Study Of The Mechanisms Of Cucurbitacin E-induced ER Stress

Objective: To mimic cytosolic protein degradation process, a split green fluorecent protein(GFP)-based assay system was establishe, through which we can screen anti-cancer chemical candidates influencing functions of endoplasmic reticulum(ER). The anti-cancer effects of chemical drugs used in this study were analysed via different molecular technlogies, such as flow cytometry and western blotting. This split GFP-based assay system could be further utilized for new drug screening.Methods: The split S-1 domain fused misfolded protein was stably expressed in Hela cells. Such cells were then transfected with plasmids containing S-10 sequence. The fluorecent intensity could increase when these transgenic cells were treated with protesome inhibitors(MG132). The MG-132 pretreated cells were then co-cultured with different concentrations of chemical candidates for 6 hours followed by microscopy analysis, which could determine the effects of such drugs. We chose one drug named Cucurbitacin E for further experiements to investigate the molecular mechanisms of its anti-cancer function. Flow cytometry technique was used. Hela cells were transfected for another misfolded protein stable expression. Western blotting was used study the protein burden in ER which could trigger ER stress。Results: Using this fluorecent assay system, we chose eleven chemical compounds which could decrease GFP fluorecent intensity by 50% percentage. This decreasing was drug dose dependent. These drugs also influenced the expression level of ER-stress related proteins. Our further study using Cucurbitacin E revealed that Cucurbitacin E could induce cellular apoptosis in a dose dependent manner. The results of western blotting showed that Cucurbitacin E could block protein transporting from ER to cytosols.Conclusions: We established a biological assay system to screening anti-cancer chemical compounds which could induce cellular apoptosis through ER stress pathway, providing a new platform to investigate the molecular mechanisms of anti-cancer therapy.