The Expression And Clinical Significance Of TAK1 In Ovarian Epithelial Carcinoma & Its Increasing Of Chemosensitivity To Taxol On SKOV3 And OVCAR3 Cells

Backgroud Ovarian epithelial carcinoma is one of the most common malignant tumor among all female reproductive system cancers.Its incidence ranks the third in malignant tumor of female reproductive organs,and only lower than that of cervical cancer and endometrial cancer.240,000 cases were diagnosed with ovarian cancer each year all around the world,with 150,000 death cases.During these years,TC(Paclitaxel,carboplatin),TP(Paclitaxel,cisplatin),DC(docetaxel,carboplatin)were proved to be effective in the treatment of ovarian cancer,while the survival rate was still low,between 25%-30%.Until now the treatment of ovarian epithelial cancer is still lack of qualitative leap,compared with malignant ovarian germ cell tumors,so its death rate is still on top rank among all female cancers.Because of the resistance to chemotherapeutics,cells will have self replication and lead to tumor recurrence,and chemotherapy resistance is still the hot and challenge topics in research field.Transforming growth factor beta activated kinase 1(TAK1or MAP3K7)is the molecular center of the cells.According to the trial in 1995,CDNA library screening and protein fragments complementary analysis,TAK1 was proved to be effectively in the control of NF-κB and mitogen activated protein kinase(MAPK)signaling pathway,which played key roles in cell survival,immune response,metabolism and cancer occurrence.It was mainly through the TAK1-Nf-κB pathway to suppress the activation of apoptotic protease and then prevented apoptosis.Recent studies showed that TAK1 abnormal expression in various malignant tumors,which could indicate its key role in malignant cancers.However,little information is reported about TAK1 expression and its role in the occurrence and development of ovarian cancer,especially for its role in chemotherapy resistance.Our trial was mainly to test the expression of TAK1 in ovarian epithelial cancer and recurrent ovarian cancer after the treatment of Taxol.And then re-tested the expression of TAK1 in SKOV3 and OVCAR3 cell lines,which were treated with RNAi technology and TAK1 specific inhibitors 5Z-7-oxozeaenol.The expression of TAK1 was evaluated and its effect on cell proliferation,apoptosis and invasion ability was analyzed at the same time.Female mouse transplantation model was set up to evaluate the response of ovarian cancer cells to Taxol treatment after suppression of TAK1.All of these were trying to evaluate the prognostic value of TAK1 and antiproliferation effect of inhibiting the expression of TAK1 in Taxol chemotherapy regimens.We do hope all the laboratory data could help to change the clinical treatment effect of ovarian epithelial cancer.Part One the Differential Expression and Clinical Significance of TAK1 in Ovarian Epithelial Cancer Tissue and Its Cancer cellsObjective: This trail was mainly to observe the expression of TAK1 in ovarian epithelial cancer tissues and recurrent group,to explore the expression of TAK1 and its relationship between clinic pathological parameters and prognosis of ovarian cancer,and observe TAK1 expression features in SKOV3 and SKOV3 PR cells.Methods: Western blotting were used to detect the expression of p-TAK1 in SKOV3 PR common and tolerance paclitaxel cell lines.Immuno histochemistry semiquantitative analysis was made to test the TAK1 and p-TAK1 expression in primary and recurrent ovarian cancer tissues.Survival information was followed up.Result: The final result showed that the expression of TAK1 was high in recurrent group compared with primary group(p=0.0076).And the activity of p-TKA1 in recurrent group was higher compared with primary group(p=0.021).There was no significant correlation of TAK1 with FIGO staging,p53 expression and lymph node metastasi.While high expression of TAK1 has obvious significant correlation with poor overall survival(p=0.031)according to