The Synthetic Lethal Effect And Mechanism Of PARP Inhibitor And Gemcitabine Combined Treatment On Non-small Cell Lung Cancer DNA Damage

Objective: To investigate the effect of hydrogen-rich saline combined with PI3 K inhibitor LY294002 on the proliferation and apoptosis of non-small cell lung cancer A549 cells.To explore the possible mechanisms of related signal transduction pathways in combination.Methods: A549 cells were selected and divided into four groups,DMSO(control),LY294002,hydrogen-rich saline,LY294002+hydrogen-rich saline combination.A549 cells were treated with PI3 K inhibitor LY294002 alone,hydrogen-rich saline alone or LY294002+hydrogen-rich saline combination.Cell proliferation was detected by MTT.The effect of cell oxidation and anti-oxidation of superoxide dismutase(SOD)activity and malondialdehyde(MDA)were detected.Apoptotic rates of A549 were determined by flow cytometry analysis using an Annexin V/PI double staining.The protein expression of HO-1,p65,AKT and p-AKT were analyzed by western blot.The effect of HO-1 and p65 m RNA was estimated by RT-PCR.Results:(1)Compared with the DMS0 group,LY294002,hydrogen-rich saline monotherapy and combination of LY294002 with hydrogen-rich saline show potent inhibition of cell viability.The proliferation of combination of LY294002 with hydrogen-rich saline was significantly inhibited compared with monotherapy group(P<0.05).(2)Compared with control and the monotherapy group,the SOD activity in the combined treatment of hydrogen-rich saline increased,and the MDA level increased significant compared with control(P<0.05).(3)Flow cytometry analysis using annexin V/propidium iodide staining showed that after LY294002/hydrogen-rich saline combination treatment the proportions of apoptotic cells were markedly increased in A549(22.1 + 1.7)% than single drug LY294002 group(9.5 + 4.7)% and hydrogen-rich saline group(15.7+ 0.9)%A549.The rate of apoptosis was significantly higher than control(13.4+ 1.2)%(P<0.05).(4)Western blot results showed that compared with DMSO and monotherapy,protein expression of NF-r B p65 and HO-1 decreased(P<0.01).(5)RT-PCR results showed that compared with DMSO and monotherapy,the m RNA expression of NF-r B p65 and HO-1 decreased.(6)The protein and m RNA expression of p-AKT/AKT was significantly decreased(P<0.01)in Western blot and RT-PCR.Conclusion: This combination therapy may be more effective than separate drug treatment.It decreased the malondialdehyde level and increased the super oxide dismutase activity.The combination therapy also enhanced the efficacy of anti-proliferation and apoptosis.Similarly,the results of the present study demonstrated that administration of the two agents in combination may inhibit p-Akt activity,and reduce expression of heme oxygenase-1 and nuclear factor-kB p65.The results further suggested that the combination therapy may reduce cell proliferation and promote cell apoptosis through downregulating Akt phosphorylation and inhibiting the PI3 K pathway in NSCLC cell lines.Therefore,the present study provided evidence that combined therapy is a novel therapeutic option for patients with NSCLC.ObjectiveIn this research,we focus on synthetic lethal of the combination of PARP inhibitors with the common first-line NSCLC chemotherapeutic gemcitabine in a panel of NSCLC cell lines and the mechanism of effect on DNA damage repair.Method1.A series of non-small cell lung cancer cell lines weretreated with gemcitabine,BMN 673,gemcitabine combined with BMN 673 and DMSO.Cell number was estimated by the sulforhodamine B(SRB)assay.Combination index,CI,which is drug combination treatments effect,was designed according to the Chou–Talalay equation,which accounts forsynergismof PARP inhibitors with gemcitabine in a panel of 3 NSCLC cell lines.2.Non-small cell lung cancer cell were grouped in gemcitabine,BMN673,gemcitabine combined with BMN 673 and DMSO.Apoptotic rates of NCI-H23 were determined by flow cytometry analysis using an Annexin V-FITC Apoptosis Kit.3.The expression of cellular proteinscleaved caspase3 ? cleaved PARP1 was evaluated by Western blotting according to detect the effect of combination and single drug of BMN673 and gemcitabine on apoptosis.4.The expression of Rad 51 which is the marker of HR repair was described with western blot and p CBASce I plasmid was used for detecting HR repair efficiency.5.Cell cycle progression of NCI-H23 cell was determined by Flow Cytometry(FCM)after PI staining in order to study the effect of cell cycle of combination and single drug of BMN673 and gemcitabine.6.To study the effect on DNA damage/DNA damage repair of combination and single drug of BMN673 and gemcitabine,the protein expression of?-H2 AX,p-RPA,and RAD17 in NCI-H23 and SK-MES-1cells were verified by Western Blot.?-H2 AX and Brd U,which were the markers of DSBs and SSBs separately,were determined by immunofluorescence staining.7.Xenograft tumor model of NCI-H23 cell in nude mice was established to examined whether the combination of BMN673 and gemcitabine can influent the growth of tumor.Immunohistochemistry was used to detect differentially expressed?