The Growth-Inhibitory Effects Of Berberine On Human Ovarian Cancer Cells

Ovarian cancers account for about3percent of the malignancies in women. Ovarian cancer is the leading cause of gynecological cancer-related mortality. Since there are usually no clinical symptoms in the early stage of ovarian cancer, the early diagnosis of ovarian cancer is very difficult. Surgery cannot remove the tumors completely, therefore the combination of surgical removal and chemotherapy is usually resorted. Cisplatin-based chemotherapy is commonly used to treat patients after surgery. However, a significant number of patients develop resistance and experience severe side effects. Identification of anti-tumor compounds in berbal plants has become widely pursued in recent years.Berberine is a natural compound derived from the roots and rhizomes of numerous herbal plants, such as Berberis, Hydrastis canadensis and Coptis chinensis. It is an isoquinoline alkaloid component. Berberine has been known to exhibit multiple pharmacological activities, such as anti-inflammation, anti-hypertensive, anti-cholinergic, anti-diabetic, anti-protozoal. Accumulative evidence in both in vitro and in vivo studies using berberine demonstrated anti-tumor properties in different cancer cells established from cervical cancers, breast cancers, esophageal cancers, oral cancers, colonic cancers, prostate cancers, leukemia and glioblastoma. Many studies showed that the berberine inhibits the growth of tumor cells by inducing the arrest of cell cycle or inducing the apoptosis of tumor cells. However, little is known about whether berberine can inhibit ovarian cancer cells.Our study evaluated the anti-tumor effects of berberine and cisplatin on human ovarian cancer cells and characterized the mechanisms underlying the anti-tumor effects of berberine, as shown in the following four parts:PART ONEThe inhibitory effects of berberine on the proliferation of human ovarian cancer cells and immortalized ovarian epithelial cellsWe analyzed the inhibitory effects of berberine and cisplatin on ovarian cancer cell lines HEY, SKOV3and immortalized ovarian cells by MTT assay.(1)Berberine significantly inhibited the proliferation of HEY cell in a time-and dose-dependent manner.(2)Cisplatin inhibited growth of SKOV3cells, but has weaker effect on HEY cells.(3)Immortalized ovarian cells appeared to be more resistant to berberine than the human ovarian cancer cells.The results in this part showed that berberine exhibited a time-dependent and dose-dependent growth-inhibitory effect on human ovarian cell lines. Low dose berberine has weaker inhibitory effect on the growth of nonmalignant ovarian epithelial cells.PART TWOCell cycle analysis of human ovarian cancer cell lines in response to berberineOne of the most important mechanisms underlying the anti-tumor effects is through inducing cell cycle arrest. We analyzed the effect of berberine and ciaplatin on cell cycle progression and underlying the mechanism by flow cytometry and Western Blotting,1. Cell cycle distribution of HEY, SKOV3cells treated with berberine(1) In HEY,SKOV3cells treated with berberine, the percentages of cells in G1phase were increased.(2) In HEY,SKOV3cells treated with cisplatin and berberine, the percentages of cells in G1phase were higher than in cells treated with cisplatin alone.2. Responses of cell cycle effectors to berberine treatment by Western Blotting:Berberine down-regulated the expression of cyclin E in HEY、SKOV3cells, up regulated the expression of p21.The results above suggested that berberine induced HEY, SKOV3cells arrest at G1phase through down regulation of cyclin E and up regulation of p21. G1phase arrest induced by cisplatin could enhance by berberine.PART THREEInduction of apoptosis in human ovarian cancer cells by berberineIn addition to cell cycle arrest, apoptosis has also been reported to be responsible for inhibition of tumor cell proliferation. In this part, we studied the pro-apoptotic effect of berberine on human ovarian cancer cells by Annexin V-PI staining followed by flow cytometry analysis, and examined apoptosis-related proteins by Western Blotting.1. After being treated with berberine, the HEY、SKOV3cells were analyzed by Annexin V-PI and flow cytometry.(1) In HEY,SKOV3cells treated with berberine, the percentages of apoptosis cells were increased over the control groups.(2) In HEY,SKOV3cells receiving combined treatment of cisplatin and berberine, the percentages of apoptotic cells were higher than in cells solely treated with cisplatin.2. Examination of apoptosis-related protein by Western Blotting.Berberine up-regulated the expression of bax protein and down regulated the expression of bcl-2protein in a dose-dependent manner.PRAT FOURThe effect of berberine on the expression of homologous recombination protein RAD51RAD51is the key protein during homologous recombination. We analyzed the expression level of RAD51in human ovarian cancer cells after they were treated with berberine and cisplatin by Western Blotting.The expression of RAD51in HEY cells were up regulated in response to cisplatin while berberine down regulated the expression of RAD51.The inhibitory effect of cisplatin on human ovarian cells can be enhanced by berberine.