Effect And Action Mechanism Of Combination Harringtonine And PARP Inhibitor Treatment In Promoting Ferroptosis In Ovarian Cancer Cells

BackgroundOvarian cancer is one of the gynecological malignancies with a high mortality rate.Because of the lack of effective procedures for early screening,70%of patients are already in the advanced disease stage at the time of diagnosis.The odds of recurrence within 2-3 years are up to 70%,and the 5-year survival rate is less than 50%.Treatment methods include surgery,chemotherapy,targeted therapy,and other comprehensive therapies.In recent years,poly-(ADP)-ribose polymerase inhibitors(PARPi)such as olaparib and niraparib,have been approved for the maintenance treatment of ovarian cancer.However,tumor resistance to these drugs remains a problem.Therefore,there is a need to find efficacious drugs with mild adverse effects that can also reverse the drug resistance phenomenon.The alkaloid agent Harringtonine(HT),which can effectively inhibit the synthesis of proteins and thus have a potent cytotoxic effect,has mainly been used against hematological tumors.The effect of a combination of HT and PARPi therapy has not been reported.Ferroptosis is a unique type of cell death that is characterized by excessive lipid peroxidation.The use of ferroptosis inducers in combination with other anticancer agents is an important strategy in antitumor therapy.Nuclear factor erythroid 2-related factor 2(NRF2)is a key transcription factor that regulates the ferroptosis pathway.Therefore,targeting NRF2 would induce ferroptosis.The objectives of this study were to investigate the therapeutic effect and mechanism of action of combining HT and PARPi administration,explore the roles of NRF2 and ferroptosis in ovarian cancer cells,and offer novel therapeutic approaches.PurposeIn this study,the effect and action mechanism of combination HT and PARPi administration in promoting ferroptosis in ovarian cancer cells were investigated to develop a novel combination PARPi therapy for the disease.MethodsA2780 and Hey-A8 ovarian cancer cells in the logarithmic growth phase were treated with HT(at 0,0.1,0.2,0.5,1,or 2 μmol/L)or olaparib or niraparib(at 0,1,2,5,or 10 μmol/L)for 24 h,following which their viability was tested using the MTT assay.The drug treatments were performed in the presence or absence of one of the following:5 μ mol/L of ferroptosis inducer(RSL3),5 μ mol/L of ferroptosis inhibitor(Fer-1),or 5 μ mol/L of apoptosis inhibitor(Z-VAD-fmk;to exclude the effect of apoptosis).The A2780 cells were exposed to the drugs for 6 h,whereas the Hey-A8 cells were exposed for 12 h and the cells were determined using the MTT assay to observe the induction of ferroptosis.The A2780 and Hey-A8 ovarian cancer cells were treated with different concentrations of HT(0,0.1,0.2,0.5,1,and 2 μ mol/L)for 24 h,after which their levels of NRF2 protein expression were measured using the western blot assay.According to the concentration gradient,at 0.5 μ mol/L HT concentration,NRF2 protein level decreased by half;therefore,this concentration was chosen for the time gradient measurements.For this experiment,the A2780 and Hey-A8 cells were treated with 0.5μ mol/L HT for different time periods(0,3,6,12,and 24 h),following which their NRF2 expression levels were determined using the western blot assay.Additionally,the viability of the cells in the presence of the various drugs was tested.NRF2 protein expression after 24 h treatment of the cells with either one of the three drugs or HT combined with olaparib or niraparib was also determined using the immunoblot assay.Propidium iodide staining was performed on the various drug-treated cell groups.The drug treatments were performed in the presence or absence of 5 μ mol/L of RSL3,5 μmol/L of Fer-1,or 5 μ mol/L of Z-VAD-fmk.The drugs were added to the A2780 cells for 6 h and to the Hey-A8 cells for 12 h.Results1.The western blot results showed that HT downregulated the level of NRF2 protein expression in both the A2780 and Hey-A8 cells.With an increase in the HT concentration,the NRF2 protein level gradually decreased.However,the time gradient did not change significantly at the HT concentration of 0.5 μmol/L.2.The western blot results showed that the NRF2 protein expression level was significantly downregulated by HT,but it was not significantly reduced by olaparib or niraparib.NRF2 protein expression in the cells treated with HT combined with either olaparib or niraparib was only marginally downregulated compared to that in the cells treated with HT alone.3.The MTT results showed that cell viability was significantly reduced in the various drug-treated groups co-treated with RSL3 compared with that in the drug-treated groups without RSL3 exposure or the groups treated with RSL3 alone.Cell viability was restored after Fer-1 treatment but was not significantly changed by the addition of ZVAD-fmk.4.Consistent with the MTT results,the propidium iodide staining results showed that cell death was significantly increased in the drug-treated groups co-treated with RSL3 compared with that in the groups without RSL3 exposure or the groups treated with RSL3 alone.Moreover,cell death was significantly reduced in the groups co-treated with both RSL3 and Fer-1 compared with that in the groups treated with Fer-1 alone or without RSL3 exposure.The addition of Z-VAD-fmk had no significant effect on cell death.Conclusions1.HT downregulated NRF2 protein expression in a concentration-dependent manner,with no time dependency observed.HT induces ferroptosis in ovarian cancer cells mainly through the NRF2 pathway.2.Considering that the PARPi reported in the current study induce ferroptosis in the cells mainly via the SLC7A11-inhibiting pathway,PARPi induce this death process in ovarian cancer cells without going through the NRF2 pathway.That is,combinations of HT and PARPi induced ferroptosis in ovarian cancer cells in two different ways.3.The synergy of HT and PARPi with RSL3 enhanced the induction of ferroptosis in ovarian cancer cells.