-H2 AXandcleaved caspase3 in tumor tissue.Result1.PARP inhibitors synergize with gemcitabine in NSCLC cell lines:In H23 cells,combination of either BMN673 or AZD2281 with gemcitabine show potent inhibition of cell viability similar effects was observed in additional lung cancer cell lines H522 and SK-MES-1.In all tested cell lines,significant synergy was observed between PARP inhibitors and gemcitabine based on the calculated CI values.In addition,compared with cells treated with combinations of cisplatin and gemcitabine,combinations of PARP inhibitors and gemcitabine have the same CI values with the primary standard lung cancer chemotherapy combination.2.Combination PARP inhibitor and gemcitabine induces apoptosisFlow cytometry analysis using annexin V/propidium iodide staining showed that after BMN673/gemcitabine combination treatment for 48 h the proportions of early and late apoptotic cells were markedly increased in NCI-H23 and SK-MES-1 cell lines as caspase-3 cleavage expression which was observed by western blot suggesting that combination was able to induce the lung cancer cell apoptosis better than either single drug.3.Gemcitabine does not suppress HR repairWe used fluorescent reporter constructs in which a functional GFP gene is reconstituted following an HR event.The functionality of the HR reporter cassette has been confirmed by isolating the GFP+ cells by flow cytometry.We found that gemcitabine increased GFP+ cells suggesting increased HR repair in both H23 and SK-MES-1cells.To verify this,we further analyzed expression of Rad51,a marker of HR repair.Western blot analysis showed no alterations in total protein level.Using single cell immunofluorescence staining for Rad51,we also observed no loss of formation in Rad51 foci after 48 hours of treatment in combination treated H23 samples versus either single treatment or untreated.4.Combination PARP inhibitor and gemcitabine alters cell cycle dynamicsAnalysis of the combination therapy revealed aspects of both monotherapies,producing s-phase arrest at early time points which transitions into G2/M phase arrest as treatment continues,and by 72 hours combination-treated cells show nearly 2-fold increase as seen in BMN673 monotherapy.5.Combination PARP inhibitor and gemcitabine induces synergistic DNA damage mediated by increased single-strand breaksWe found that in both cell lines H23 and SK-MES-1,protein expression of histone 2AX(H2AX)phosphorylation on Serine 139(?-H2 AX,a marker for DNA double-strand breaks)increased with combination of gemcitabine and PARP inhibitor BMN673.However,we observed even larger synergistic increases in expression of phosphorylation of RAD17 and RPA,markers for DNA single-strand breaks.To further confirm this observation,we examined the formation of DNA damage foci by staining for ?H2AX as a double-strand break marker and native Brd U as a marker of single-stranded breaks indicative of replication stress.Quantification indicated that the intensity of ?H2AX cells was greater in combination treatment versus monotherapy and untreated control(P <0.05),consistent with the Western blotting data.In contrast,levels of single-strand breaks as indicated by native Brd U,were increased much more dramatically(combination via gemcitabine,P < 0.01;combination via BMN673,P < 0.05).Immunohistochemical analysis of H23 tumor tissues at the termination of the study demonstrated that this phenotype was also observed in vivo,with large increases in ?-H2 AX staining intensity in gemcitabine/BMN673 combination treated tumors.6.Combination PARP inhibitor and gemcitabine results in tumor regression in NSCLC xenograft modelsFor the H23 xenografts,BMN673 or gemcitabine exposure delayed tumor growth compared with the vehicle group,and the combination treatment was the most effective at slowing tumor growth.Nonetheless,the BMN and gemcitabine combination was superior to BMN(P=0.019)or gemcitabine alone(P=0.033)in reducing tumor growth.Analysis of H23 tumors at the termination of the study indicated tumors treated with combination therapy had increased apoptosis,as indicated by immunohistochemical staining of cleaved caspase 3.This was consistent with the cleavage of caspase-3 observed in vitro,where the largest increase was observed in cells treated with the gemcitabine/BMN673 combination.CONCLUSION:The synthetic lethal of combination of PARP inhibitor with gemcitabine was shown in the treatment in non-small cell lung cancer in vivo and vitro in our study.The combination can effectively inhibit the tumor growth,increase the single-strand breaks and double-strand breaks of DNA,and induce the cancer cell apoptosis.The reason of synthetic lethal is not the change of HR repair,but stalling of replication forks by gemcitabine.The stalling of replication forks need PARP to restart and it will continue stall because of PARP inhibitor and inducemore double-stranded DNA breaks damage and lead to cell death finally.