The Molecular Mechanisms Of Berberine Induced GRP78 Cell-surface Translocation Contributes To Apoptosis In Cancer Cells

Berberine,an important isoquinoline alkaloid,is mainly extracted from Coptis chinensis.Due to its significant effects on antimicrobial and antioxidant,Coptis chinensis,a kind of traditional Chinese medicine,was applied to the treatment of gastrointestinal infectious diseases such as diarrhea,dysentery and so on.Berberine was first been reported that it has anticancer activity in 1959.And since the 1990 s,a large number of researches suggested that berberine played the anti-tumor activity mainly by inhibiting the proliferation of cancer cells,inducing them apoptosis,and blocking the cell cycle.Glucose regulation protein 78(GRP78)is a kind of molecular chaperone located in endoplasmic reticulum.In tumor cells,GRP78 is regulated by the tumor microenvironment.It is an important factor of stress induced response in the cells.When the tumor cells are under stressed conditions,the expression of GRP78 will be changed,at the same time,the location of GRP78 will also be altered.According to the recent researches,the translocation of GRP78 in tumor cells induced by the drugs may contributes to cell apoptosis.In order to detect the change of expression and the location of GRP78 in tumor cells,we treated colon cancer cells HCT-116 and breast cancer cells MCF-7 with different doses of berberine.The results showed that the expression of GRP78 did not have obvious change,while more GRP78 from cytoplasm tanslocated to the cell membrane.And the translocation may closely related to berberine-induced tumor cells apoptosis.In order to explore the role of GRP78 in the berberine-induced tumor cells apoptosis and its mechanism,this research mainly carry out the following research contents:1.MTT assay and plate clone formation assay were carried out to detect the effects on the tumor cell viability and proliferation by berberine treatment.The results revealed that berberine exhibited inhibition effects in both cell viability and proliferation.Flow cytometry demonstrated that the tumor cellsappeared obvious apoptosis phenomenon when treated with berberine.Western blotting and real-time quantitative PCR results showed that Caspase12 and CHOP expression levels were significantly increased after treated with berberine.2.The location of GRP78 in tumor cells after treated with berberine were detected by immunofluorescent.Western blotting was applied to assess the cytoplasmic and membrane expression of GRP78 in extracts of HCT-116 cells and MCF-7 cells that treated with different doses of berberine.After using the corresponding antibody to block the membrane GRP78,berberine-induced apoptosis was alleviated significantly,suggesting that the tumor cell surface GRP78 mediated the berberine-induced tumor cell apoptosis.3.Furthermore we explored the mechanism of translocation of GRP78 in tumor cells.The results showed that the ROS level in tumor cells was increased significantly after treated with berberine.While adding the ROS inhibitor,the translocation of GRP78 was suppressed in berberine-treated tumor cells.These results provide a new target for cancer therapy.In summary,berberine could induce GRP78 translocated from endoplasmic reticulum to cell membrane via elavating the level of ROS in tumor cells.And the translocation of GRP78 was contributed to tumor cells apoptosis.This study will provide a new thought in berberine-induced GRP78 targeted antitumor mechanism,and has important theoretical significance and application value.