Lanthanum Chloride Enhancesthe Sensitivity Of Cisplatin-resistant Ovarian Cancer Cells And Research About The MolecularMechanism

Objective:Ovarian cancer is the most malignant tumor in female reproductive system.Clinical studies have shown that chemotherapy after ovarian cancer cell cytoreductive surgery is irreplaceable.Platinum-based chemotherapy is considered a standard program and widely used in clinic.In recent years,despite the emergence of new chemotherapeutic drugs,the 5-year survival rate of patients with advanced ovarian cancer has not been significantly improved,mainly due to chemotherapy resistance.Based on the clinical practice of chemotherapeutic resistance,and preliminary work of the group(lanthanum chloride can enhance the chemotherapeutic sensitivity of cisplatin-resistant strains in vitro at the cellular level),this study verified the results of in vitro experiments at the animal level,and by using differential proteomics to study differentially expressed proteins,immunohistochemistry and western-bloting techniques to verify the results,and explore the related mechanisms,for the clinical treatment of cisplatin-resistant ovarian cancer,and provide scientific basis for the development of medicinal value of rare earth lanthanum chloride.Methods:1.The transplanted tumors of the two cells(Sensitive cell line,drug-resistant cell line)were set as control group,cisplatin group,lanthanum chloride group,lanthanum chloride + cisplatin group.The length(a),width(b)and height(c)of tumor nodules in each experimental group were measured with vernier caliper.The approximate volume(V)of the tumor was calculated by the formula,V =abc *?/6(unit cm3).2.TUNEL was used to detect the apoptosis of COC1 and COC1/DDP cells transplanted tumors in different groups(control group,cisplatin group,lanthanum chloride group,lanthanum chloride + cisplatin group).3.Immunohistochemical method was used to detect the expression of tumor-associated proteins in COC1 and COC1/DDP cells transplanted tumors of different groups(control group,cisplatin group,lanthanum chloride group,lanthanum chloride + cisplatin group).4.Free quantitative proteomic analysis was used to determine differentially expressed proteins in cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after cisplatin application.Using GO analysis,the proteins were divided into different subgroups based on their cellular components,biological processes,and molecular functions.KEGG pathway analysis was used to identify key signaling pathways involved in these proteins.5.The COC1/DDP cells in logarithmic growth phase were divided into control group,cisplatin group,lanthanum chloride group,lanthanum chloride + cisplatin group.After 48 h of culture,the expression of 5 proteins(including ERCC1,Ki67,CDK6,c-Cbl and EB1)in different experimental groups were detected by Western Blot.Results:1.Effects of lanthanum chloride on tumor growth in vivo: the combination of cisplatin and lanthanum chloride can inhibit the growth of tumor in vivo.Compared with the control group,cisplatin alone could significantly reduced tumor weight and volume by 45% and 54%,respectively(p<0.001).The lanthanum chloride + cisplatin group resulted in a 54% and 61% reduction in tumor weight and volume compared to the control group(p<0.001).In contrast,lanthanum chloride had no statistically significant effect on tumor growth(p>0.005).The results were basically consistent between COC1 and COC1/DDP cell.The results showed that lanthanum chloride combined with cisplatin inhibited the growth of ovarian tumors in vivo.2.TUNEL method was used to detect the apoptosis of transplanted tumors in every group: compared with the control group,Tunel positive cells in COC1 cells treated with cisplatin were significantly increased,with statistically significant differences(p<0.001).Compared with the lanthanum chloride+cisplatin group,the addition of lanthanum chloride further promoted the apoptosis of tumor cells,so the proportion of apoptotic cells in the lanthanum chloride + cisplatin group group was significantly increased(p<0.001).However,there was no significant effect on apoptosis of tumor cells treated with lanthanum chloride alone(P > 0.05).The results in the COC1/DDP cell were consistent with that of the COC1 cell.3.Immunohistochemistry was used to detect the protein expression in the transplanted tumors of every group: compared with the control group,the expression level of Ki67 in the cisplatin group was decreased,and the expression level of Ki67 in the lanthanum chloride+cisplatin group was further decreased.However,lanthanum chloride alone has little effect on the expression of Ki67.The expression of oncogenes and anti-apoptotic protein BCL-2 was similar to that of Ki67.In contrast,expression levels of the human tumor suppressor genes BRCA1 and BRCA2 were oposite to those of Ki67 and BCL-2.In other words,cisplatin increased the expression levels of BRCA1 and BRCA2,while co-treated with lanthanum chloride and cisplatin further improved the expression levels of BRCA1 and BRCA2.The expression level of DNA repair gene ERCC1 was basically consistent with that of Ki67 and BCL-2,and the expression level of ERCC1 was reduced in the group of cisplatin group and lanthanum chloride+cisplatin group.The positive rate of BCL-2 and Ki-67 in the control group was significantly higher than that in lanthanum chloride + cisplatin group and cisplatin group(P<0.01).The positive rates of BRCA1 and BRCA2 were opposite to those of ERCC1,BCL-2 and Ki-674.The results of proteomics research have shown that 3537 qualitative and quantitative proteins were detected in the experiment,and GO annotation analysis was performed on these proteins.There were 710 proteins differentially expressed between COC1 and COC1/DDP,and 783 proteins differentially expressed between COC1+cisplatin and COC1/DDP + cisplatin,917 proteins differentially expressed between COC1+ lanthanum chloride and COC1/DDP + lanthanum chloride,775 proteins differentially expressed between COC1/DDP + cisplatin and COC1/DDP + cisplatin + lanthanum chloride.Before and after cisplatin addition,there are 411 proteins differentially expressed between COC1 and COC1/DDP cell.Before and after adding cisplatin,there were 536 different proteins between COC1/DDP and COC1/DDP+lanthanum chloride.Among them,14% were located on the cell membrane,and KEGG analysis divided the differentially expressed proteins into 21 subgroups.5.Western blot was used to verify the expression of functional proteins.The results showed that the expression of five functional proteins(ERCC1,Ki67,CDK6,c-Cbl and EB1)in COC1/DDP treated with cisplatin,lanthanum chloride and cisplatin combined with lanthanum chloride.The results showed that the expressions of EB1 and c-Cbl in cisplatin combined with lanthanum chloride group were significantly higher than those in control group and lanthanum chloride group,while the expressions of ERCC1,Ki67 and CDK6 in drug-resistant strains were contrary to those in EB1 and c-Cbl.It is suggested that lanthanum chloride may down-regulate the expression of ERCC1,Ki67 and CDK6 in various ways,and up-regulate the expression of EB1 and c-Cbl to enhance the anti-tumor effect of cisplatin.Conclusion:Lanthanum chloride down-regulates the expression of BCL2,Ki-67,ERCC1 and CDK6 through multiple pathways,in addition,up-regulating the expression of BRCA1,BRCA2,EB1 and c-Cbl to enhance the sensitivity of cisplatin in ovarian cancer,and that combined with cisplatin,the apoptosis of xenograft tumor cells in COC1 and COC1/DDP was greatly promoted in vivo,thus enhancing the antitumor effect of cisplatin,and inhibiting tumor growth in vivo,but the specific regulatory mechanism needs to be further studied.