Synergistic Radiation Sensitivity Of Prostate Cancer Cell By Genistein And AG1024

Purpose:Radiotherapy is one of the most important clinical therapies for PC patients,especially for castration refractory prostate cancer(CRPC).Although the efficacy of radiotherapy is satisfactory initially,it is gradually weaken during cancer progression because of the radio-tolerance.Therefore,to find a suitable radiosensitizer has become a hot issue in the area of prostate cancer.Our team has been engaged in radiosensitization studies of prostate cancer and found that blocking EGFR or IGF1 R can increased the sensitivity of the X-ray to prostate cancer cells.However,the study found that the role of a single drug is limited.Currently,some studies have found that when two or more radiosensitizers are used in combination,synergistic effects between drugs may occur.Genistein,also known as genistein,has multiple anti-prostate cancer activity effects.We previously found that in bladder cancer,genistein can inhibit the activation of the key kinase ATM of DNA damage repair and reconstruction,inhibit DNA damage repair and reconstruction,and promote apoptosis.At the same time,Genistein can also inhibit the activation of EGFR and other tyrosine kinases.Therefore,Genistein is a very useful adjunct to other radiosensitizers.Therefore,based on our previous studies,we intend to investigate whether Genistein can synergistically inhibit the DNA damage repair with IGF1 R inhibitor and increase the sensitizing effect of radiotherapy.We aim to clarify the mechanism of such synergies and lay the foundation for clinical application of Genistein and IGF1 R inhibitors.Methods:1.Radiation dose and drug concentration settings: Prostate cancer cells(PC-3 and DU145)were irradiated with 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy,10 Gy and 20 Gy X-rays for 24 hours respectively,and then the cell viability was determined by CCK-8 assay.PC-3 and DU145 cells were pre-treated with Genistein at 10 ??,20 ??,30 ??,50 ?? and 100 ?? for 1 h,respectively,and then cell viability was measured with CCK-8 after 24 h X-ray irradiation.2.In vitro experiments:The experiment was divided into 4 groups as control group,Genistein group,AG1024 group and Genistein combined with AG1024 group.Prostate cancer cells were pretreated with inhibitor or Genistein for 1 h and then irradiated with 4 Gy X-rays.After 24 hours,the cell viability was detected by CCK-8 assay,cell proliferation was detected by cloning experiments,cell cycle and apoptosis were detected by flow cytometry.Laser confocal test was used to detect the focal formation of ?H2AX after irradiation,and the DNA damage after X-ray irradiation was determined.Western-blot was used to detect apoptosis and DNA repair-related gene expression.3.In vivo experiments: DU145 cells were implanted subcutaneously in the right flank of male rats aged 6-8 weeks.Then the mice were divided into four groups and then treated with Genistein or AG1024.And the tumor size was monitored over time.Results:1,CCK-8 experiments and colony formation experiments showed that prostate cancer cells treated with genistein or AG1024 given can inhibit prostate cancer cell proliferation and colony formation(P <0.05)and the combined effect is more significant(P <0.05).2.Flow cytometry assay showed that the apoptosis of prostate cancer cells was significantly increased by using Genistein or AG1024(P <0.05),and synergistic effect was observed when combined with Genistein and AG1024.3.Flow cytometry showed that Genistein and AG1024 could block the cell cycle of prostate cancer cells.Genistein mainly blocked PC-3 and DU145 cells in S phase,while AG1024 mainly blocked prostate cancer cells in G2/M phase.What's more,the S phase cells increased significantly when Genistein combined with AG1024.4.Western-blot showed that X-ray irradiation of prostate cancer cells alone or in combination with Genistein and AG1024 increased the expression of pro-apoptotic factors BAX and acaspase-3 and decreased the expression of Bcl-2.5.Laser confocal experiments showed that the use of Genistein and AG1024 increased the focus of ?H2AX in prostate cancer cells(P <0.05),and the combined use of both of them increased the focus of ?H2AX more significantly(P <0.05).6.Western-blot experiments showed that Genistein can inhibit both Ra51 and ku70,DNAPKcs,while AG1024 only inhibited Rad51.It is suggested that Genistein can inhibit DNA repair through both Homologous recombination(HR)and Non-homologous end joining(NHEJ)pathways,whereas AG1024 inhibits DNA repair only through the NHEJ pathway.However,the combination of the two can produce a synergistic effect to enhance DNA repair inhibition.7.Genistein or AG1024 inhibited the growth of tumor(P <0.05),and the synergistic effects were found in combination(P <0.05).Conclusions:1.Genistein and AG1024,alone or in combination,can decrease proliferation,increase apoptosis and arrest cell cycle in prostate cancer cells after radiotherapy.2.Genistein and AG1024,alone or in combination,can increase DNA double strand breaks in prostate cancer cells after radiotherapy.3.Genistein and AG1024 synergistically inhibit the DNA repair of prostate cancer cells through the non-homologous end-joining(NHEJ)pathway.4.Genistein and AG1024 provide new options for sensitization to clinical radiotherapy...