Effect And Mechanism Of PARP Inhibitor Rucaparib Combined Therapy Against Glioblastoma

Objective:Glioblastoma is a type of tumor with extremely high mortality and recurrence rate.At present,surgery is combined with postoperative radiotherapy and chemotherapy,and the 5-year survival rate is only about 10%.Glioblastoma faces a great challenge in treatment,which is reflected in its extremely prone to primary or secondary resistance to radiotherapy and chemotherapy,resulting in limited choices of radiotherapy and chemotherapy drugs that can be used for clinical treatment.More than 20 years have passed,temozolomide is still the “golden first choice” for chemotherapy for patients with glioblastoma.Compared with the recent emergence of chemotherapeutics and targeted drugs for other cancers such as lung cancer and breast cancer,the development of therapeutic drugs for glioblastoma appears to be very slow and inefficient.As the only marketed anti-cancer drug developed based on the concept of“synthetic lethality”,PARP inhibitors have set off a “turbulent wave” in the field of cancer treatment,giving hope to many cancers that had no targeted drug options before.It is worth mentioning that PARP inhibitors have shown relatively ideal effects in combination with the chemotherapy drug temozolomide and adjuvant radiotherapy,and have entered the clinical trial stage.However,their role is only an auxiliary for the time being.Even if it is finally promoted to the clinic,its application in the treatment of glioblastoma is actually very limited.This article aims to further promote and expand the applicatio n of PARP inhibitors in the treatment of glioblastoma based on the “first appearance” of the application of PARP inhibitors in glioblastoma.To interpreting the “synthetic lethal” theory and to explore the current other targeted inhibitors such as PI3 K inhibitors,BET inhibitors in glioblastoma,through causing homologous recombination deficient phenotypes to enhance the anti-tumor efficiency of PARP inhibitors.The combination of low-toxic targeted drugs can provide strong preclinical experimental data,improving the cure rate and survival rate of patients with glioblastoma ultimately.Methods:1.The effects and synergistic effects of PARP inhibitor Rucaparib and PI3 K inhibitor on the proliferation of glioblastoma cells were detected by MTT experiment,and the effects of Rucaparib and BKM120 on the formation of glioblastoma cells were detected by cloning formation assay.Comet experiment was used to detect the DNA damage of glioblastoma cel s.2.Cell cycle test was used to detect the effect of Rucaparib co mbined with BKM120 on cell cycle;using HR reporter assay to determine the effect of the combination on homologous recombination repair efficiency;Immunofluorescence was used to evaluated DN A damage and repair after combination;Western blot was used to study the effect of the combination on the expression of apoptosis-related protein,homologous recombination repair related protein,drug efflux transporter,etc.The models of transplanted tumor(subcutaneous tumor and in situ tumor)and zebrafish in situ transplanted tumor were constructed to study the antitumor effect,survival time and toxicity of BKM120 and Rucaparib in vivo.3.Western blot and immunohistochemistry were used to detect the main markers PAR level and drug efflux transporters after entering the brain of nude mice without tumor when Rucaparib combined with BKM120.4.The effects and synergistic effects of PARP inhibitor Rucaparib and BET inhibitor on the proliferation of glioblastoma cells were detected by MTT experiment,and the effects of Rucaparib and OTX015 on the formation of glioblastoma cells were detected by cloning formation assay.Immunofluorescence and Western blot were used to detect the DNA damage of the cel s.5.The final effect of Rucaparib and OTX015 on cell cycle was detected by cell cycle experiment;the effect of the combination on homologous recombination repair efficiency was detected by HR reporter experiment;and the effect of the combination on the expression of homologous recombination repair related proteins was detected by Western blot.The key gene levels of homologous recombination repair were detected.The anticancer effect of OTX015 and Rucaparib were detected in zebrafish orthotopic tumor model.6.The effects of Rucaparib and OTX015 on the clonal formation of glioblastoma cells were investigated by clonal formation assay;Annexin V/PI double staining and Western blot were used to investigate the effects of different drug delivery order on apoptosis;Cell cycle test was used to determine the final effect of the combination on cell cycle.7.Effects of sequential administration and continuous administration on the DNA injury of glioblastoma and normal cells were investigated by comet assay,and the effect of sequential administration and continuous administration on the formation of normal cell clone was investigated.The antitumor effect of sequential administration and continuous administration in nude mice was studied by in situ transplantation model.Results:1.Rucaparib in combination with BKM120 significantly inhibited cell proliferation in U251 and U87 MG cells,significantly reduced the clonal formation of tumor cells;The combination significantly co-induced apoptosis,increased expression of apoptotic protein cleaved PARP and cleaved caspase-3,reduced BKM120-induced PAR upregulation,reduced resistance to apoptosis;A combination of the