Combination Of Rad001(Everolimus) And Propachlor Synergistically Induces Apoptosis Through Enhanced Autophagy In Prostate Cancer Cells

Purpose:The purpose of this study is to compare the important and relevant features of the two most commonly used prostate cancer cell lines, LNCaP and PC-3, with prostatic adenocarcinoma and SCNC. Through a high throughout screen of5,000widely used small molecules, we identified couples of compounds that can synergize with Rad001to induce cytotoxicity of prostate cancer cells and probed into the mechanism.Methods:Xenograft tumors of LNCaP and PC-3were prepared and their features compared with human prostatic adenocarcinoma and SCNC for the expression of key signaling molecules by immunohistochemistry and western blot analysis. The synergistic effect of combinational treatment of Rad001and Propachlor was determined with Chou TC model; FACS assay was also performed to qualify the apoptotic prostate cancer cells. The expression of certain key signaling molecules (LC-3, Beclin1, Atg5-Atg12, PARP) was detected by the western blot analysis. The autophagosomes were detected by the immunofluorescence and electronic microscopy. The NOD-SCID mice bearing PC-3xenograft, were administered Rad001, Propachlor, and combination. The tumor size and body weight were mearsured.Results:LNCaP cells express AR and PSA and their growth is inhibited by androgen withdrawal, similar to human prostatic adenocarcinoma. PC-3cells do not express AR and PSA and their proliferation is independent of androgen, similar to SCNC. Adenocarcinoma cells and LNCaP cells are negative for neuroendocrine markers and stem cell-associated marker CD44while SCNC and PC-3cells are positive. LNCaP cells have identical cytokeratin profiles to adenocarcinoma while PC-3cells have cytokeratin profiles similar to SCNC. The combination of Rad001and Propachlor can induce more cytotoxicity of prostate cancer PC-3or C4-2cells, a large amout of Combination Index (CI) less than0.9, suggesting the synergistic cytotoxicity. The combinational treatment can also induce more apoptotic cell death and autophagosomes. The combinational treatment resulted in upregulation of LC3-2, Beclin1, Atg5-Atgl2and Beclinl mRNA transcription activity. The proliferation of PC-3xenograft tumor was complete regression as administered the combination of Rad001and Propachlor in vivo.Conclusion:LNCaP cells share common features with adenocarcinoma while PC3cells are characteristic of SCNC. The Rad001and Propachlor can synergize with each other to cause the death of castration-resistance prostate cancer cells (PC-3and C4-2), providing a novel therapeutic possibility for this incurable disease. The induction of autophagy may be as a novel direction of prostate cancer therapy.