Down-regulation Of PAK5 Mediates Paclitaxel-chemoresistance Of Epithelial Ovarian Cancer With P-gp Inhibition

Part 1 The Clinical Study of PAK5 and P-gp in Epithelial Ovarian CancerObjective To determinate the expression of PAK5 and P-gp in epithelial ovarian cancer(EOC)tissue,and investigate the clinical factors correlation of PAK5 and P-gp in EOC,to provide guidance to predict disease progress and prognosis of EOC patients.Methods Research Objects were the patients who accepted surgery in Department of Gynecology and were diagnosed epithelial ovarian cancer definitely by pathological diagnosis in Xinhua Hospital affiliated to Shanghai Jiaotong university school of medicine from January 2013 to June 2015.We carried out a retrospective study of the clinical factors,such as,pathological type,FIGO stages,cell differentiation,chemoresisitance and so on.Immunohistochemistry(IHC)staining was performed to investigate the expression of PAK5 and P-gp.Finally,we analyzed the expression of PAK5 and P-gp in EOC tissue and the correlation of PAK5 and P-gp and the above clinical factors.Results 1.The positive rate of PAK5 in FIGO advanced group was higher than in FIGO early group(91.2%vs 55.6%),the positive rate of PAK5 in poor cell differentiation group was higher than in well and moderate cell differentiation group(81.8%vs 47.3%),differences were statistically significant in the above factors(P<0.01).However,there was not significant difference in positive expression rate of PAK5 when grouped by pathological type(P>0.05).The positive rate of P-gp in FIGO advanced group was higher than in FIGO early group(97.0%vs 44.4%),the positive rate of P-gp in poor cell differentiation group was higher than in well and moderate cell differentiation group(90.9%vs 52.6%),differences were statistically significant in the above factors(P<0.01).However,there was not significant difference in positive expression rate of P-gp when grouped by pathological type(P>0.05).2.The positive rate of PAK5 in chemoresistance group was higher than in chemosensitivity group(59.0%vs 20%),differences were statistically significant in the above factors(P<0.05).The positive rate of P-gp in chemoresistance group was higher than in chemosensitivity group(68.1%vs 0%),differences were statistically significant in the above factors(P<0.001).Conclusion 1.PAK5 and P-gp were high-expressed in epithelial ovarian cancer.2.The positive expression of PAK5 and P-gp were related to the FIGO late stages,poor cell differentiation,chemoresistance.PAK5 and P-gp could be used to predict the disease progression and prognosis of patients in epithelial ovarian cancer.Part 2 Establish EOC chemoresistant cell lines and determine the paclitaxel chemosensitivity correlation with PAK5 and P-gpObjective 1.Establish stable and reliable EOC cells resistant to different paclitaxel concentrations.And set experimental model for the study of drugresistance mechanisms of EOC.2.Determinate the paclitaxel chemosensitivity correlation with PAK5 and P-gp.And investigate the paclitaxel-chemoresistance mechanism of EOC.Methods 1.Establish paclitaxel-chemoresistance cell line remaining greater than 90% survival rate by the concentration of 150 ug / ml and 200 ug / ml dose gradient.Cells chemoresisitance was measured by MTT method in SKOV3-TR1 and SKOV3-TR2.2.Examine the expression of PAK5 and P-gp in paclitaxel-drugresistance cells by using Western Blot technique.Results 1.Successfully construct paclitaxel resistance line SKOV3-TR1 and SKOV3-TR2.The chemoresistance was measured by MTT method.2.PAK5 and P-gp expressions are significantly highly in paclitaxel-chemoresistance cells than paclitaxel-chemosensitivity cells.Conclusion The expression of PAK5 and P-gp in paclitaxel-chemoresistant cells were significantly higher than paclitaxel-chemoresistant cells,and the expression of PAK5 and P-gp are positively correlated with chemoresistance in EOC.Part 3 Down-regulation of PAK5 inhibits the expression of P-gp in EOCObjective P-gp and PAK5 play the important role in the chemoresistance of EOC.Our study aims to investigate whether PAK5 mediates paclitaxel chemoresistance through P-gp,in order to examined the association of PAK5 and P-gp in the chemoresistance of EOC.Methods 1.Using the specific expression of lentivirus-PAK5 expressing vetors to transfect into SKOV3-TR1 and SKOV3-TR2.Using both Western Blotting and Real-Time techniques to determinate the expression of PAK5.The differences were significant,P<0.01.2.Using Western Blotting technique to determinate the expressions of P-gp after down-regulation of PAK5 in SKOV3-TR1 and SKOV3-TR2.Results 1.The expression of PAK5 was knockdown with short hairpin RNA(sh RNA)mediated by lentivirus.2.The expression of P-gp was reduced after we knocked down PAK5 in SKOV3-TR1 and SKOV3-TR2.Conclusion Knockdown of PAK5 significantly increased sensitivity of EOC to paclitaxel treatment with P-gp downexpression...