Chloroquine reverses chemoresistance via upregulation of p21(WAF1/CIP1) and autophagy inhibition in ovarian cancer

Abstract

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of gamma H2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21(WAF1/CIP1) was also increased in CQ-CDDP-treated cells. Knockdown of p21(WAF1/CIP1) by shRNA reduced the expression of gamma H2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21(WAF1/CIP1) partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21(WAF1/CIP1) overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21(WAF1/CIP1) in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of gamma H2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21(WAF1/CIP1) expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of gamma H2AX and p21(WAF1/CIP1) in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21(WAF1/CIP1) expression and autophagy inhibition in CDDP-resistant EOC.