| Objective:To investigate the inhibition effect of Chloroquine and the inhibition effect of autophagy on ovarian cancer cells,and to further study the inhibition effect of Chloroquine combined with PARP inhibitor-olaparib on ovarian cancer platinum insensitive cells in vitro and in vivo,so as to provide new ideas and experimental basis for the difficult treatment of drug-resistant ovarian cancer.Methods:1.CCK8 method was used to detect the inhibitory effect of chloroquine,Olaparib and cisplatin on ovarian cancer cells A2780S,a2780CP,OV2008 and C13*,calculate the drug IC50,and verify the drug resistance index of A2780CPand C13*.2.CCK8 method was used to detect the inhibitory effects of Chloroquine,Olaparib,Chloroquine+Olaparib on ovarian cancer cells A2780CP,C13*.3.Detection of apoptosis of A2780cp cells by flow cytometry.4.WB method was used to detect the effect of different concentrations of Chloroquine on the inhibition of autophagy in ovarian cancer cell line A2780CP,and the effect of Olaparib+Chloroquine on the autophagy proteins Beclin 1,P62,LC3B.5.A2780CP cells were used to establish subcutaneous tumor model in nude mice.The control group,Chloroquine group,Olaparib group,Cisplatin group and Chloroquine+Olaparib group were set.The effect of drug combination was compared by tumor volume.6.HE staining was used to observe the morphological changes of tumor tissue in each group.7.Autophagy structure of tumor tissue was observed by transmission electron microscope.8.Drug safety test:observe the activity,weight change and HE staining of liver and kidney sections of nude mice to observe the structural change.Results:1.Chloroquine has a certain anti ovarian cancer effect and can block the autophagy of ovarian cancer cells at a concentration of 5μg/ml.2.For drug-resistant ovarian cancer cell lines A2780cp,C13*,the combined anti-tumor effect of Chloroquine and Olaparib is better than that of single drug,and has a slight synergistic or additive effect at a certain concentration.3.In vitro experiment,Olaparib can affect the number of autophagic proteins in ovarian cancer cells,and the overall protein change shows an increase in autophagy level;in vivo experiment,the ultrastructural analysis of tumor sections in Olaparib group suggests that autophagy structure increases.4.Flow cytometry showed that the apoptosis of the combination group of chloroquine and Olaparib was mainly late apoptosis,and the total apoptosis rate was higher than that of the single drug group,which was statistically significant.5.In vivo experiments showed that the tumor volume of group control was 2929±131mm~3,group CQ was 2042±128mm~3,group OLA was 830±219mm~3,group DDP was 520±185mm~3,group CQ+Ola was 349±160mm~3.between Group CQ+Ola、DDP、Ola and group control have significant difference,between CQ+OLA and OLA have significant difference(P=0.037);between CQ+OLA and CQ have significant difference(P=0.000);between CQ+OLA and DDP have no significant difference(P=0.272);between CQ and group control have no significant difference(P=0.646)6.HE staining showed that the tissues of nude mice in each group were epithelial ovarian tumors with obvious mitosis.7.Under transmission electron microscope,autophagy was enhanced in group Olaparib,lysosomes were accumulated,and the ratio of autophagy vesicles/autophagy lysosomes of group Chloroquine and group Chloroquine+Olaparib was lower than other groups.8.There was no significant difference in the body weight of nude mice in each group,and no significant damage in the structure of liver and kidney compared with the control groupConclusion:Chloroquine has the effect of blocking autophagy on ovarian cancer cells,and has a better single drug anti-tumor effect.Olaparib can enhance the autophagy level of ovarian cancer cells.The drug combination effect of Chloroquine and Olaparib is better than that of single drug in vitro and in vivo. |
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