Effects Of Herceptin Combined With Oxaliplatin On SKOV3 Biological Behavior Of Ovarian Cancer Cells Through P53-P21/P27 Pathway

Objective:To investigate the effects of Herceptin combined with Oxaliplatin on the biological behavior of HER2-positive ovarian cancer cells SKOV3 by regulating the P53-P21/P27 pathway,providing a new idea for the treatment of ovarian cancer.Methods:According to the experimental requirements,it was divided into four groups,Control,Herceptin group,Oxaliplatin group and two-drug combination group.MTT assay was used to detect the inhibitory effect of single drug and sequential combination of two drugs on the proliferation of ovarian cancer SKOV3 cells,and the IC₅₀ values of Oxaliplatin and the combination of two drugs were calculated.Cell clone formation assay was used to detect the proliferation of ovarian cancer SKOV3cells.The protein expressions of P53、P21、P27、CDK1、Cyclin B1、P-CDK1、CDK4、Cyclin D1、Pro-Caspase3、Cleaved-Caspase3 and ERCC1 were detected by Western blot.The apoptosis of SKOV3 cells and the changes of mitochondrial membrane potential were detected by flow cytometry.Results:1.Detected by the MTT experiment:Compared with the Control,Herceptin group（10μg/m L）、Oxaliplatin group（3.125、6.25、12.5、25、50μg/m L）and the combination of the two drugs could inhibit the proliferation of SKOV3 cells,and the difference was statistically significant（P<0.05）.Compared with the Oxaliplatin group,the combination of the two drugs at the same concentration could inhibit the proliferation of SKOV3 cells,and the difference was statistically significant（P<0.05）.The IC₅₀ of Oxaliplatin was 129.03μg/m L.Oxaliplatin IC₅₀ of the combined group was 38.89μg/m L.2.Detected by cell clone formation experiment:Compared with Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）,the number of cell clones formation in the same concentration combination group was less.3.Detected by Western blot method:3.1.Western blot method detected P53-P21/P27 pathway:P53-P21/P27 related protein expression:The expression levels of P53、P21 and P27 proteins in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group at the same concentration increased successively,and the difference was statistically significant（P<0.05）.3.2 Western blot method detected Cycle-related proteins:Expression of CDK1 and Cyclin B1proteins:The expression levels of CDK1 and Cyclin B1 proteins in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group decreased successively,and the difference was statistically significant（P<0.05）.P-CDK1 protein expression:The expression of P-CDK1 protein increased successively in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and two-drug combination group,and the difference was statistically significant（P<0.05）.CDK4 and Cyclin D1 proteins expression:The expressions of CDK4 and Cyclin D1 proteins increased sequentially in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group,and the difference was statistically significant（P<0.05）.3.3Western blot method detected Apoptosis-related protein:Pro-Caspase3 protein expression:There was no significant difference in pro-caspase3 protein expression between Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group（P>0.05）.Cleaved-Caspase3 protein expression:Cleaved-Caspase3 protein expression increased sequentially in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group,and the difference was statistically significant（P<0.05）.3.4.Western blot method detected Drug-resistant associated proteins:Expression of ERCC1protein:The expression levels of ERCC1 protein in Control、Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the combination group decreased successively,and the difference was statistically significant（P<0.05）.4.Cell apoptosis was detected by flow cytology:Compared with Control,Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the same concentration of the two drug combination group,cell apoptosis showed an increasing trend,and the same concentration of the two drug combination group had the highest percentage of apoptosis,and the difference was statistically significant（P<0.05）.5.Cell mitochondrial membrane potential changes were detected by flow cytology:Compared with Control,Herceptin group（10μg/m L）、Oxaliplatin group（25μg/m L）and the same concentration of the two drug combination group,mitochondrial membrane potential decreased,and the loss of membrane potential in the combination group was the most significant,and the difference was statistically significant（P<0.05）.Conclusions:1.Herceptin combined with Oxaliplatin can inhibit the proliferation of HER2 positive ovarian cancer SKOV3 cells.2.Herceptin combined with Oxaliplatin may regulate the expression of CDK1,Cyclin B1,P-CDK1,CDK4 and Cyclin D1 proteins by activating the P53-P21/P27 pathway,and block the ovarian cancer SKOV3 cell cycle in S and G2/M phases.3.Herceptin combined with Oxaliplatin can increase mitochondrial membrane potential loss and up-regulate apoptosis protein cleaved-caspase3 expression to initiate the internal apoptosis pathway.4.Herceptin combined with Oxaliplatin increases the cytotoxicity of Oxaliplatin by down-regulating the expression of drug-resistant protein ERCC1.