Research On The Synergistic Antitumor Efficacy Of Hyperthermia And Olaparib In Ovarian Cancer

Background Ovarian cancer is the most lethal gynecologic malignancy.About 75% of patients are diagnosed at an advanced stage.Although,>80% of these patients have response to initial chemotherapy,the majority ultimately relapse with chemotherapy-resistant disease.The overall 5-year survival rate for ovarian cancer remains at 47% and new treatments are urgently needed.The poly(ADP-ribose)polymerase(PARP)inhibitors bring new hope to the treatment of ovarian cancer.PARP1/2 play an important role in repair of DNA single-strand breaks(SSBs).When the function of PARP is inhibited,the unrepaired SSBs are converted into double-strand breaks(DSBs)which predominantly depend on homologous recombination(HR)DNA repair pathway.However,in HR-deficient cells,the DSBs cannot be accurately repaired and then result in potentially lethal accumulation of DNA lesions and eventually lead to synthetic lethality.Thus,cancer cells with BRCA mutation are particularly sensitive to PARP inhibition.Of the PARP inhibitors,Olaparib has been approved by FDA for BRCA1/2-mutated advanced ovarian cancer treatment.However,BRCA mutation rate only accounts for 15%-25% of ovarian cancer in China,and how to improve the sensitivity of BRCA wild-type ovarian cancer to PARP inhibitors has become a focus of research.ROS are oxygen-containing derivatives composed of highly unstable oxygen free radicals.Excessive ROS can enhance the level of intracellular oxidative stress,causing severe damage to fat,protein and DNA,leading to cell death.Recently,PARP inhibitors has been reported to exert antitumor effect through increased reactive oxygen species(ROS)inducing DNA oxidative damage in ovarian cancer cells.Increased oxidative stress may be one of the mechanisms of antitumor effects of PARP inhibitor.Hyperthermia is an anti-cancer treatment in which tumours are heated using an exogenous energy source.It is usually used as a “sensitizer” in combination with chemotherapy or radiotherapy to have greater potency.HT can inhibit HR via targeting the BRCA2 protein for proteasomal degradation in both human cancer cells and tissues.Furthermore,HT can increase generation of ROS in cells,which is part of the mechanism by which PARP inhibitors kill tumors.However,the antitumor efficacy of HT combined with PARP inhibitor in ovarian cancer remains to be determined.In this study,we speculated that HT impairs HR and increased oxidative stress in ovarian cancer cells and thus enhances antitumor efficacy of PARP inhibitor.Objectives 1.To evaluate the effect of HT on HR pathway and oxidative stress in ovarian cancer cells.2.To confirm the synergistic antitumor efficacy of hyperthermia and Olaparib in ovarian cancer cells.Materials and Methods In this study,human ovarian cancer cell line A2780(BRCA wild-type)was used as the experimental object.RNA-seq was firstly performed to detect the expression of genes in HR pathway and genes related to oxidative stress changed after HT treatment.Secondly,immunofluorescence assay was used to detect the formation of RAD51 foci,a key step of HR pathway,to observe HR pathway in treated ovarian cancer cells.Next,the ROS level in ovarian cancer cells and the expression of 8-OHd G,a marker of DNA oxidative damage,were separately detected by oxidation sensitive fluorescent probe(DCFH-DA)and Western Blot.Then,alkaline comet assay was used to measure the level of DNA damage in ovarian cancer cells.Meanwhile,the expression of γ-H2 AX,a marker of DSBs,was detected by Western Blot and immunofluorescence assay.Finally,CCK8 assay and clonogenic assay were performed to evaluate the antitumor efficacy of HT and Olaparib in ovarian cancer.Results 1.The expression of genes in HR pathway and genes related to oxidative stress greatly changed in HT-treated ovarian cancer cells.2.HT impaired the formation of RAD51 foci in ovarian cancer cells.3.Combined HT and Olaparib synergistically increased oxidative stress in ovarian cancer cells.4.Combined HT and Olaparib synergistically increased DSBs of DNA in ovarian cancer cells.5.Combined HT and Olaparib synergistically inhibited the growth of ovarian cancer cells.Conclusion HT impairs HR and increased oxidative stress in ovarian cancer cells and thus enhances antitumor efficacy of PARP inhibitor.