our survival analysis curve.The western blot result showed that the expression of TAK1 and p-TAK1 in SKOV3 were significantly lower than that in SKOV3 PR cell lines(p=0.008 and 0.009).Conclusions: TAK1 Participated in the occurrence and development of ovarian cancer.Taxol-resistant ovarian cells lines had over-expression of TAK1 and p-TAK1.It may be the molecular markers of poor prognosis of ovarian cancerPart Two Inhibition of TAK1 Expression and its influence on biological behavior in SKOV3,OVCAR3 and Taxol-resistant ovarian cells linesObjective: The expression of TAK1 were inhibited with si RNA in SKOV3 cell lines.The proliferation,apoptosis,invasion ability of SKOV3 cell lines were analyzed and cytotoxic effect of Taxol on the cells were evaluated after successfully suppression of TAK1.Methods: The transfected cells were established with two independed target si RNA(signal Silence（？）siRNA Ⅰ#6317andⅡ #6318),and the control group were formed at the same time(signal Silence（？）control siRNA #6201).The expression of TAK1 were tested with western blotting method.The changes of proliferation,apoptosis and invasion ability in TAK1 suppressed group,paclitaxel treated group and 5Z-7-oxozeaenol treated group were calculated with CCK-8 test,Annexin V-FITC/PI double marking experiment and flow cell experiment.Results: Silence TAK1 could significantly improve the sensitivity of taxol in SKOV3 and OVCAR3 cells.It is confirmed that 5Z-7-oxozeaenol was in a dose-dependedon manner in inhibition of TAK1 phosphorylation.In SKOV3 cell lines the Toxol IC50 value was 2.5 with 5Z-7-oxozeaenol treatment,while the IC50 value changed to 236 without 5Z-7-oxozeaenol.Paclitaxel could increase the ratio of G2 / M phase cells,the SKV03 RP had no similar response.However this kind of phenomenon would reappear after re-used of 5Z-7-oxozeaenol.In addition the number of cancer cell apoptosis was higher in Taxol group compared with control group(p=0.02).However the apoptosis in Taxol+5Z-7-oxozeaenol group had no significant difference with other test groups(p=0.001).Conclusions: The Silence TAK1 could improve the cytotoxic effect of paclitaxel on ovarian cancer cells.5Z-7-oxozeaenol was in a dose-dependedon manner in cytotoxic effect of paclitaxe.The combination usage of Taxol and TAKI inhibitor 5Z-7-oxozeaenol could make cancer cells stop in the period of G2/M and lead to apoptosis.Part Three the effection of Combined utilization of TAK1 Inhibitor 5z-7-oxozeaenol & taxol on Planting Tumor Cells Growth in Athymic MiceObjective: The combined regimen TAK1 Inhibitor 5z-7-oxozeaenol & paclitaxel was used in ovarian transplantation tumor mice models.The influence effect and safty on growth of transplanted tumor cells and nude mice were observed at the same time.Methods: 60 nude mice were divided into four groups(control group,Taxol group,5z-7-oxozeaenol group and 5z-7-oxozeaenol&Taxol group),with 15 nude mice in each group.Ovarian cancer transplantation models were established through the injection of SKOV3 cell lines(5*106 cells for each injection).The treatment of Taxol(8mg/kg)and 5z-7-oxozeaenol(6.5mg/kg)were started when a subcutaneous tumor could be touched.Peritoneal cavity injection was carried 2 hours before decapitated the mice. The weight of mice body,spleen and tumor were recorded.The total number of tumor cells and positive staining tumor cells were calculated with flow cytometry instrument.Results: The weigh of tumor mass in Taxol&5z-7-oxozeaenol group was significant lower than other three groups(p<0.01).Brd U test showed the percentage of tumor cells was also obvious lower in this group(p<0.01).The ratio of p-TAK1/TAK1 was even significant lower in 5z-7-oxozeaenol group(p<0.01).While the body weight and spleen weigh showed no significance among all test groups.Conclusion: The combination of Taxol and 5z-7-oxozeaenol could effectively suppress the growth of ovarian epithelial cancer cells,and the effective dose could be highly tolerant in transplantation mice models.