Effects Of Ixabepilone Combined With Silybin On The Angiogenesis Of Human Ovarian Carcinoma Cell Line SKOV3

Background:Ovarian cancer is one of the most common gynecologic malignant tumors.The mortality rate of ovarian cancer ranks first in gynecological cancer.Ovarian cancer causes great harm to women’s health and life. Surgery is the primary treatment for ovarian cancer. But because the specificity and sensitivity of diagnosis of ovarian cancer is not high, the ovarian cancer patients are diagnosed at advanced stage with losing the optimal timing of surgery. Therefore, chemotherapy plays an important role in the treatment of ovarian cancer. First-line chemotherapy for ovarian cancer is PT(Paclitaxel+ Cisplatin) chemotherapy. But the final tumor drug resistance and recurrence in patients with advanced ovarian cancer after chemotherapy and 5 years survival rate is less than 30%. People are eager to find new chemotherapy drugs and exploration of the mechanism of drug resistance.With the deepening of research, people get the preliminary results of the Paclitaxel resistant mechanism. The main reason of Paclitaxel resistance is due to over expression of tumor tubulin changes and P- glycoprotein. Natural cytotoxic drug Epothilone is a medicine which acts on tubulin macrolides. The Epothilones are developed to resolve the resistance in the Paclitaxel. The drugs’ antitumor mechanisms are similar to Paclitaxel’s. They are resistant to various tumor cell lines and the effects are stronger than Paclitaxel’s. Bristol Myers Squibb Company created a new derivation Ixabepilone which is also the first used as clinical medicine of new Epothilone anticancer drugs.The medicine is approved by Food and Drug Administration(FDA) In the United States in October 2007. It can cause the unbalance between the tubulin polymerization and depolymerization through efffects on the tubulin binding,resulting in apoptosis of tumor cells during Mitotic division. Its mechanism is similar to the effects of Paclitaxel on tumor cells. But the binding sites are different between the two medicines. P-glycoprotein can’t be applied to Ixabepilone when Ixabepilone goes through the cell membrane. Therefore, it explains why Ixabepilone is complementary resistance with Paclitaxel. The drug in Europe showed potent antitumor activity after being the listing,letting people see hope on the road of overcoming cancer again.PI3K/AKT/m TOR signaling pathway plays an important role in the occurrence and development of ovarian cancer. It also plays an important role in the downstream pathway of HIF-1 alpha and VEGF in tumor angiogenesis and cancer cells’ invasion and metastasis. Experiments show that low concentration of Paclitaxel causes anti angiogenesis effect.However,few studies of Ixabepilone’ effects on apoptosis and anti angiogenesis effects have been shown in ovarian cancer.Objective:To testify effects of Ixabepilone and Silybin on ovarian cancer cells and do research on the PI3K/AKT/m TOR pathway.Method:1. Ovarian cancer cell lines SKOV3 was cultured in vitro under the hypoxia environment. Different concentrations of Ixabepilone and other different concentrations of traditional Chinese medicine were added in the logarithmic phase cells of 24 hours. study the effect on proliferation of SKOV3 cells through the CCK-8 experiment.2. Ovarian cancer cell lines SKOV3 was cultured in vitro under the hypoxia environment. Study the effects of Ixabepilone and Silybin alone and combination on ovarian cancer cell apoptosis via flow cytometry experiment.3. Detect the effects of Ixabepilone and Silybin alone and combination on the secreted VEGF via enzyme linked immunosorbent assay(ELISA)experiments and RT-PCR experiment.4. Detect the effects of Ixabepilone and Silybin alone and combination on PI3K/AKT/m TOR signaling pathway and HIF-1α protein via Western blot(WB)experiment.Result:1. Different concentrations of Ixabepilone were added into SKOV3 cells24 h in the logarithmic phase. CCK-8 reagent detected the processing. The results showed that Ixabepilone can inhibit the proliferation of SKOV3 cells.With the increasing of drug concentration, inhibition rate increased significantly and the IC50 was 9.84umol/L.The effects of Silybin on the proliferation of SKOV3 cells were also detected and the IC50 was365.23umol/L.The combined effect of Silybin and Ixabepilone on cells’ proliferation inhibitation was improved significantly.2. Different concentrations of Ixabepilone were added into SKOV3 cells24 h in the logarithmic phase. The results showed that Ixabepilone and Silybin on SKOV3 cells apoptosis was obvious and the combined action of the two on cell apoptosis were also significantly improved through flow cytometry experiment.3. Simulating the hypoxic environment, using enzyme immunoassay(ELISA)test and RT-PCR detection to test the effect of Ixabepilone and Silybin.The results showed that the combined effect of the both medicine Inhibited SKOV3 cells create and secrete vascular growth factor VEGF obviously.4. Simulating the hypoxic environment, different concentrations of Ixabepilone and Silybin alone or in combination were added in logarithmic phase of SKOV3 cells for 24 h.PI3K and m TOR inhibitors being compared to detecteIxabepilone and Silybin has inhibitory effect on PI3K/AKT/m TOR pathway and HIF-1α protein through Western blot experiment, and the combined action effect is remarkable.Conclusion:1. In a certain range of concentration, Ixabepilone and Silybin can effectively improve the inhibition rate of SKOV3 cells. With the increase of concentration, the ability of Ixabepilone inhibiting cell proliferation and promoting apoptosis is strong. The combined effect of Ixabepilone and Silybin which inhibit the proliferation and promote the apoptosis effect is significantly enhanced.2. Under simulated hypoxic environment, Ixabepilone and Silybin in a certain range of concentration can affect PI3 K / Akt / m TOR pathway and its downstream of HIF-1 alpha 、EVGF protein factor levels in SKOV3 cells. When the two medicine were added together the effects were stronger.Experimental results show that the Ixabepilone in addition to kill cancer cells and on vascular generation process, an important growth factor also has inhibitory effect.