ABSTRACTP73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73a overexpression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of DNp73a over-expression was variable. P73a downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73a steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73a downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73a in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca2þ]i) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca2þ-ATPase inhibitor, caused this response and calpain activation, p73a processing and apoptosis in both cell types. CDDP-induced [Ca2þ]i increase in OV2008 cells was not effected by the elimination of extracellular Ca2þ, but this was attenuated by the depletion of internal Ca2þ store, indicating that mobilization of intracellular Ca2þ] stores was potentially involved. These findings demonstrate that p73a and its regulation by the Ca2þ-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca2þ/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.
REFERENCESAbedini MR, Muller EJ, Bergeron R, Gray DA, Tsang BK. (2010). Akt promotes chemoresistance in human ovarian cancer cells by modulating cisplatin-induced, p53-dependent ubiquitination of FLICE-like inhibitory protein. Oncogene 29: 11–25.
Abedini MR, Muller EJ, Brun J, Bergeron R, Gray DA, Tsang BK. (2008). Cisplatin induces p53-dependent FLICE-like inhibitory protein ubiquitination in ovarian cancer cells. Cancer Res 68: 4511–4517.
Aqeilan RI, Pekarsky Y, Herrero JJ, Palamarchuk A, Letofsky J, Druck T et al. (2004). Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci USA 101: 4401–4406.
Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook T, Lu X. (2004). ASPP1 and ASPP2: common activators of p53 family members. Mol Cell Biol 24: 1341–1350.
Bernassola F, Karin M, Ciechanover A, Melino G. (2008). The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14: 10–21.
Bernassola F, Salomoni P, Oberst A, Di Como CJ, Pagano M, Melino G et al. (2004). Ubiquitin-dependent degradation of p73 is inhibited by PML. J Exp Med 199: 1545–1557.
Chen CL, Ip SM, Cheng D, Wong LC, Ngan HY. (2000). P73 gene expression in ovarian cancer tissues and cell lines. Clin Cancer Res 6: 3910–3915.
Di Sano F, Ferraro E, Tufi R, Achsel T, Piacentini M, Cecconi F. (2006). Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism. J Biol Chem 281: 2693–2700.
Eltabbakh GH, Awtrey CS. (2001). Current treatment for ovarian cancer. Expert Opin Pharmacother 2: 109–124.
Fontemaggi G, Kela I, Amariglio N, Rechavi G, Krishnamurthy J, Strano S et al. (2002). Identification of direct p73 target genes combining DNA microarray and chromatin immunoprecipitation analyses. J Biol Chem 277: 43359–43368.
Fraser M, Bai T, Tsang BK. (2008). Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function. Int J Cancer 122: 534–546.
Fraser M, Leung B, Jahani-Asl A, Yan X, Thompson WE, Tsang BK. (2003a). Chemoresistance in human ovarian cancer: the role of apoptotic regulators. Reprod Biol Endocrinol 1: 66.
Fraser M, Leung BM, Yan X, Dan HC, Cheng JQ, Tsang BK. (2003b). p53 is a determinant of X-linked inhibitor of apoptosis protein/Akt-mediated chemoresistance in human ovarian cancer cells. Cancer Res 63: 7081–7088.
Gao G, Dou QP. (2000). N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death. J Cell Biochem 80: 53–72.
Grob TJ, Novak U, Maisse C, Barcaroli D, Luthi AU, Pirnia F et al. (2001). Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53. Cell Death Differ 8: 1213–1223.
Gunes DA, Florea AM, Splettstoesser F, Busselberg D. (2009). Coapplication of arsenic trioxide (As(2)O(3)) and cisplatin (CDDP) on human SY-5Y neuroblastoma cells has differential effects on the intracellular calcium concentration ([Ca(2+)](i)) and cytotoxicity. Neurotoxicology 30: 194–202.
Holcakova J, Ceskova P, Hrstka R, Muller P, Dubska L, Coates PJ et al. (2008). The cell type-specific effect of TAp73 isoforms on the cell cycle and apoptosis. Cell Mol Biol Lett 13: 404–420.
Irwin MS. (2004). Family feud in chemosensitivity: p73 and mutant p53. Cell Cycle 3: 319–323.
Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC, Kaelin Jr WG. (2003). Chemosensitivity linked to p73 function. Cancer Cell 3: 403–410.
Kartasheva NN, Contente A, Lenz-Stoppler C, Roth J, Dobbelstein M. (2002). p53 induces the expression of its antagonist p73 Delta N, establishing an autoregulatory feedback loop. Oncogene 21: 4715–4727.
Kobayashi S, Yamashita K, Takeoka T, Ohtsuki T, Suzuki Y, Takahashi R et al. (2002). Calpain-mediated X-linked inhibitor of apoptosis degradation in neutrophil apoptosis and its impairment in chronic neutrophilic leukemia. J Biol Chem 277: 33968–33977.
Liu G, Nozell S, Xiao H, Chen X. (2004). DeltaNp73beta is active in transactivation and growth suppression. Mol Cell Biol 24: 487–501.
Marabese M, Marchini S, Sabatino MA, Polato F, Vikhanskaya F, Marrazzo E et al. (2005). Effects of inducible overexpression of DNp73alpha on cancer cell growth and response to treatment in vitro and in vivo. Cell Death Differ 12: 805–814.
Melino G, Bernassola F, Ranalli M, Yee K, Zong WX, Corazzari M et al. (2004). p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation. J Biol Chem 279: 8076–8083.
