by cytochrome C release from mitochondria in to cytosol and mitochondrial membrane depolarization using pifithrin-α and z- VDVAD.fmk, the irreversible p53 and caspase-2 inhibitors, respectively.

38 p53 and caspase-2 activation mediates silibinin-induced mitochondrial membrane depolarization and apoptosis *The inhibitory effect of pifithrin-α was more than that of z-vdvad.fmk suggesting a possible caspase-2-independent effect of p53 on mitochondrial apoptosis Cy ytochrome c Silibinin VDVAD Pifithrin Fig. 6. Silibinin induces translocation of p53 and Bid protein in to mitochondria. (B) RT4 cells were pre-treated with pifithrin-α (24 h) or z-vdvad for 2 h and then treated with or without silibinin (150 µm) 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 reprobing the same membranes for tubulin.

39 p53 and caspase-2 activation mediates silibinin-induced mitochondrial membrane depolarization and apoptosis 1. Overall, these results suggested the role of p53 and caspase-2 in causing mitochondrial membrane disruption and cytochrome C release in to cytosol by silibinin. 2. This also, in part, involved a caspase-2-independent d effect of p53 on mitochondrial apoptosis. Fig. 6. Silibinin induces translocation of p53 and Bid protein in to mitochondria. (C) cells were harvested and stained with JC-1 dye, and percentage of cells positive for JC-1 monomers was analyzed by FACS analysis as described under methods.

40 12 10 ph2a.x(ser139) Silibinini patm(ser1981) pchk2(thr68) Cytochro ome c Silibinin VDVAD Pifithrin pp53(ser15) Caspase-2 Caspase-8??? tbid Cytochrome c release The JNK pathway is activated rapidly by distinct extra cellular stimuli, such as ultraviolet irradiation, oxidative stress, Apoptosis DNA damaging agents, inflammatory cytokines and growth factors. Oncogene,, 1999 Caspase-9 Caspase-3

41 Silibinin activates JNK, which mediates caspase-2 activation, cytochrome C release and apoptosis Fig. 7. Silibinin causes p-53-mediated activation of JNK for apoptosis. RT4 cells were treated with either DMSO alone (control) or 150 µm 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 & C) Cells were pre-treated with the SP for 24 h and then added with fresh media containing SP with or without silibinin for 12 h.

42 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspase-8? pjnk1/2 tbid Cytochrome c release In order to determine the relationship between JNK1/2 activation and p53 in this cell system, we next pre- treated cells with pifithrin-α which completely Apoptosis reversed silibinin-induced phosphorylation of JNK1/2. Caspase-9 Caspase-3

43 Silibinin activates JNK, which mediates caspase-2 activation, cytochrome C release and apoptosis These results provided the evidence that JNK1/2 is activated by p53 in response to silibinin treatment. Fig. 7. Silibinin causes p-53-mediated activation of JNK for apoptosis. (D & E) Cells were pre-treated with the g p p p ( ) p pifithrin-α for 24 h and then added with fresh media containing pifithrin-α 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 JNK1/2 by substrate phosphorylation of cjun as described under method.

44 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspase-8 pjnk1/2 tbid Cytochrome c release Next, we defined the role of JNK1/2 activation in cytochrome C release from mitochondria in to cytosol. Apoptosis Caspase-9 Caspase-3

45 Silibinin activates JNK, which mediates caspase-2 activation, cytochrome C release and apoptosis These data lend further support to the notion that p53-jnk1/2 are active upstream of cytochrome C; however, p53 can also partially activate caspases, independent of JNK1/2 pathway. Fig. 7. Silibinin causes p-53-mediated activation of JNK for apoptosis. (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 pre-treatments with pifithrin-α and SP followed by silibinin for 12 h and western blotting was carried out for cleaved caspase-3.

46 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 did apoptosis assay using p53 and pan-caspase inhibitors.

47 Silibinin activates JNK, which mediates caspase-2 activation, cytochrome C release and apoptosis Overall, these results suggested the major role of p53 in caspase-mediated apoptosis, and JNK1/2 activation as a connecting link for p53-mediated of activation of caspase- 2 and subsequent mitochondrial apoptosis. Fig. 7. Silibinin causes p-53-mediated activation of JNK for apoptosis. (H) Cells were pretreated with pifithrin-α and z-vad followed by silibinin and processed for flow cytometric analysis of annexin V/PI- stained apoptotic cells as described under methods.

48 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspase-8 pjnk1/2 tbid Cytochrome c release Apoptosis Caspase-9 Caspase-3

49 p21 as a modulator of multiple biological functions p21 Biological Function Cell cycle / DNA synthesis Stress response / Apoptosis Stem Cell commitment t Differentation *By far the most widely studied of these is p21waf1/cip1, a potent inhibitor of a wide array of CDK cyclin complexes and which is responsible for G1/S arrest induced d by p53 in response to DNA damage. Cancer Surveys: Checkpoint Controls and Cancer, 1997 *Recently, it has been observed that caspase mediated cleavage of Cip1/p21 is an important event in apoptosis. Oncogene, 1999 Biochimica et Biophysica Acta, 2000

50 *Recently, the cleavage of Cip1/p21 during apoptosis has been reported in response to various stimuli. Carcinogenesis, 2000 *For example, TNF-α-induced apoptosis of human cervical carcinoma cells, growth factor-deprived ed human endothelial cells, butyrate-induced apoptosis s in colorectal cancer cells, and DNA damage by γ-irradiation as well as other DNA-damaging agents are shown to involve Cip1/p21 cleavage. Oncogene, 1999; Carcinogenesis, 2000; Mol. Cell,1998 Therefore, Cip1/p21 cleavage could possibly be a critical event in driving arrested cells for their apoptotic death. *However, it is still not clear how Cip1/p21 induction causes both G1 arrest but then apoptosis as a function of its caspase-dependent cleavage. 1. It is more likely that the cells have to release form the cell cycle arrest in order to turn on the apoptotic mechanisms. 2. 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. Carcinogenesis, 2000

51 Silibinin causes caspasedependent cleavage of Cip1/p21 These results suggest that caspase-2 activated both extrinsic and intrinsic caspase pathways to cleave Cip1/p21 in response to silibinin. Fig. 8. Silibinin causes caspase-dependent cleavage of Cip1/p21. RT4 cells were treated with either DMSO alone (control) or 150 µm 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 pre-treated 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.

52 Silibinin causes caspasedependent cleavage of Cip1/p21 These results suggest that caspase-mediated cleavage of Cip1/p21 plays critical role in driving cells for apoptotic death by silibinin. Fig. 8. Silibinin causes caspase-dependent cleavage of Cip1/p21. (E) Cells were transfected with Cip1/p21siRNA and then treated with silibinin as described under methods. (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 under methods.

53 Conclusion

54 *The central findings in the present study are that silibinin activates p53 via ATM-Chk2 pathway and causes caspase-dependent apoptosis in human bladder transitional-cell ii ll 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 i apoptosis. pt *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 caspase-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.

55 Silibinini patm(ser1981) ph2a.x(ser139) pchk2(thr68) pp53(ser15) Caspase-2 Caspase-8 pjnk1/2 tbid Cytochrome c release Apoptosis Cleaved Cip/p21 Caspase-9 Caspase-3

56 Thanks for your attention!

57 pp53 site