Phenethyl Isothiocyanate Enhances The Cytotoxic Effects Of PARP Inhibitors In High-Grade Serous Ovarian Cancer Cells

1.Research Background Ovarian cancer is the second leading cause for women who die from gynecological cancer worldwide.Ovarian cancer is usually diagnosed at an advanced stage(stage III or IV)due to numerous causes and atypical early clinical symptoms.In advanced ovarian cancer,cancer cells often have spread outside the ovary.The peritoneum is the first-place cancer cells spread,and it is here that fluid builds up the sites.In some cases,the first apparent symptom of ovarian cancer is the appearance of ascites.Ovarian cancer,a highly heterogeneous disease,comprises several subtypes with different biological and molecular characteristics(even in the same histological subtype).Early treatment of ovarian cancer mainly relies on cytoreductive surgery and combined platinum/paclitaxel chemotherapy.Maintenance therapy is recommended for patients with stage II and above or breast cancer susceptibility gene(BRCA)-mutated ovarian cancer who achieve clinical remission after early treatment.Poly(ADP-ribose)polymerase(PARP)inhibitors and anti-angiogenic agents are common maintenance therapy drugs.Although these patients’ conditions can be relieved by those methods,the recurrence rate of ovarian cancer is as high as 85%.The drug resistance of recurrent ovarian cancer is significantly increased.Recurrent ovarian cancer has a remission rate of less than 30% after treatment with chemotherapeutic medicines.Anti-cancer drugs have significant toxic and side effects,which limit their doses.In the past thirty years,despite the great progress in frontier research,screening,surgery,and treatment methods involving ovarian cancer,patients’ survival rate has not changed much.Therefore,there is an urgent need to find a new,effective,specific and patient-tolerable targeted therapy for ovarian cancer.Epithelial ovarian cancer(EOC)accounts for more than 95% of ovarian cancer.High-grade serous ovarian cancer(HGSOC)is the most common histological subtype of EOC,accounting for about 70%.TP53 mutation,BRCA1/2 mutation,and homologous recombination repair(HRR)deficiency may be occur in HGSOC.Notably,PARP inhibitors are mainly used for maintenance therapy in patients with ovarian cancer after responding to platinum-based chemotherapy.PARP1/2 effectively repair DNA single-strand breaks(SSB)by participating in the base-excision repair(BER)pathway.PARP1 is firstly recruited to DNA SSB sites,and then with the participation of cofactors NAD+,it promotes the process of BER by PARylation itself and other proteins involved in DNA damage repairs such as XRCC1,CHD4,APLF or CHFD.In addition,PARP1 is also involved in the repair of replication fork damage.PARP inhibitors not only impair the repair of DNA SSB by inhibiting the BER process but also prevent the release of PARP1 by inhibiting PARylation,thereby causing persistent DNA double-strand breaks(DSB).These DNA DSBs are often not accurately repaired in HRR-deficient cells,resulting in genomic instability and cell death.HGSOC can be divided into four subtypes,namely germline mutation of BRCA1/2(g BRCA),somatic mutation of BRCA1/2(s BRCA),HRR deficiency and wild-type.DNA DSBs due to directly present or replication fork stalling are not accurately repaired in HGSOC harboring BRCA mutations or HRR deficiency.Even in wild-type HGSOCs in the absence of HRR-deficiency-associated mutations,normal HRR pathways may not occur,resulting in DNA DSBs being repaired in a low-fidelity manner,and that ultimately renders HGSOCs sensitive to PARP inhibitors.Currently,the U.S.Food and Drug Administration(FDA)has approved three PARP inhibitors(Olaparib,Niraparib,and Rucaparib)for the maintenance treatment of recurrent ovarian cancer that initially responded to platinum therapy.Although PARP inhibitors have shown good therapeutic effects in the treatment of HGSOCs,the phenomenon of primary resistance or acquired resistance to PARP inhibitors often occurs.Therefore,there is an urgent need to further develop combination therapies based on PARP inhibitors to help more HGSOC patients benefit from PARP inhibitor therapy.Previous studies have shown that PARP inhibitors inhibit the repair of intracellular reactive oxygen species(ROS)-induced DNA damage by inhibiting the activity of PARP enzymes.In addition,PARP inhibitors can also upregulate the level of intracellular ROS through NOX1/4.Comprehensive genome analysis revealed that approximately 97% of HGSOCs have TP53 mutation.Functional loss caused by TP53 mutation is related to redox imbalance,which may cause an increase in ROS in cells and promote tumor cell growth.More and more research evidences show that oxidative stress regulation is an important factor in tumor development and anti-cancer response.Cells with high ROS levels may be more sensitive to ROS activators.Phenethyl isothiocyanate(PEITC),a natural compound found in cruciferous vegetable plants,quickly binds to glutathione(GSH),leads to GSH consumption,and induces the production of ROS in cells.In addition,PEITC exerts antitumor effects by regulating multiple mechanisms(such as EGFR/AKT,NAD+/ATP,and PARP).Clinical research on PEITC is also being actively carried out.It is worth noting that due to various factors participating in redox regulation and stress response,redox changes in cancer cells are very complicated,and the simple addition of ROS activators