Melino G, De Laurenzi V, Vousden KH. (2002). p73: friend or foe in tumorigenesis. Nat Rev Cancer 2: 605–615.
Melino G, Lu X, Gasco M, Crook T, Knight RA. (2003). Functional regulation of p73 and p63: development and cancer. Trends Biochem Sci 28: 663–670.
Million K, Horvilleur E, Goldschneider D, Agina M, Raguenez G, Tournier F et al. (2006). Differential regulation of p73 variants in response to cisplatin treatment in SH-SY5Y neuroblastoma cells. Int J Oncol 29: 147–154.
Moncoq K, Trieber CA, Young HS. (2007). The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump. J Biol Chem 282: 9748–9757.
Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. (2007). Calcium and cancer: targeting Ca2+ transport. Nat Rev Cancer 7: 519–530.
Muller M, Schilling T, Sayan AE, Kairat A, Lorenz K, Schulze- Bergkamen H et al. (2005). TAp73/Delta Np73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma. Cell Death Differ 12: 1564–1577.
Muller M, Schleithoff ES, Stremmel W, Melino G, Krammer PH, Schilling T. (2006). One, two, three—p53, p63, p73 and chemosensitivity. Drug Resist Updat 9: 288–306.
Munarriz E, Bano D, Sayan AE, Rossi M, Melino G, Nicotera P. (2005). Calpain cleavage regulates the protein stability of p73. Biochem Biophys Res Commun 333: 954–960.
Muscolini M, Cianfrocca R, Sajeva A, Mozzetti S, Ferrandina G, Costanzo A et al. (2008). Trichostatin A up-regulates p73 and induces Bax-dependent apoptosis in cisplatin-resistant ovarian cancer cells. Mol Cancer Ther 7: 1410–1419.
Nyman U, Sobczak-Pluta A, Vlachos P, Perlmann T, Zhivotovsky B, Joseph B. (2005). Full-length p73alpha represses drug-induced apoptosis in small cell lung carcinoma cells. J Biol Chem 280: 34159–34169.
Ozaki T, Nakagawara A. (2005). p73, a sophisticated p53 family member in the cancer world. Cancer Sci 96: 729–737.
Perrin BJ, Huttenlocher A. (2002). Calpain. Int J Biochem Cell Biol 34: 722–725.
Pietsch EC, Sykes SM, McMahon SB, Murphy ME. (2008). The p53 family and programmed cell death. Oncogene 27: 6507–6521.
Ramadan S, Terrinoni A, Catani MV, Sayan AE, Knight RA, Mueller M et al. (2005). p73 induces apoptosis by different mechanisms. Biochem Biophys Res Commun 331: 713–717.
Righetti SC, Perego P, Carenini N, Zunino F. (2008). Cooperation between p53 and p73 in cisplatin-induced apoptosis in ovarian carcinoma cells. Cancer Lett 263: 140–144.
Rossi M, De Laurenzi V, Munarriz E, Green DR, Liu YC, Vousden KH et al. (2005). The ubiquitin-protein ligase Itch regulates p73 stability. EMBO J 24: 836–848.
Sabatino MA, Previdi S, Broggini M. (2007). In vivo evaluation of the role of DNp73alpha protein in regulating the p53-dependent apoptotic pathway after treatment with cytotoxic drugs. Int J Cancer 120: 506–513.
Sang M, Ando K, Okoshi R, Koida N, Li Y, Zhu Y et al. (2009). Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. Genes Cells 14: 775–788.
Sayan AE, Paradisi A, Vojtesek B, Knight RA, Melino G, Candi E. (2005). New antibodies recognizing p73: comparison with commercial antibodies. Biochem Biophys Res Commun 330: 186–193.
Shaw TJ, Senterman MK, Dawson K, Crane CA, Vanderhyden BC. (2004). Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer. Mol Ther 10: 1032–1042.
Splettstoesser F, Florea AM, Busselberg D. (2007). IP(3) receptor antagonist, 2-APB, attenuates cisplatin induced Ca2+-influx in HeLa-S3 cells and prevents activation of calpain and induction of apoptosis. Br J Pharmacol 151: 1176–1186.
Sullivan A, Lu X. (2007). ASPP: a new family of oncogenes and tumour suppressor genes. Br J Cancer 96: 196–200.
Toyota H, Yanase N, Yoshimoto T, MoriyamaM, Sudo T, Mizuguchi J. (2003). Calpain-induced Bax-cleavage product is a more potent inducer of apoptotic cell death than wild-type Bax. Cancer Lett 189: 221–230.
Vayssade M, Haddada H, Faridoni-Laurens L, Tourpin S, Valent A, Benard J et al. (2005). P73 functionally replaces p53 in Adriamycin-treated, p53-deficient breast cancer cells. Int J Cancer 116: 860–869.
Vikhanskaya F, Marchini S, Marabese M, Galliera E, Broggini M. (2001). P73a overexpression is associated with resistance to treatment with DNA-damaging agents in a human ovarian cancer cell line. Cancer Res 61: 935–938.
Yoshida K, Ozaki T, Furuya K, Nakanishi M, Kikuchi H, Yamamoto H et al. (2008). ATM-dependent nuclear accumulation of IKKalpha plays an important role in the regulation of p73-mediated apoptosis in response to cisplatin. Oncogene 27: 1183–1188.