The Research On Inhibitor Of Deubiquitinating Enzyme USP7 P5091 Inducing Inhibition In Ovarian Cancer

Ovarian cancer is one of the three most common malignant tumor in the female reproductive system.Because of the early diagnosis of ovarian cancer being difficult and the lack of effective prevention and control measures,ovarian cancer mortality is in the first place.The epithelial ovarian cancer accounts for 85%~90% of ovarian malignant tumors[1-3].At present,the mainly treatment for ovarian cancer are effective surgical treatment and the chemotherapy of platinum combined taxol.But,there are a large number of advanced ovarian cancer patients facing the problem of platinum or taxol resistant,leading to poor treatment effect.So,it's very important to explore the new drugs of targeted therapy of ovarian cancer.There are some researches reported that deubiquitinating enzyme USP7 has high expression in the epithelial ovarian cancer tissue[4-5].It has been confirmed that P5091 can against the role of tumor cell proliferation in the research on the drugs about the inhibitor of USP7.P5091 is a selective and efficient inhibitor of deubiquitinating enzyme USP7.There is a study reported that P5091 can inhibit the growth of multiple myeloma and prolong the life of mice[6].Now,the research of P5091 using in the ovarian cancer has not been reported at home and abroad.First of all,we through immunohistochemical method to detect that the epithelial ovarian cancer tissue samples relating to the normal ovarian tissue and benign ovarian tissue has high expression of deubiquitinating enzyme USP7.Secondly,through the relevant experiment we found that P5091 acting on the ovarian cancer cells can inhibitcell proliferation and induce cell death,has the inhibition.Finally,we explore mechanisms about the cell inhibition of P5091.USP7 may become a new target of therapy on the ovarian cancer in the future.Inhibitor of deubiquitinating enzyme USP7P5091 may become a new drug of treating ovarian cancer.Objective:1.To explore that deubiquitinating enzyme USP7 has high expression in the epithelial ovarian cancer tissue.2.To explore that P5091 acting on the ovarian cancer cells can inhibit cell proliferation and induce cell death,has the inhibition.3.To explore the mechanisms about the cell inhibition of inhibitor of deubiquitinating enzyme USP7 P5091.Methods:1.We detect the collected clinical tissues by immunohistochemical method,analysis the results of the experiment.We need to make sure that deubiquitinating enzyme USP7 has high expression in the epithelial ovarian cancer tissue.2.We select two ovarian cancer cell lines.One is Hey A8 cell line,which is wild-type p53 cell.Another one is OVCAR-8 cell line,which is mutated p53 cell.P5091 is set to six concentrations,there are 5mM,10mM,20mM,30mM,40mM and 50mM.We observe whether the drug has inhibitory effect on cells by MTT experiments.3.Through microscope and Hoeschst 33342 experiments,we detect if P5091 can effect the cells.4.Through the flow cytometry cell cycle experiment,we test whether P5091 roles in cell cycle arrest.5.Through the flow cytometry apoptosis and western blotting experiments,we tect whether P5091 roles in cell apoptosis and necrosis.6.Through western blotting experiment,we test the protein expressions of USP7,HDM2 and p53.We want to explore the relationship between the inhibition of P5091 acting on the cells and p53-HMD2 pathway.Results:Through immunohistochemical method,we found that the epithelial ovarian cancer tissue samples relating to the normal ovarian tissue and benign ovarian tissue has high expression of deubiquitinating enzyme USP7.We select two ovarian cancer cell lines.One is Hey A8 cell line,which is wild-type p53 cell.Another one is OVCAR-8 cell line,which is mutated p53 cell.Through the MTT experiment,we discover that P5091 can inhibit ovarian cancer cell proliferation.Through microscope,we observe that cells morphology change significantly after conducted with P5091.Through Hoechst 33342 experiment,we observe that cells morphology change and become bright after conducted with P5091.These explain P5091 induces the cell death.Through the flow cytometry cell cycle experiment,we test cells treated with P509118 h,P5091 roles in cell cycle arrest.Cell cycle arrest is probably one of the reasons for cell death.Through the flow cytometry apoptosis experiment and western blotting experiment detecting apoptosis related proteins,we found P5091 roles in ovarian cell apoptosis and necrosis.Through western blotting experiment,we test the protein expressions of USP7,HDM2 and p53.When P5091 roles in the early stage of Hey A8 cell,HDM2 plays a main role in the inhibition to p53,when P5091 roles in the late stage of Hey A8 cell,USP7 plays a main role in the stability to p53.The results explain P5091 roles in Hey A8 cell is associated with p53-HDM2 pathway.The correlation between P5091 roles in OVCAR-8 cell and p53-HDM2 pathway may be not big.Conclusion:1.Deubiquitinating enzyme USP7 has high expression in the epithelial ovarian cancer tissue.2.P5091 acting on the ovarian cancer cells can inhibit cell proliferation and induce cell death,has the inhibition.These may be associated with the cell cycle arrest.3.Inhibitor of deubiquitinating enzyme USP7 P5091 roles in cell apoptosis and necrosis.4.P5091 roles in wild-type p53 Hey A8 cell is associated with p53-HDM2 pathyway.The correlation between P5091 roles in mutated p53 OVCAR-8 cell and p53-HDM2 pathyway may be not big.