Figure 6

ARHI re-expression enhanced cisplatin-induced apoptosis and ARHI-mediated autophagy-associated cell death enhanced the cytotoxicity of cisplatin in cell culture. (a) Cisplatin-induced apoptotic cell death in ARHI-induced autophagic ovarian cancer cells could be partially blocked by Z-VAD in short-term cell cultures. SKOv3-ARHI cells were treated with DOX, chloroquine and cisplatin as described in Figure 5a. Z-VAD (40 μM) and DOX were added simultaneously. Cell viability was measured with SRB assays. Data were obtained from three independent experiments. The columns indicate the mean, and the bars indicate the S.E. (**P<0.01, DOX+ versus DOX−; *P<0.01 Cis versus no Cis). (b) Treatment of ARHI-induced autophagic ovarian cancer cells with chloroquine and cisplatin induced activated caspase-3 release and increased PARP cleavage. SKOv3-ARHI cells were pretreated with 5 μM chloroquine and 1 μg/ml DOX for 24 h. Cells were then treated with 5 μM chloroquine, 1 μg/ml DOX and 5 μM cisplatin for 48 h. Cell lysates were collected for western analysis. (c) Treatment of ARHI-induced autophagic ovarian cancer cells with chloroquine and cisplatin downregulated ERK and HER2 activity and the expression of XIAP and Bcl-2. Experiments were performed as described in (b). (d) ARHI-induced growth inhibition was blocked by shATG5 knockdown and partially blocked by Nec-1, but not by Z-VAD. ARHI-enhanced cell death by Cis was blocked by both Z-VAD and Nec-1 in shATG5 knockdown cells. SKOv3-ARHI-shcontrol and SKOv3-ARHI-shATG5 cells were treated with DOX and/or cisplatin as described in Figure 5a. Z-VAD (40 μM) and/or Nec-1 (40 μM) were added with DOX. The figure shows the combined values of three independent experiments. The columns indicate the mean, and the bars indicate the S.E. (*P<0.01, DOX+ versus DOX−; ^#P<0.01, Cis versus Cis+Z-VAD or Cis+Nec-1 or Cis+Z-VAD+Nec-1) and the numbers indicate the ratio of DOX− versus DOX+