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Effect of dual inhibition of apoptosis and autophagy in prostate cancer
Author(s) -
Saleem Ahamed,
Dvorzhinski Dmitri,
Santanam Urmila,
Mathew Robin,
Bray Kevin,
Stein Mark,
White Eileen,
DiPaola Robert S.
Publication year - 2012
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.22487
Subject(s) - autophagy , cytotoxicity , in vivo , lncap , apoptosis , cancer research , biology , cancer cell , microbiology and biotechnology , pharmacology , in vitro , cancer , biochemistry , genetics
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. Prostate 72:1374–1381, 2012. © 2012 Wiley Periodicals, Inc.