Research On The Function Of P-gp In Taxol-resistant Ovarian Cancer Cell And The Reversal Of Drug Resistance

Ovarian cancer is one of the three most common malignant tumors in female reproductive system, which threatens the women's health seriously. Chemotherapy is the most consideration therapy after surgery. And taxol plays a very important role in chemotherapy. But after several courses of treatment, tumor cells can show drug resistance which leads to the failure of post chemotherapy. Thus, how to surmount the multidrug resistance of ovarian cancer has been the hot point for the past few years.There has been a lot of mechanism that cells are resistant to taxol , such as the high expression of P-gp ( P-glyco-protein),the change of eitherβorαsubunit of microtubule,and the change of apoptosis pathway . P-gp that encoded by MDR1 gene(multidrug resistance gene 1), can pumps multiple types of drugs out of the cell using the energy generated from ATP, and confers multidrug resistance on cancer cells. So we cultivated Taxol-resistant ovarian cancer cell line A2780/Taxol and detected the expression of P-gp and PKC (protein kinase C) in it. And we studied the effects of activation and inhibition agents of PKC on the function and expression of P-gp in A2780/Taxol. Meanwhile constructed shRNA expression vector of P-gp to silence the expression of MDR1, after that we explored the drug accumulation, so as to provide evidence for the reversal of drug resistance of ovarian tumor.AIM:1. To Cultivante Taxol-resistant ovarian cancer cell line A2780/Taxol and detecte the expression of p-gp and PKC-αin it.2. To study the effects of activation and inhibition agents of PKC on the function and expression of P-gp in A2780/Taxol.3. Use RNAi to silence the expression of MDR1 and explore the drug accumulation to identify whether the silencing of MDR1 can reverse drugresistance.METHODS:1. Immunocytochamical and double-labeled immunofluorescent methods were used to determine the expression of P-gp and PKC-αin A2780/Taxol and A2780.2. Westernblot was used for assessing the expression of P-gp after A2780/ Taxol was co-cultured with PMA or SP.3. R123 (Rohdamin 123) was used as fluorescent probe to determine the drug accumulation in A2780/ Taxol so as to evaluate the effects of PMA and SP on the function of P-gp .The drug accumulation was measured by flow cytometry.4.Two different shRNA targeting the coding sequence of MDR1 were designed, and the pWH1-MDR1 was constructed by inserting the designed shRNA to the eukaryotic expression vector pWH-1.The vector was identified by restriction endonuclease digestion .The ovarian cancer cell strain A2780/Taxol was transfected by pWH1-MDR1. After selected by G418, the MDR1 expression in the positive clones was detected by Westernblot. MTT assay was carried out to observe the effect on proliferation ability.Chose the most efficient one, and then observeed the silence effect of MDR1 by Western blot and flow cytometry.RESULTS:1. P-gp had positive staining on A2780/Taxol and negative staining on A2780. And there was co-expression of P-gp and PKC-αin A2780/Taxol, but not in A2780.2. The expression level of P-gp in A2780/Taxol was not affected by PMA and SP.3. When treated by PMA, A2780/Taxol cells showed decreasing of drug accumulation. When treated by SP, A2780/Taxol cells showed increasing of drug accumulation.4.It was verified by restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of MDR1 was correct.The constructed eukaryotic vector pWH1-MDR11,pWH1-MDR12 were successfully transfected into A2780/Taxol and the positive clone has been got; The expression of P-gp in A2780/Taxol cells was significantly suppressed by pWH1-MDR12.5. MTT showed that there were no differences in Cell Proliferation Assay among the A2780/Taxol, A2780/Taxol/pWH1 group, A2780/ Taxol/ pWH1-negtive group and A2780/Taxol/pWH1-MDR12 group. Drug accumulation in A2780/Taxol/pWH1-MDR12 group exceeded that in A2780/ Taxol significantly.CONCLUSION:1. P-gp plays important roles in taxol-resistance ovarian cancer cell A2780/Taxol. 2. The change of PKC's activity can not alter the expression of P-gp, but can modulate its function.3. Drug accumulation in ovarian cancer cells can be increased significantly after using RNAi to silence MDR1.4. Using PKC inhibition agent to suppress the function of P-gp or using RNAi to decrease the expression of P-gp can increase drug accumulation and reverse drugresistance in a certain extent.