The Role and Regulation of p73 in Cisplatin-Induced Apoptosis in Human Ovarian Cancer Cells in vitro

Abstract

Chemoresistance is a primary concern in chemotherapyand is an obstacle to successful treatment of human ovarian cancer (OVCA). P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in OVCA is poorly understood. Furthermore, how the deregulation of p73-mediated apoptosis confers resistance to CDDP is unclear. In the present studies, we found that TAp73alpha over-expression enhanced CDDP-induced PARP cleavage and apoptosis in the chemosensitive OVCA cells OV2008 and A2780s and their resistant counterpart (C13* and A2780cp) in addition to a chemoresistant (Hey cells; the effect of Deltap73alpha over-expression was to be found variable. P73alpha down-regulation attenuated CDDP-induced PUMA and NOXA up-regulation and apoptosis in OV2008 cell. CDDP decreased p73alpha steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced TAp73alpha and DeltaNp73alpha down-regulation in OV2008 was not affected by the presence of proteasome inhibitors (Epoximicin and Lactacystine), nor by the forced expression of Xiap, an anti-apoptotic protein. CDDP-induced, p73alpha down-regulation was calpain-dependent. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73alpha in OV2008, but not C13* cells. Calcium (Ca 2+ homeostasis plays an important role in apoptosis. We have evaluated the contribution of the Ca2+-mediated, calpain activation and its influence on TAp73alpha and DeltaNp73alpha contents in CDDP-induced apoptosis. We found that CDDP increased the intracellular calcium concentration ([Ca2+]i) in OV2008 but not C13* while cyclopiazonic acid (CPA), a Ca2+-ATPase inhibitor, caused this response and calpain activation, p73alpha processing and apoptosis in both cell types. CDDP-induced [Ca2+]i increase in OV2008 cells was not affected by the elimination of extracellular Ca2+, but stopped by the depletion of internal Ca2+ store, indicating that mobilization of intracellular Ca2+stores was potentially involved. These findings demonstrate for the first time that p73alpha and its regulation by the Ca2+-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that disregulation of Ca 2+/calpainlp73 signalling 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.