Effect of dual inhibition of apoptosis and autophagy in prostate cancer

Purpose: Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that Autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits Autophagy, will increase cytotoxicity of ABT-737.

Experimental design: Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of Autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential Autophagy gene Beclin1. ROS was measured by flow cytometry, and Mitophagy assessed by the mCherry-Parkin reporter.

Results: Induction of Autophagy by ABT-737 was a mechanism of resistance in prostate Cancer cell lines. Therapeutic inhibition of Autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity.

Conclusions: We demonstrated that Autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of Apoptosis and Autophagy in future clinical studies.