Screening And Evaluation Of Anti-tumor Activity Of PARP Inhibitors

Breast cancer is the second leading cause of cancer death in women.Breast cancer susceptibility genes 1(BRCA 1)and BRCA 2 gene mutations were up to45-80% in breast cancer,and caused a very low survival rate.At present,there is no ideal cure for BRCA mutant breast cancer,so it is rather urgent to develop effective therapeutic drugs.BRCA1 and BRCA2 genes mutations are highly correlated with the incidence of breast cancer,and they play an important role in maintaining genomic stability during cell growth.Poly(ADP-ribose)polymerase(PARP)is a ribozyme which is involved in DNA repair in eukaryotic cells.PARP has 17 subtypes,PARP is mainly involved in base excision repair(BER)in the repair of DNA single strand damage.When PARP is inhibited,BER defects lead to a large number of DNA single strand breaks that can not be repaired in time that results in a large number of DNA double strand breaks.These DNA double strand lesions can be repaired in normal cells by BRCA1/2 and other factors;while in the BRCA gene deficient cells,due to the lack of HR repair pathway,the DSB can not be repaired or repaired by high error rate NHEJ repair,which greatly increases the probability of cell death.PARP inhibitors play a therapeuticrole in BRCA-deficient ovarian cancer and breast cancer through synthetic lethality mechanism.In this study,previouly obtained 26 new compounds with strong PARP inhibitory activity(analogs of PARP inhibitor AZD2281)were screened and evaluated in vivo and in vitro for their antitumor activity and mechanisms.The aim of this study was to obtain PARP inhibitors with curative effect in the treatment of BRCA mutant cancer,and to provide clues for the independent development of anti-tumor effect of PARP inhibitors.1.Screening and evaluation of antitumor activity of PARP Inhibitors in vitro In this part,we investigated the efficacy of PARP inhibitors alone on BRCA mutant tumor cells and the efficacy of PARP inhibitors in combination with DNA alkylating agent temozolomide on BRCA non-mutant tumor cells.The study tried to screen compounds with better activity from 26 new structural compounds for subsequent screening and evaluation.1.1 The effect of PARP inhibitor alone on BRCA mutant tumor cells In this part,we investigated the effects of PARP inhibitors on BRCA mutant tumor cells by detecting cell viability and apoptosis,and screened the compounds with good efficacy.1.1.1 Effects of PARP inhibitor on proliferation of BRCA mutant tumor cells In this study,the effect of PARP inhibitors Olaparib and 26 compounds such as XML120104-C(compound 5),XML12021502(compound 7),XML12022901(compound 13)and XML12032104(compound 19)on the survival of human BRCA mutated breast cancer cell line MDA-MB-436 were detected by CCK-8 method.The results showed that 15 compounds,such as compounds 5,7and so on could significantly inhibit the proliferation of MDA-MB-436 cells,and the IC50 was between 15-70 ?M,inhibitory effects of 15 compounds were close to AZD2281.1.1.2 Effects of PARP inhibitors on apoptosis of BRCA mutant tumor cells In this study,flow cytometry was used to observe the effect of 15 PARP inhibitors with strong inhibitory effect on proliferation of MDA-MB-436 cells.It was found that 9 compounds could induce advanced cell apoptosis and showed concentration-dependent,such as compounds 5,7,19 and so on.1.2 Effects of PARP inhibitors and temozolomide on proliferation of non BRCA mutant tumor cells In addition,26 compounds were used in combination with cytotoxic agent temozolomide to determine cell viability in human lung cell line A549 cells.The study found that there are seven compounds with a synergistic effect with temozolomide,such as compounds 5,7,19 and so on.Taking into account the results of the compound used alone and in combination with temozolomide ? structural diversity ? the difficulty of drug synthesis and other factors,two compounds(compound 5 and 19)with better anti-tumor activity in vitro,were selected to evaluate anti-tumor activity in vivo.2.Evaluation the antitumor activity of PARP inhibitor in vivo This part mainly studied whether the new PARP inhibitors can play an anti-tumor effect in vivo by synthetic lethality mechanism,and establish BALB/C nude mouse xenograft tumor model with human BRCA mutant breast cancer cell line MDA-MB-436 The results showed that after continuous oral administration in 23 days,AZD2281(50mg/kg)and compound 