CUDC-907诱导胶质瘤细胞DNA损伤与自噬的实验研究
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摘要
目的:探讨新型组蛋白去乙酰化酶(histone deacetylase,HDAC)/磷脂酰肌醇3-激酶(phosphatidyl-inositol 3-kinase,PI3K)双靶点抑制剂CUDC-907对人胶质瘤U251细胞DNA损伤、细胞周期及自噬的影响。方法:采用不同浓度的CUDC-907处理U251细胞24 h,MTT法检测细胞活力的变化;激光共聚焦显微镜观察DNA损伤标志物γ-H2AX在细胞内的分布;流式细胞术分析CUDC-907对细胞周期的影响;Western blot实验检测细胞内相关蛋白表达水平的变化。结果:CUDC-907能够抑制U251细胞活力。在CUDC-907处理的细胞中,蛋白激酶B(PKB)/Akt和p70核糖体蛋白S6激酶(p70s6K)的磷酸化水平降低,γ-H2AX的焦点数量与蛋白表达显著升高(P <0.05);U251细胞经CUDC-907作用后,G2/M期细胞数量增多;Western blot实验结果表明,CUDC-907促进p21的表达,同时抑制细胞周期素B1(cyclin B1)的蛋白表达和细胞分裂周期蛋白2(Cdc2)的磷酸化水平(P <0.05);另外,CUDC-907能够诱导细胞自噬,抑制自噬可促进CUDC-907诱导的DNA损伤。结论:CUDC-907能够显著抑制PI3K/Akt信号通路,诱导胶质瘤细胞发生DNA损伤,并阻滞细胞于G_2/M期,同时可诱导胶质瘤细胞发生保护性自噬。
AIM:To investigate the effect of CUDC-907,a dual histone deacetylase(HDAC)and phosphatidylinositol 3-kinase(PI3K)inhibitor,on the DNA damage,cell cycle distribution and autophagy in human glioma U251cells.METHODS:U251 cells were treated with CUDC-907 of different concentrations,and the cell viability was detected by MTT assay.The quantitativeγ-H2AX foci were determined by laser scanning confocal microscopy.The cell cycle distribution of U251 cells was examined by flow cytometry.The protein expression was determined by Western blot analysis.RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase(p70s6K)in the U251 cells(P<0.05).In CUDC-907-treated cells,the number ofγ-H2AX foci and protein expression ofγ-H2AX were increased significantly(P<0.05).CUDC-907 also induced cell arrest in theM phase by up-regulating the expression of p21,and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein2(Cdc2).In addition,CUDC-907 triggered cell autophagy,and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells.CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway,induces DNA damage and arrests cell cycle inM phase.Blockage of autophagy promotes CUDC-907-induced DNA damage of U251cells.
AIM:To investigate the effect of CUDC-907,a dual histone deacetylase(HDAC)and phosphatidylinositol 3-kinase(PI3K)inhibitor,on the DNA damage,cell cycle distribution and autophagy in human glioma U251cells.METHODS:U251 cells were treated with CUDC-907 of different concentrations,and the cell viability was detected by MTT assay.The quantitativeγ-H2AX foci were determined by laser scanning confocal microscopy.The cell cycle distribution of U251 cells was examined by flow cytometry.The protein expression was determined by Western blot analysis.RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase(p70s6K)in the U251 cells(P<0.05).In CUDC-907-treated cells,the number ofγ-H2AX foci and protein expression ofγ-H2AX were increased significantly(P<0.05).CUDC-907 also induced cell arrest in theM phase by up-regulating the expression of p21,and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein2(Cdc2).In addition,CUDC-907 triggered cell autophagy,and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells.CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway,induces DNA damage and arrests cell cycle inM phase.Blockage of autophagy promotes CUDC-907-induced DNA damage of U251cells.
引文
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[2]Wang X,Qiu Y,Yu Q,et al.Enhanced glioma therapy by synergistic inhibition of autophagy and tyrosine kinase activity[J].Int J Pharm,2018,536(1):1-10.
[3]Hu Q,Kang T,Feng J,et al.Tumor microenvironment and angiogenic blood vessels dual-targeting for enhanced anti-glioma therapy[J].ACS Appl Mater Interfaces,2016,8(36):23568-23579.
[4]Liu Y,Mei L,Xu C,et al.Dual receptor recognizing cell penetrating peptide for selective targeting,efficient intratumoral diffusion and synthesized anti-glioma therapy[J].Theranostics,2016,6(2):177-191.
[5]Sun K,Atoyan R,Borek MA,et al.Dual HDAC and PI3K inhibitor CUDC-907 downregulates MYC and suppresses growth of MYC-dependent cancers[J].Mol Cancer Ther,2017,16(2):285-299.
[6]Kotian S,Zhang L,Boufraqech M,et al.Dual inhibition of HDAC and tyrosine kinase signaling pathways with CUDC-907 inhibits thyroid cancer growth and metastases[J].Clin Cancer Res,2017,23(17):5044-5054.
[7]To KKW,Fu LW.CUDC-907,a dual HDAC and PI3Kinhibitor,reverses platinum drug resistance[J].Invest New Drugs,2018,36(1):10-19.
[8]Fei HR,Tian H,Zhou XL,et al.Inhibition of autophagy enhances effects of PF-04691502 on apoptosis and DNAdamage of lung cancer cells[J].Int J Biochem Cell Biol,2016,78:52-62.
[9]Liu C,Wu H,Li Y,et al.SALL4 suppresses PTEN expression to promote glioma cell proliferation via PI3K/AKTsignaling pathway[J].J Neurooncol,2017,135(2):263-272.
[10]LoRusso PM.Inhibition of the PI3K/AKT/m TOR pathway in solid tumors[J].J Clin Oncol,2016,34(31):3803-3815.
[11]Peserico A,Simone C.Physical and functional HAT/HDAC interplay regulates protein acetylation balance[J].J Biomed Biotechnol,2011,2011:371832.
[12]李家庆,韩潇.二氢吡啶酮类组蛋白去乙酰化酶抑制剂的设计、合成及抗肿瘤活性[J].药学学报,2016,51(11):1734-1744.
[13]崔海娟,张亚云,焦鹏,等.GDC-0032抑制U251胶质瘤细胞活力并诱导DNA损伤[J].中国病理生理杂志,2017,33(10):1858-1863.
[14]Shimizu S.Biological roles of alternative autophagy[J].Mol Cells,2018,41(1):50-54.
[15]张翠,姜文艳,吴玉梅,等.抑制自噬促进MK-2206诱导SGC-7901胃癌细胞发生DNA损伤[J].中国病理生理杂志,2015,31(9):1545-1549.