Regulation of apoptosis-inducing factor-mediated, cisplatin-induced apoptosis by Akt

British Journal of Cancer (2008) 98, 803 – 808 & 2008 Cancer Research UK All rights reserved 0007 – 0920/08 $30.00 www.bjcancer.com Regulation of apoptosis-inducing factor-mediated, cisplatin-induced apoptosis by Akt X Yang1,2,3,4, M Fraser1,2,3, MR Abedini1,2,3, T Bai1,2,3 and BK Tsang*,1,2,3 1 Cisplatin is a first-line chemotherapeutic for ovarian cancer, although chemoresistance limits treatment success. Apoptosis, an important determinant of cisplatin sensitivity, occurs via caspase-dependent and -independent mechanisms. Activation of the protein kinase Akt, commonly observed in ovarian tumours, confers resistance to ovarian cancer cells via inhibition of caspase-dependent apoptosis. However, the effect of Akt on cisplatin-induced, caspase-independent apoptosis remains unclear. We show that in chemosensitive ovarian cancer cells, cisplatin induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF), a mediator of caspase-independent apoptosis, and AIF-dependent apoptosis. Cisplatin failed to induce these effects in the chemoresistant variant cells. Overexpression of AIF sensitised resistant cells to cisplatin-induced apoptosis. Finally, activation of Akt attenuated the cisplatin-induced mitochondrial release and nuclear accumulation of AIF and apoptosis in chemosensitive cells, whereas inhibition of Akt activity facilitated these effects and sensitised chemoresistant cells to AIF-dependent, cisplatin-induced apoptosis. These results suggest that cisplatin-induced apoptosis proceeds, in part, via a caspase-independent mechanism involving AIF, and that Akt activation confers resistance to cisplatin-induced apoptosis by blocking this pathway. These results provide insights into the molecular mechanism of chemoresistance, and suggest that inhibition of Akt activity may represent a novel therapeutic approach to the treatment of cisplatin-resistant ovarian cancer. British Journal of Cancer (2008) 98, 803 – 808. doi:10.1038/sj.bjc.6604223 www.bjcancer.com Published online 19 February 2008 & 2008 Cancer Research UK Keywords: Akt; apoptosis; cisplatin; chemoresistance; AIF Chemoresistance is a significant barrier to the successful treatment of human ovarian cancer. While many factors affect the sensitivity of cancer cells to chemotherapy, the regulation of a number of key mediators of apoptosis is frequently altered in chemoresistant cells (Asselin et al, 2001; Fraser et al, 2003; Dan et al, 2004). Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is released from the mitochondria and translocates to the nucleus in response to specific death signals (Susin et al, 1999; Daugas et al, 2000). Nuclear AIF causes large-scale DNA fragmentation and chromatin condensation in a caspaseindependent manner (Cregan et al, 2002), and recent studies have shown that mitochondrial AIF release is regulated by p53 and the Bcl-2 family (Cregan et al, 2002; MacFarlane et al, 2002; Kandasamy et al, 2003; Majewski et al, 2004; Vyas et al, 2004). Akt (protein kinase B) mediates the cell survival action of growth factors and cytokines in a variety of cell types and blocks apoptosis induced by multiple apoptotic stimuli. It is a major downstream target of phosphatidylinositol 3-OH-kinase (PI3K) (Cheng et al, 2002; Franke et al, 2003; Fresno Vara et al, 2004). *Correspondence: Dr BK Tsang, Chronic Disease Program, Ottawa Health Research Institute, Civic Campus, 725 Parkdale Avenue, Ottawa, Ontario K1Y 4E9, Canada; E-mail: Revised 12 December 2007; accepted 7 January 2008; published online 19 February 2008 Both PI3K and Akt are frequently activated and/or overexpressed in human ovarian cancer (Fraser et al, 2003; Dan et al, 2004). Akt activation promotes cell survival, suppresses apoptotic death and regulates cis-platinum (II) diammine dichloride (CDDP) sensitivity in human ovarian cancer cells (Asselin et al, 2001; Fraser et al, 2003; Dan et al, 2004). We previously demonstrated that activated Akt is an important regulator of both X-linked inhibitor of apoptosis protein and p53 levels after cisplatin (CDDP) challenge and that p53 mutational status is a determinant of Akt-mediated chemoresistance (Fraser et al, 2003; Dan et al, 2004). Recent data from our laboratory have shown that p53 triggers the Akt-sensitive release of mitochondrial Smac into the cytosol and induces apoptosis via a caspasedependent pathway (Yang et al, 2006). However, other groups have reported that mitochondrial AIF translocation to the nucleus induces caspase-independent apoptosis (Cregan et al, 2002). Whether this process contributes to CDDP-induced apoptosis and is regulated by Akt remain unclear. In the current study, we demonstrate that CDDP-induced mitochondrial AIF translocation to nucleus is a determinant of chemosensitivity in ovarian cancer cells. Decreased mitochondrial AIF release is one mechanism by which Akt-mediated chemoresistance occurs. Our results suggest that modulation of these key cell fate regulators may be an effective means of overcoming chemoresistance in human ovarian cancer. Translational Therapeutics Reproductive Biology Unit and Division of Gynaecologic Oncology, Department of Obstetrics & Gynaecology, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6; 2Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; 3Chronic Disease Program, Ottawa Health Research Institute, Civic Campus, Ottawa, Ontario K1Y 4E9, Canada; 4Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China Regulation of mitochondrial AIF translocation by Akt X Yang et al 804 MATERIALS AND METHODS Western blot analyses Reagents Western blotting was performed as described previously (Fraser et al, 2003). Membranes were incubated overnight at 41C in primary antibodies (anti-AIF, 1 : 500; anti-actin, 1 : 2000; anti-Cox4, 1 : 1000; anti-LDH, 1 : 1000), followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1 : 2000) at room temperature for 1 h. Peroxidase activity was visualised with the enhanced chemiluminescence kit. Results were scanned and analysed using Scion Image software (Scion Inc.). Translational Therapeutics Cells were cultured at 371C with 5% CO2 in RPMI (Roswell Park Memorial Institute, Buffalo, NY, USA)-1640 (OV2008 and C13*) or DMEM (Dulbecco’s modified Eagle’s medium)/F-12 (A2780s, A2780cp; Invitrogen Inc., Burlington, ON, Canada). Medium was supplemented with 10% fetal bovine serum, streptomycin (100 mg ml
1), penicillin (100 U ml
1), and fungizone (0.625 mg ml
1). Digitonin, CDDP, dimethyl sulphoxide, and Hoechst 33258 were supplied by Sigma (Oakville, ON, Canada). Apoptosis-inducing factor small inhibitory RNA was from Santa Cruz Biotechnology (cat. no. sc-29193; Santa Cruz, CA, USA). Negative control siRNA was from Dharmacon Inc. (Chicago, IL, USA). Ribojuice siRNA transfection reagent was from Novagen (...truncated)