The Role And Mechanism Of ClC-3 Chloride Channel In Paclitaxel Resistance Of Ovarian Cancer

Objectives:EPithelial Ovarian Carcinoma（EOC）accounts for 95%of all ovarian malignancies,and its 5-year survival rate is about 44%,which is the highest mortality rate of gynecological malignant tumors.Paclitaxel（PTX）is the first-line chemotherapy in ovarian cancer,which ultimately affects its clinical application due to chemotherapy resistance.But its mechanism is still unclear.In recent years,the role of chlorine channels in chemotherapy resistance has attracted the attention of researchers.There are differences in ClC-3 expression between PTX-sensitive and-resistant lung cancer cells.These results suggest that ClC-3 chloride channel may be the potential mechanism of PTX resistance.1)We intend to investigate that the difference of the expression,function and distribution of ClC-3 chloride channel exists in ovarian cancer drug-sensitive and-resistant cells.2)Does changing ClC-3reverse the drug resistance of PTX in ovarian cancer?3)How does ClC-3 mediate drug resistance?Does ClC-3 participate in PTX resistance by influencingβ-tubulin polymerization?This study provides a new viewpoint and experimental basis for further obtaining the mechanism of chlorine channel in chemotherapy resistance and interfering with PTX resistance in ovarian cancer.Methods:All experiments were performed on the human ovarian cancer drug-sensitive cell A2780 and drug-resistant cell A2780/PTX.1)Using MTT assay to detect the cell proliferation of A2780 cells and A2780/PTX cells treated with PTX;2)Appling Flow Cytometry（FCM）to measure the cell cycle and apoptotic of A2780cells and A2780/PTX cells treated with PTX;3)Appling whole-cell patch method to record the chlorine current under different conditions;4)Using RT-PCR method to detect the expression of target gene mRNA;5)Using siRNA interference technology to down-regulate chloride channel and tubulins gene;6)The expression and distribution of chloride channel protein and tubulins were detected by immunofluorescent analysis using confocal microscopy;7)Westernblot method was used to detect the expression level of related proteins;8)Statistical analysis and data processing using SPSS and SigmaPlot software.Results:1)The number of A2780 cell treated with PTX decreased significantly with the increase of concentration,the IC₅₀0 value was（31.23±2.03）nM;however,the A2780/PTX cell viability did not decrease at the same concentration,Its IC₅₀0 value was（297.14±4.15）nM,drug resistance index was 9.53±0.53.Based on this,we select30nM and 300nM,which are close to IC₅₀,to carry out the corresponding experimental study.2)After A2780 and A2780/PTX cells treated with PTX for 24 hours,A2780 cells in G2/M phase increased from（6.06±0.61）%to（35.53±1.25）%,while A2780/PTX cells increased from（6.85±0.63）%to（12.77±0.06）%.3)There was no obvious apoptosis in A2780 and A2780/PTX cells treated with PTX for 24 hours.4)Effect of PTX on chlorine channel function of ovarian cancer cells:（1）PTX（30nM）inhibited background chloride currents of A2780 cells,but not A2780/PTX.At the clamp voltage of±80mV,the inhibitory effect of PTX on the background chloride currents of A2780 cells was（64.38±5.25）%and（42.05±2.31）%,respectively.However,the inhibitory effect of PTX on background chloride current was not observed in A2780/PTX cells,and low permeability still activated chloride current.（2）Chloride channel activity in A2780/PTX cells was significantly higher than that in A2780 cells.PTX（30 nM）inhibited the activated chloride current in A2780 cells,but not in A2780/PTX.5)The mRNA screening showed that only ClC-2 and ClC-3 mRNA were expressed in ovarian cancer cells.The expression of ClC-3 mRNA in PTX-resistant cells was significantly higher than that in A2780 cells,and the expression of ClC-3 protein in A2780/PTX cells was significantly higher than that in A2780 cells.The expression of ClC-3 protein was（4.95±0.62）times higher than that of the latter.The distribution of ClC-3 was observed by immunofluorescence.It was found that ClC-3 was mainly located in the membrane and nucleus of A2780 cells,while in A2780/PTX cells,ClC-3 was mainly distributed in cytoplasm.6)PTX is a classical microtubule protein inhibitor.We found that both ovarian cancer cells expressedα-tubulin andβ-tubulin proteins,but their expression level in A2780/PTX cells was significantly higher than that in A2780 cells.The ratio ofβ-tubulin polymerization in A2780/PTX was lower than that in A2780 cells.7)After down-regulating the expression of ClC-3 protein in drug-resistant cells:（1）The sensitivity of drug-resistant cells to PTX was increased,and the IC₅₀0 value decreased from（260.67±16.59）to（89.75±13.78）;（2）The expression of MDR gene also decreased significantly;（3）The reactive recovery of chloride channels to PTX.PTX（30 nM）inhibited the background,hypoosmotic or cisplatin-activated chloride current in ClC-3siRNA-treated cells,but still had no obvious inhibitory effect in NC siRNA-treated cells;（4）The expression ofβ-tubulin protein in drug-resistant cells was decreased,and the ratio ofβ-tubulin polymerization in drug-resistant cells was increased.8)After down-regulating the expression ofβ-tubulin protein in A2780/PTX cells,it had no effect on the expression of ClC-3 protein.Conclusion:1)There are significant differences in mRNA expression,protein expression,distribution and channel function of ClC-3 chloride channels between PTX-sensitive and drug-resistant ovarian cancer cells.2)After down-regulating ClC-3 of drug-resistant cells,the sensitivity of drug-resistant cells to PTX was increased,the relative expression of MDR was decreased,and the function of chloride channel was restored,which proved that ClC-3 was the key target of drug-resistance.3)There is a correlation between ClC-3 and tubulins,which may be involved in PTX drug resistance by affecting the expression of tubulins and its free form of polymerization.