may not always lead to the preferential killing of cancer cells.Therefore,it is urgent to explore the combination of ROS activators and other drugs to expand the patient population benefiting from such drugs.2.Research purposes PARP inhibitors have been approved for the clinical treatment of ovarian cancer,but resistance to PARP inhibitors in HGSOC is widespread.There is increasing evidence that the use of ROS activators may improve the therapeutic response of drugs or overcome drug resistance.This study aims to explore the role and mechanism of ROS in promoting the antitumor activity of PARP inhibitors and provide a potential candidate therapeutic strategy for the treatment of HGSOC.3.Research methods Ovarian cancer resistant to PARP inhibitor therapy occur frequently so there is an urgent need to develop effective combination strategies.This research aimed to investigate the efficacy and mechanism underlying how PEITC enhances PARP inhibitor in the treatment of HGSOC.In this study,four HGSOC cell lines(OVSAHO,SNU119,COV362 and OVCAR4)and two cervical cancer cell lines(Ca Ski and ME180)were selected.We conducted the following experiments:(1)Cell proliferation experiments were used to detect the growth-inhibitory effects of PEITC or BMN 673 in the four HGSOC cell lines.(2)Based on the Chou-Talay method,the Calcu Syn software was used to calculate the drug combination index of PEITC and BMN 673 in the four HGSOC cell lines.(3)Long-term clonogenic assays detect the effects of hydrogen peroxide(H2O2),PEITC,BMN 673 or antioxidant N-acetyl cysteine(NAC)on the ability of the cells to proliferate.(4)Flow cytometry detection assesses the level of ROS in the four HGSOC cell lines,which were treated with H2O2,PEITC,BMN 673,or antioxidant NAC for 48 hours and stained with redox-sensitive fluorescent probe H2-DCFDA.(5)Four HGSOC cell lines were treated with H2O2,PEITC,or BMN 673 for 48 hours,and then stained with Annexin V/PI.Flow cytometry was used to detect the percentage of Annexin V+/PI-and Annexin V+/PI+ cells,and to evaluate the level of apoptosis in different treatment groups.(6)The cells to be tested were first cultured in a serum-free medium for 8-12 hours and then in a medium containing PEITC,BMN 673,or NAC for 24 hours,then collected,fixed,and stained with propidium iodide(PI).Flow cytometry was used to measure DNA content in cells to determine the effect of different treatments on cell cycle distribution.(7)Transient plasmids [p DR-GFP(addgene#26475)and p CBA-Sce I(addgene#26477)for HRR;EJ5-GFP(addgene#44026)and p CBA-Sce I(addgene#26477)for non-homologous end joining(NHEJ)] into the four HGSOC cell lines by using LipofectaminTM 3000 reagent.Flow cytometry was used to detect the percentage of GFP-positive cells in different treatment groups after PEITC or BMN 673 treatment for a certain period of time.The higher the percentage of GFP-positive cells,the stronger the HRR or NHEJ ability of cells in the treatment group.(8)Immunofluorescence assay analysis of γH2AX(a protein marker of DNA damage)foci in the four HGSOC cell lines treated with PEITC and BMN 673 for 48 hours.(9)Western blot analysis of the protein levels of Cleaved PARP(a protein marker of apoptosis),γH2AX(a protein marker of DNA damage)or PAR(reflecting the catalytic activity of PARP)in the four HGSOC cell lines treated with H2O2,PEITC,BMN 673,or antioxidant NAC for 48 hours.(10)After treating the four HGSOC cell lines with PEITC and BMN 673 for a certain period of time,the chromatin fractions of cells in different treatment groups were extracted.PARP1 protein levels in different treatment groups were analyzed by western blot.The stronger the PARP1 signal,the higher the PARP1 trapping ability.(11)Four HGSOC cell line tumor spheroids cultured in Matrigel were treated with PEITC and BMN 673 for a certain period of time,and then photographed under an inverted phase-contrast microscope,scored according to structural integrity.(12)Cells from fresh clinical samples(ascites drained from HGSOC patient or tumor tissue from cervical squamous cell carcinoma patient)were collected,separated,extracted,and used for the derivation of organoids.These constructed organoids were used to further verify the combined efficacy of PEITC and BMN 673.4.Research results Oxidative stress induced by H2O2 sensitizes HGSOC cells to PARP inhibitors.Furthermore,PEITC,a ROS activator,showed synergistic cytotoxicity with the PARP inhibitor BMN 673.Mechanistically,PEITC combined with BMN 673 synergistically induced the overproduction and accumulation of ROS,and enhanced DNA damage,G2/M arrest and apoptosis in HGSOC cells,all of which were significantly reversed by the antioxidant NAC.Although PEITC did not further enhance the PARP1-trapping ability of BMN 673,the combined effect of PEITC and BMN 673 was partially dependent on the presence of PARP1.Importantly,PEITC/BMN 673 combination abolished the growth potential of HGSOC tumor spheroids and patient-derived organoids of HGSOC and cervical cancer.5.Research conclusions Our findings demonstrate that the combined use of the ROS inducer PEITC and PARP inhibitor can produce cytotoxicity by synergistically inducing excessive accumulation of ROS,resulting in the effective killing of HGSOC and cervical cancer cells.This study will provide a basis for further exploring the combination regimen based on PARP inhibitors and expanding the scope of clinical application of PARP inhibitors.