The Efect Of Novel Histone Deacetylase Inhibitor AR-42 On The Proliferation And Apoptosis Of Human Retinoblastoma Cells Y79 In Vitro

Purpose:To study the effect of novel histone deacetylase inhibitor AR-42 on the proliferation and apoptosis of human retinoblastoma cells Y79 in vitro Method:The human retinoblastoma cell line Y79 was selected as the research object and interceded by AR-42.Firstly,Y79 was categorized into the experimental group and the control group.The experimental group was treated with different concentrations of AR-42(0.05-0.8μM,0.05/0.1/0.2/0.4/0.8μM).The control groups were treated with an equal volume of DMSO adjuvant(final DMSO concentration < 0.5%).Then,the following experiments were performed: MTT assay was used to observe the cell growth activity and concentration dependence of the cells in the experimental group and the control group after 24 hours,48 hours,and 72 hours,and the IC50 of AR-42 on Y79 cells was determined.The soft agar colony formation assay evaluated the colony formation of Y79 cells at different concentrations of AR-42,to evaluate the proliferation of Y79 cells.Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis of Y79 cells of the experimental group and the control group after 48 h.Western blot was used to detect the apoptosis effect of different concentrations of AR-42 on Y79 cells.After 48 hours,the level of histone H3 acetylation and the expression of apoptosis-related protein caspase 3/9,cleaved-caspase 3/9 and substrate PARP activation levels were evaluated.Finally,AR-42 combined with cisplatin(DDP)drug-assisted experiment was used to evaluate the inhibitory effect of combination therapy on the proliferation of Y79 cells.Result:AR-42 can effectively inhibit the growth and proliferation activity of Y79 cells in vitro.The results of MTT assay showed that the degree of inhibition of Y79 cells was proportional to the drug concentration and the reaction time of AR-42,and the half inhibitory concentration of AR-42-activated Y79 cells at 48 h was about 0.4 μM.Under these conditions,AR-42 was effective.Anti-Y79 cell proliferation will not cause serious side effects on normal cells.Soft agarose colony formation assay results showed that 0.05-0.8μM AR-42 inhibited the colony formation of Y79 cells and showed a significant inhibitory effect on proliferation,compared with the control group(DMSO group),the difference was statistically significant(P <0.05).The results of flow cytometry showed that AR-42 increased the apoptosis rate of Y79 cells,among which,the apoptosis rate of 0.2,0.4,and 0.8μM treated groups was significantly higher(P<0.05).Western blot results showed that AR-42 increased the level of histone H3 acetylation in Y79 cells,and activated the expression of cleaved-caspase 3/9 and cleaved-PARP.AR-42 combined with DDP drug synergistic experiments showed significant synergistic inhibition,and AR-42 concentration of0.2μM,cisplatin concentration of 0.125μM inhibition effect is most obvious(CDI<0.7).Conclusion:AR-42 can effectively inhibit the growth and proliferation of human retinoblastoma cell line Y79 cells,and has a synergistic inhibitory effect in combination with the traditional chemotherapy drug cisplatin(DDP);AR-42 induces apoptosis in Y79 cells by activating the caspase-dependent apoptosis pathway.