two significantly increased DNA damage in tumor cel s.2.The combination of Rucaparib and BKM120 reversed the G2/M block caused by partial Rucaparib,reducd the efficiency of homologous recombination repair of U251 and U87MG cells,The combination caused increased γ-H2 AX “foci” and decreased RAD51 “foci”,BKM120 inhibited Rucaparib-induced expression of homologous recombinant repair-related protein BRCA1/2、RAD51、RRM2,possibly through its inhibition in levels of ATR and CHK1 phosphorylation in homologous recombination repair pathways;BKM120 inhibited Rucaparib expression of drug efflux transporter P-gp and BCRP;Rucaparib suppressed BKM120-induced PAR elevation.From the zebrafish orthotopic tumor model,we can see that while Rucaparib in single use in vivo is poor,after combined with BKM120,the tumor volume decreased significantly.We further discovered by establishing models of subcutaneous and orthotopic xenograft tumors in nude mice,Rucaparib significantly inhibited the growth of xenograft tumor in nude mice,Homologous recombination repair related protein BRC A1/2、RAD51 level decreased significantly,DNA damage sign γ-H2 AX level rises,The P-gp and BC RP levels of drug efflux protein transporter in tumor mess decreased significantly.BKM120 is toxic to mice with subcutaneous and in situ transplanted tumors,and Rucaparib addition does not al eviate this toxicity.3.Rucaparib significantly reduced the P-gp and BC RP levels of drug efflux transporters in the brain region under the cooperation of BKM120,from which made the Rucaparib play an effective role in the bra in region and inhibited the increase of PAR.4.The combination of Rucaparib and OTX015 inhibited the proliferation of U251 and U87MG cells,induced apoptosis,increased the expression of apoptotic protein cleaved PARP,increased the damage of tumor cells and caused the up-regulation ofγ-H2 AX expression.5.The combination of Rucaparib and O TX015 can reverse the G2/M block caused by partial Rucaparib,Reducing the efficiency of homologous recombination repair of U251 and U87 MG cells,OTX015 inhibited Rucaparib-induced expression of homologous recombinant repair-related protein BRCA1/2、RAD51、RRM2、MYC,reversing PARP inhibitor-induced homologous recombination repair ac tivation,combination leads to “synthetic death”.In the zebrafish orthotopic tumor model,A combination of the two significantly inhibited the growth of U87 MG transplanted tumors.6.The Rucaparib and OTX015 of sequential administration can effectively inhibit the formation of glioblastoma U251 and U87 MG cell cloning,from the levels of apoptosis and apoptosis-related proteins,The sequential administration of first Rucaparib and then OTX015 was significantly better than that of individual administration,To induce apoptosis,To achieve the same effect as continuous administration;From the cell cycle results,sequential administration BET inhibitor can effectively restrict the activation of G2/M cell cycle checkpoints dominated by PARP inhibitors.As WB can see,PARP inhibitor response to homologous recombinant repair proteins is more pronounced under sequential administration,And sequential administration of BET inhibitor can effectively inhibit homologous recombination repair related proteins,Consistent with continuous administration,as BRCA1/2、RAD51 expression.7.Colony formation assay showed that the sequential effect of Rucaparib and OTX015 was the same as that of continuous effect in tumor cell U251 and U87 MG,while the damage degree of sequential administration to normal cells was significantly lower than that of continuous administration in normal cell BV-2 and HUVEC.Comet experiments show that sequential administration can effectively induce DN A damage of tumor cells,while for normal cells,continuous administration also causes DNA damage to normal cells,while the DN A damage is significantly reduced by sequential administration.7.The synergistic anti-tumor effect of Rucaparib and O TX015 has been verified in vivo.From the intervention results of the in situ glioma model,it can be seen that the growth of tumor under sequential administration is well inhibited,even better than that of continuous administration,which may be related to the long-term continuous drug resistance.Importantly,the survival time of sequential administration was significantly prolonged compared with that of single Rucaparib.Conclusion:The combination of Rucaparib and BKM120 can play the role of synergistic antiglioblastoma cell proliferation and promote apoptosis.The DNA single strand damage to glioblastoma by BKM120 which inhibit homologous recombination repair process is irreversible,that is,“synthetic death”;Through the verification of three glioblastoma models in vivo,we proved that the combination of the two can significantly inhibit the growth of tumor,inhibit the process of homologous recombination repair.Besides,BKM120 can reduce the level of drug efflux protein in vivo and in vitro,which can improve the efficiency of Rucaparib anti-glioblastoma;The combination of Rucaparib and OTX015 can pla y a powerful synergistic role in the proliferation of glioblastoma cells under continuous and sequential administration.The sequential administration of first Rucaparib and then OTX015 can effectively target tumor cells in vitro and do less harm to normal cells.The combination can effectively inhibit tumor growth and prolong survival time,showing a comparable efficacy to continuous administration with lower doses.