Ferroptosis Involvement In The Treatment Of Apatinib Combined With Olaparib

Background: The death rate of ovarian cancer ranks first among gynecological malignant tumors.Most patients were in advanced stage at the time of diagnosis.Although first-line treatment programs such as surgery and chemotherapy have been developed in recent years,the five-year survival rate of ovarian cancer patients is still less than 50%,which brings a huge burden to patients and society.Therefore,it is necessary to find new treatment methods for ovarian cancer patients.Brent R Stockwell et al.defined ferroptosis in 2012,which is different from autophagy,apoptosis and necrosis.Ferroptosis is a type of programmed cell death mode which accompanied by iron accumulation and lipid peroxidation.Recent studies have shown that induced ferroptosis could inhibit the growth of a variety of cancer cells,including ovarian cancer cells.Induction of ferroptosis may be an effective anti-cancer strategy.Olaparib is one of the most classic and effective inhibitors of poly(ADP-ribose)polymerase(PARP),and has been approved by the Food and Drug Administration(FDA)for first-line maintenance treatment of advanced ovarian cancer patients with breast cancer susceptibility gene(BRCA)mutations.Apatinib is an anti-angiogenic drug that can selectively bind to vascular endothelial growth factor receptor-2(VEGFR-2),and is a new small molecule tyrosine kinase inhibitor.In addition to inhibiting angiogenesis,it can also inhibit tumor proliferation by promoting apoptosis and inhibiting epithelial mesenchymal transformation.Clinical studies have found that olaparib combined with anti-angiogenesis drugs can improve the overall survival(OS)and progression-free survival(PFS)of patients with BRCA wild-type ovarian cancer,and can improve the efficacy of drugs,but its mechanism is still unclear.Some studies have shown that olaparib can inhibit the proliferation of ovarian cancer cells by inducing ferroptosis in BRCA wild-type ovarian cancer cells,and apatinib can also inhibits cancer cells proliferation,inducing ferroptosis in a variety of cancer cells.Therefore,it is very important to understand the role of ferroptosis in the treatment of ovarian cancer with apatinib and olaparib.Objective: The purpose of this study is to investigate the role of ferroptosis and its specific mechanism in the treatment of ovarian cancer with apatinib and olaparib,and to provide a theoretical basis for clinical use of the combination of the two drugs.Methods: In this study,human ovarian cancer cell lines A2780 and OVCAR3 were used as experimental objects,and the expression of ferroptosis related protein GPX4 after apatinib combined with olaparib treatment was detected by Western blot.The effects of ferroptosis induced by apatinib combined with olaparib on cell survival and migration were observed by cloning formation and wound healing experiments.The Super Pred database was used to predict the possible targets of apatinib combined with olaparib and the predicted results were verified by Western blot experiment.ML385 and RTA408 were used to inhibit or activate the expression of Nrf2,and the effect of Nrf2 on cell migration in the treatment of ovarian cancer cells with apatinib and olaparib was determined by wound healing assay.The expression of autophagy index LC3 II/I,p62 and ferroptosis related protein GPX4 was detected by Western blot to explore the role of Nrf2 in autophagy and ferroptosis.3MA and Rapa were used to intervene autophagy,and the expression of GPX4 was detected by Western blot to explore the role of autophagy in ferroptosis.Using the p53 activator RITA,the expression of Nrf2 and GPX4 was detected by Western blot assay,and the role of p53 in the treatment of ovarian cancer with apatinib and olaparib was analyzed.Results: 1.Apatinib combined with olaparib inhibited cell survival and migration by inducing ferroptosis in A2780 cells.In OVCAR3 cells,ferroptosis was inhibited.2.In A2780 cells,apatinib combined with olaparib reduced the expression level of Nrf2.In OVCAR3 cells,apatinib combined with olaparib increased the expression level of Nrf2.3.RTA-408 was used in A2780 cells to activate Nrf2,which promoted cell migration,increased the ratio of autophagy flux index LC3II/I,decreased the expression of p62,and increased the expression of ferroptosis negative regulatory protein GPX4.ML385 was used in OVCAR3 cells to inhibit Nrf2,inhibit cell migration,reduce the ratio of autophagy flux index LC3II/I,promote the expression of p62,and reduce the expression of GPX4.It shows that Nrf2 promotes autophagy and inhibits the occurrence of ferroptosis.4.Using autophagy activator Rapa to promote autophagy in A2780 cells increased the expression of GPX4.The use of autophagy inhibitor 3MA in OVCAR3 cells inhibited autophagy and reduced the expression of GPX4.It showed that autophagy inhibited ferroptosis.5.Using p53 activator RITA to activate p53 in the p53 mutant cell line OVCAR3 cells reduced the expression of Nrf2 and GPX4,indicating that p53 promotes ferroptosis.Conclusion: 1.Apatinib combined with olaparib inhibits the proliferation of p53wild-type ovarian cancer cells,and its mechanism is to promote ferroptosis by inhibiting the expression of Nrf2 and inhibiting autophagy.2.Apatinib combined with olaparib did not significantly inhibit the proliferation of p53 mutant ovarian cancer cells,and the combination of Nrf2 inhibitor or autophagy inhibitor could improve the effect of apatinib combined with olaparib by promoting ferroptosis.3.The ferroptosis induced by apatinib combined with olaparib depends on the p53 status of cells.