5(50mg/kg)and compound 19(50mg/kg)could significantly inhibit MDA-MB-436 tumor growth,and tumor inhibitory rates were 80.6%,73.6% and 88.8%,tumor volumes of administration groups were significantly smaller than control group.There was no significant decline in body weight and no tumor metastasis in each administration group.3.Study on the mechanism of PARP inhibitors In this part,the effects of PARP inhibitors on BRCA mutant and non-mutant tumor cells were investigated.The effects of PARP inhibitors on antitumor efficacy were studied by synergistic lethal effects.The selected compounds are four positive compounds and AZD2281,results are as follows.3.1 Effects of PARP inhibitors on BRCA mutant and non mutant tumor cell proliferation The effects of compounds 5,7,13 and 19 on the proliferation of BRCA mutant breast cancer cell line(MDA-MB-436)?non BRCA mutant breast cancer cell line(MDA-MB-231 and MCF-7 cells)were observed by CCK-8 method.It was found that the proliferation of MDA-MB-436 cells were highly inhibited,while MDA-MB-231 and MCF-7 cells was weakly inhibited by the four compounds.Results suggested that four compounds have stronger inhibitory effect on BRCA mutant cells than non-BRCA mutant cells,suggesting that they have further value in treatment of BRCA mutant breast cancer.3.2 Effects of PARP inhibitors on BRCA mutant and non mutant tumor cell apoptosis Cytometric analysis with Annexin V-FITC/PI double staining was applied to qualify the rate of apoptosis induced by compound 5,7,13,19 and AZD2281 in MDA-MB-436 cells(BRCA mutant)and MCF-7 cells(wild type).The results showed that compound 5,7 and 19 could induce the late apoptosis of MDA-MB-436 cells in a concentration-dependent manner.Moreover,these three inhibitors could cause 80% apoptosis at the concentration of 100 ?M.When it came to MCF-7 cells,these four PARP inhibitors caused less than 25 percent apoptosis even at the concentration of 100 ?M.Compound 13 had no influence on the cell cycle of both tumor cells.It suggested that the above three compounds can play an anti-tumor effect by selectively inducing apoptosis of BRCA mutant cells.3.3 Effects of PARP inhibitors on BRCA mutant and non mutant tumor cell cycle Cell cycle distribution was detected by PI labeling with flow cytometry.Results showed that compound 5 ? 7 ? 19 and AZD2281 could arrest both MDA-MB-436 cells(BRCA mutant)and MCF-7 cells(wild type)at G2 phase and S phase and their effects increased in a concentration-dependent manner(1~30 ?M).Moreover,compound 13 had no influence on the cell cycle of both tumor cells.It suggested that compounds 5?7?19 may play an anti-tumor effect by arresting tumor cells in G2 phase and S phase.3.4 Effects of PARP inhibitors on PAR protein expression in BRCA mutant tumor cells Western blotting was used to detect the expression of ADP ribose polymer(PAR)protein in human breast cancer cell line MDA-MB-436 with BRCA mutation.We found that the expression of PAR was gradually decreased with the increased concentration of PARP inhibitors(compounds 5,7,19).It suggested that compounds 5,7,19 may inhibit the expression of PAR protein,resulting in reduced DNA damage repair of tumor cells,leading to cell death.In conclusion,this paper can draw the following conclusions:1.Compound 5 and compound 19 screened from 26 PARP inhibitors with better enzymatic activity can selectively inhibite MDA-MB-436 cells proliferation and apoptosis in a concentration-dependent manner.2.PARP inhibitors compound 5 and compound 19 were able to significantly inhibit the growth of tumor-bearing mice with triple-negative breast cancer xenografts with BRCA mutation.The efficacy of compound 5 and compound 19 were close to AZD2281.3.PARP inhibitors alone have a selective inhibitory effect on BRCA mutant tumor cells,and their antitumor mechanisms may be the following:(1)induction of late apoptosis of cells;(2)PARP inhibitors may arrest cells in G2 and S phase,(3)PARP inhibitors may inhibit the expression of PAR protein,resulting in DNA damage repair of tumor cells decreased,leading to cell death.In this study,we selected compound 5 and compound 19 from 26 new synthetic compounds by in vitro experiments,and found that they had strong inhibitory effect on BRCA mutant tumor cells and nude mice transplanted tumor.This study found that PARP inhibitor anti-tumor mechanism may be through the induction of apoptosis,induced cell cycle arrest,inhibition of PAR protein expression to block DNA damage repair.