Inhibitive Effects Of Fangchinoline On Proteasome Were Involved In Its Anti-cancer Effects

Purpose:To get better use of Fangji in clinic and development of new anti-cancer drugs from Fangji, we tried to clarify the molecular mechanisms of antitumor effects of fangchinoline.Method:The enzyme activities of proteasome of PC-3cells were checked using specific fluorescent peptide substrates. The binding affinities of fangchinoline to recombinant proteasome catalytic β1subunit were assayed in vitro by Biacore, and the catalytic activity of the recombinant human proteasome β1subunit was checked by specific fluorescent peptide substrate. Cell viability of PC-3cells with or without fangchinoline treatment was measured using MTT assay. Flow cytometric analysis of cell cycle and apoptosis after propidium iodide staining was conducted. Accumulation of ubiquitinated proteins including ubiquitinated IκBα, Bax and p27in vitro were examined using Western blotting. The effect of fangchinoline on tuomor growth, apoptosis, proteasome enzymatic activities, and accumulation of ubiquitinated proteins were checked in nude mice innoculated with PC-3tumor cells. Furthermore, the proteasome-inhibting effects of fangchinoline in tumor cells other than PC-3were also checked. The proteasome-inhibting effects of tetrandrine, a compound with structure similar to fangchinoline, were also observed.Result:Fangchinoline dose-dependently inhibited activities of cellular proteasome, and exhibited the strongest inhibitory effect on C-L activity mediated by proteasome β1subunit. Fangchinoline showed direct binding affinity with recombinant proteasome β1subunit (PSMB6) and also inhibited its enzyme activity in vitro. Fangchinoline induced cell cycle arrest at G0/G1phase and cell apoptosis in PC-3cells. Inhibition of the proteasome activity by fangchinoline in PC-3cells resulted in accumulation of ubiquitinated proteins including proteins related to cell cycle control and apoptosis such as IκBα, Bax and p27. Treatment of PC-3tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts and accumulation of proteasome substrates. Fangchinolin also exhibited proteasome-inhibiting effects in tumor cells such as A549and others. Tetrandrine also exhibited considerable proteasome-inhibiting effects. Conclusion:Fangchinoline was a new natural proteasome inhibitor targeting β1subunit and proteasome inhibition was involved in its anti-tumor effects.