Effect Of Silencing G6PD On Cisplatin Sensitivity Of Ovarian Cancer Cells And Its Mechanism

Among various malignant tumors of female reproductive system,ovarian cancer is the most lethal.Due to the asymptomatic nature in the early stage,almost 75%of patients with ovarian cancer are in the advanced stage when diagnosed.Operation combined with cisplatin chemotherapy is the main treatment for ovarian malignant tumor.However,after several cycles of treatment,patients eventually develop resistance to cisplatin chemotherapy.Therefore,clarifying the mechanism of cisplatin resistance in ovarian cancer and how to reverse cisplatin resistance have become key links to improve the prognosis of patients.G6PD(glucose-6-phosphate dehydrogenase)is a key enzyme in the pentose phosphate pathway(PPP),which can reduce NADP+ to NADPH and participate in cell metabolism.According to previous tumor studies,G6 PD can promote the malignant progression of various tumors,but its role in drug resistance of ovarian cancer needs to be explored urgently.PART ONE: Proteomic analysis of cisplatin-sensitive and cisplatin-resistant cells in human ovarian cancerObjective: To analyze differentially expressed proteins in cisplatin-sensitive A2780 cells and cisplatin-resistant A2780/CDDP cells in ovarian cancer,and to search for protein targets that may cause drug resistance of ovarian cancer.Methods: 1.Cisplatin-resistant ovarian cancer cell line A2780/CDDP was established by continuous induction at low concentration.2.The differential expressed proteins in A2780 and A2780/CDDP cells were analyzed by a label-free LC/MS-based protein quantification method.3.GO functional annotation analysis and KEGG signaling pathway were used to analyze the main functions and related signaling pathways of drug-resistant differentially expressed proteins in ovarian cancer.4.The expression levels of G6 PD in A2780 and A2780/CDDP cells were detected by Western blot and RT-PCR.5.A2780 cells were treated with different concentrations of cisplatin for 24 h,and the expression of G6 PD in cells was detected by Western blot.Results: 1.According to the proteomic analysis,a total of 197 differential proteins were identified,including 95 up-regulated proteins and 102 down-regulated proteins,and G6 PD protein had the highest significance.2.Bioinformatics analysis results showed that upregulated proteins(such as G6 PD,GCLM,STT3 B,etc.)and down-regulated proteins(such as MIF,PDHA1,etc.)were mostly enriched in metabolic pathways.3.The results of Western blot and RT-PCR showed that the expression level of G6 PD in A2780/CDDP cells was higher than that in A2780 cells.4.Western blot results showed that the expression of G6 PD increased with the increase of cisplatin concentration.Conclusion: The occurrence of drug resistance in ovarian cancer may be related to metabolic pathways.The expression of G6 PD in A2780/CDDP cells is especially higher than A2780 cells.These results indicate that G6 PD may promote the process of drug resistance in ovarian cancer.PART TWO: The functional research of G6 PD in drug resistance of ovarian cancerObjective: To further explore the role and potential mechanism of differential protein G6 PD in cisplatin resistance of ovarian cancer cells.Methods: 1.The A2780/ CDDP cells were transfected either by si RNA targeting G6PD(A2780/ CDDP-si G6PD)or control si RNA(A2780/CDDP-NC).2.MTT experiment was used to detect cell proliferation ability.3.Flow cytometry was used to detect the apoptosis rate.4.The levels of ROS,MDA,GSH and lipid peroxides which correlate to ferroptosis were detected in different groups(A2780,A2780/CDDP,A2780/ CDDP-si G6 PD and A2780/CDDP-NC).Results: 1.The results of MTT assay indicated that G6 PD silencing could increase the sensitivity of A2780/CDDP cells to CDDP(p<0.05).2.The results of flow cytometry showed cisplatin treatment induced more significant cell apoptosis in A2780/CDDP-si G6 PD cells compared with A2780/CDDP-NC cells(p<0.05).3.The levels of ROS,MDA and lipid peroxides in A2780/CDDP cells were lower than in A2780 cells and the level of GSH was higher(all p<0.05).Silencing G6 PD enhanced ROS,MDA and lipid peroxidation levels in A2780/CDDP cells,while decreased GSH levels(all p<0.05).Conclusion: G6 PD knockdown by si RNA transfection in cisplatin-resistant cells could induce ferroptosis and increase cell sensitivity to cisplatin in cells.