| Objective:Ovarian cancer is one of the most common malignancies in the female reproductive system.Platinum-based chemotherapy is considered as the first-line program for the patients with postoperative chemotherapy.Although chemotherapy drugs are constantly updated,but the 5-year survival rate has not been effectively improved,the main reason is chemotherapy resistance.This study is to investigate the sensibility of rare earth lanthanum chloride to enhance ovarian cancer cisplatin resistant cells to cisplatin,to make a preliminary study of its related mechanism,and to provide new ideas and theoretical basis in treating the cisplatin-resistant ovarian cancer patients.Methods:1.The growth inhibition rate of COC1 and COC1 / DDP cells was detected by MTT assay in different concentrations of cisplatin(0.01,0.1,1,10,100 ?g/m L),and the half inhibitory concentration(IC50)and resistance index(RI)were collected.2.The respective proliferation inhibition rate of COC1 and COC1/DDP cells cultured with different concentrations of lanthanum chloride(0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 ?mol/L)and combined with IC50 concentration of cisplatin for 48 h were determined by MTT assay.MTT assay was used to detect the proliferation inhibition rate of COC1 and COC1/DDP cells which cultured 48 h with different concentrations of lanthanum chloride(0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 ?mol/L).With the above concentration of lanthanum chloride and IC50 concentration of cisplatin,culture COC1 and COC1/DDP cell for 48 h,and the proliferation inhibition rate was detected by MTT assay.3.The morphological changes were observed after COC1 and COC1/DDP cells were cultured with 0,0.5,1.5 and 4.0 ?mol/L lanthanum chloride for 48 hours under transmission electron microscope.4.The sensitive and resistant strains were set as the control group,cisplatin group,lanthanum chloride group,lanthanum chloride + cisplatin group.The apoptosis rate of COC1 and COC1/DDP cells was detected by flow cytometry.5.The COC1/DDP cells in the logarithmic growth phase were divided into control group,cisplatin group,lanthanum chloride group and lanthanum chloride + cisplatin group.After 48 h of culture,ERCC1,Ki67,CDK6 and c-Cbl protein expression in COC1/DDP cells were detected by Western Blot.Results:1.Cisplatin have significant growth inhibition on COC1 and COC1/DDP cells,and with the increase in cisplatin concentration,there is a significant dose-dependent.The IC50 of COC1 cell line was 2.06 ?g/ml,the IC50 of COC1/DDP cell line was 23.08 ?g/ml,and the resistance index was 11.19 times.2.MTT assay was detect cell proliferation inhibition rate: in COC1 cell group,the proliferation inhibition rate of cells increased with the lanthanum chloride concentration contrast to the control group(P> 0.05).When the concentration of lanthanum chloride was 1.5 ?mol/L and below,the inhibition rate of the cells was low,but when above 1.5 ?mol/L,the inhibition rate of the cells was high.In the COC1 + DDP cell group,when the concentration of lanthanum chloride was less than 1.5 ?mol/L,the inhibition rate of the drug was still lower than that of the control group(P> 0.05),but the inhibition rate increased with the increase of lanthanum chloride concentration.When the concentration of lanthanum chloride was more than 1.5 ?mol/L,the inhibition rate of the drug was significantly higher than that of the control group(P <0.05).The results in COC1/DDP cells group were similar to the group of COC1 cells.So that 1.5 ?mol/L lanthanum chloride has no obvious side effects on ovarian cancer cells,and in combination with cisplatin,can significantly enhance the role of cytotoxicity.This concentration was chosen for the best concentration of sensitization for the follow-up study.3.The morphology was Observed under transmission electron microscopy: in the lanthanum chloride groups between different concentration(0,0.5 ?mol/L),the morphology of COC1 and COC1/DDP cells was normal,the nuclei were intact,the organelles and nuclei were clear,and there was no significantly difference,In the lanthanum chloride group(1.5 ?mol/L),the morphology of COC1 and COC1/DDP cells showed slight shrinkage,the cell state was slightly worse than the former two groups.In the lanthanum chloride group(4 ?mol/L),apoptosis was serious,and apoptotic boby formed.4.The apoptotic rates were detected by flow cytometry: the apoptotic rates of COC1 cells respectively were 5.52±0.92%,25.40±4.38%,7.22±2.55%,27.54±4.03% in the control group,cisplatin group,lanthanum chloride group and lanthanum chloride + cisplatin group.The apoptotic rates of COC1/DDP cells respectively were 4.60±1.11%,18.24±2.81%,5.59±1.73%,29.12±5.38% in the control group,cisplatin group,lanthanum chloride group and lanthanum chloride + cisplatin group.The change trend is consistent with the MTT test results.5.Western Bolt detection results: the expression of ERCC1,Ki67,CDK6 and c-Cbl protein in the control group,lanthanum chloride group,cisplatin group and lanthanum chloride + cisplatin group were gradually decreased.Compared with the control group,the expression of ERCC1,Ki67,CDK6 and c-Cbl were down-regulated in the lanthanum chloride group,but there was no significant difference(P> 0.05).In the cisplatin group and lanthanum chloride + cisplatin group,the expression of ERCC1,Ki67,CDK6 and c-Cbl protein were significantly lower than that of the control group(P <0.01).The expression of ERCC1,Ki67,CDK6 and c-Cbl protein was significantly lower than that of cisplatin group(P <0.01).Conclusion:The lanthanum chloride(1.5?mol/L)has no significantly inhibition on ovarian cancer cells and drug-resistant cells,but co-culture with cisplatin could enhance the inhibition of cisplatin to COC1 and COC1/DDP cells,and promote apoptosis.The lanthanum chloride(1.5?mol/L)can enhance the killing effect of cisplatin on ovarian cancer resistant cisplatin cells in vitro,which may bemediatedt by the expression of ERCC1,Ki67,CDK6 and c-Cbl,improve the sensitivity of ovarian cancer resistant cell to cisplatin,but its specific regulatory mechanisms need more research. |
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