Role and Molecular Mechanism of Proteasome Inhibition in the Induction of Autophagy in Prostate Cancer Cells by Fangchinoline

Abstract

Purpose: Elucidation of the role and molecular mechanism of proteasome inhibition in the induction of autophagy in prostate cancer (PCa) cells by Fangchinoline. Methods: The role of Fangchinoline on the growth of PC-3 human prostate cancer cells was Explored using Methylthiazolyl Tetrazolium (MTT) assay. The role of Fangchinoline on the growth of PC-3 tumours was examined using the PC-3 Ho nude mouse model. The effect of Fangchinoline on c-Jun N-terminal kinase (JNK), Phosphorylated JNK (p-JNK), P38 and P-P38 protein expression in PC-3 tumor tissues was checked using Western Blot. The role of Fangchinoline on autophagy-related molecules Ki-67, LC3B and P62 was assessed using immunohistochemistry. The protein expression of Proteasome subunit beta type-6 (PSMB6), JNK, p-JNK, P38 and P-P38 in PC-3 cells after PSMB6-Small Interfering Ribonucleicacid (PSMB6-siRNA) was estimated using Western Blot. PSMB6, JNK, p-JNK, P38 and P-P38 Messenger RNA (mRNA) expression in PC-3 cells after Fangchinoline intervention was checked using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: MTT showed a dose- and time-dependent significant inhibition of PC-3 cell proliferation by Fangchinoline. WB showed that Fangchinoline significantly promoted the expression of JNK, p-JNK, P38, and P-P38 in PC-3 tumor tissues. Immunohistochemistry showed that Fangchinoline significantly promoted LC3B expression and significantly inhibited Ki-67 and P62 expression. WB showed that PSMB6-siRNA significantly inhibited the expression of PSMB6 in PC-3 cells and significantly promoted the expression of JNK, p-JNK, P38, and P-P38. Compared with the control group, RT-PCR results showed that PSMB6 mRNA expression was significantly down-regulated and JNK, p-JNK, P38, P-P38 mRNA expressions were up-regulated in a dose-dependent manner in the Fangchinoline group. Conclusions: Proteasome inhibition of Fangchinoline activated autophagy and apoptosis in PC-3 human prostate cancer cells, which may be related to the activation of protein expression of JNK and P38 signaling pathways.