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

Abstract

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.

Figure 1

CDDP induced mitochondrial AIF translocation to nucleus and apoptosis in chemosensitive, but not resistant, ovarian cancer cells. Chemosensitive (OV2008, A2780s) and resistant (C13^*, A2780cp) cells were treated with different concentrations of CDDP (0, 2.5, 5, and 10 μM) for 24 h. CDDP decreased mitochondrial and increased nuclear AIF contents (A) as well as induced apoptosis (B) in a concentration-dependent manner in OV2008 and A2780s cells, but not C13^* and A2780cp cells (^**P<0.01, ^***P<0.001). Cox-4 blots indicate mitochondrial loading. C23 was used as a loading control for the nuclear fraction. The reciprocal blots demonstrate purity of the preparations. CDDP-induced apoptosis was assessed by Hoechst staining.

Figure 2

CDDP induced mitochondrial AIF translocation (A) and apoptosis (B) in a time-dependent manner in chemosensitive, but not resistant, ovarian cancer cells. OV2008 and C13^* cells were treated with 10 μM CDDP for different durations (0–24 h). CDDP induced decrease in mitochondrial and increase in nuclear AIF contents in OV2008 cells in a time-dependent manner, but not in C13^* cells (^*P<0.05, ^**P<0.01, ^***P<0.001).

Figure 3

Apoptosis-inducing factor is required for CDDP-induced apoptosis in ovarian cancer cells. (A) OV2008 cells were transfected with AIF siRNA (0–400 nM; 48 h) and then treated with CDDP (10 μM; 24 h). Downregulation of AIF was confirmed by western blot. Downregulation of AIF significantly attenuated CDDP-induced apoptosis in OV2008 cells (^**P<0.01). (B) OV2008 cells were tranfected with 200 nM AIF siRNA for 48 h, followed by CDDP treatment (10 μM; 24 h). Transfection of 200 nM AIF siRNA decreased AIF content in whole cell and mitochondria and decreased AIF translocation to nucleus. Downregulation of AIF decreased CDDP-induced apoptosis in OV2008 cells (^***P<0.001). (C) C13^* cells were infected with adenoviral AIF for 48 h, followed by CDDP treatment (10 μM; 24 h; ^***P<0.001).

Figure 4

Akt regulates CDDP-induced mitochondrial AIF translocation and apoptosis in ovarian cancer cells. (A) Activated Akt2 inhibited mitochondrial AIF translocation and CDDP-induced apoptosis. A2780s-PMH6 (control) and A2780s-AAkt2 (active Akt2) cells were treated with CDDP (0–10 μM; 24 h). Compared with A2780s-PMH6 cells, A2780s-AAkt2 cells showed a significant suppression of CDDP-induced AIF translocation. Constitutively activated Akt2 also reduced the sensitivity of A2780s cells to cisplatin-induced apoptosis (^*P<0.05, ^***P<0.001) expressing DN-Akt2 in response to CDDP. (B) Inhibition of Akt2 sensitised C13^* cells to CDDP (^*P<0.05). (C) C13^* cells were infected with DN-Akt (MOI=80; LacZ control) in the presence or absence of AIF siRNA (200 nM). DN-Akt sensitised the cells to CDDP-induced apoptosis, which was attenuated by downregulation of AIF (^***P<0.001).