作者：孟瑾1,2，刘新利3，车玲1，陈明1，吴漫1，马晨珂1，赵冠人1

单位：1.解放军总医院第八医学中心 药剂科，北京 100091； 2.空军军医大学唐都医院 肿瘤科，陕西 西安 710038； 3.西北工业大学 生命学院，陕西 西安 710072

Authors:  MENG Jin1,2，LIU Xinli3，CHE Ling1，CHEN Ming1，WU Man1，MA Chenke1，ZHAO Guanren1

Unit:  1.Department of Pharmacy, The Eighth Medical Center of PLA , Beijing 100091, China； 2.Department of Oncology, Tangdu Hospital, Air Force Military Medical University, Xi'an 710072, Shaanxi, China； 3.College of life, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, China

摘要：

目的 探讨组蛋白去乙酰化酶抑制TSA对结肠癌HT29细胞增殖能力的影响及其作用机制。方法 以结肠癌HT29细胞为研究对象,采用浓度梯度TSA处理并计算其对上述细胞的IC50值,并采用MTT法观察不同浓度TSA对结肠癌HT29细胞增殖能力的影响;采用流式细胞术观察不同浓度TSA对结肠癌HT29细胞周期的影响;Western blot法观察不同浓度TSA对结肠癌HT29细胞中Ku70蛋白乙酰化水平以及对细胞周期蛋白CyclinB1、CDC25C和周期抑制蛋白P21及其上游调控蛋白P53表达的影响。结果 MTT法和流式细胞术检测结果表明,TSA可浓度依赖性抑制结肠癌HT29细胞的增殖并促进细胞发生G2/M期阻滞;此外,Western blot检测结果表明TSA可明显上调结肠癌HT29细胞中Ku70蛋白的乙酰化水平,同时上调周期抑制蛋白P21和P53的表达,并抑制细胞周期相关蛋白CyclinB1和CDC25C的表达,差异有统计学意义(P<0.05)。结论 组蛋白去乙酰化酶抑制剂(TSA)可能通过促进结肠癌HT29细胞中Ku70蛋白乙酰化,抑制其与DNA结合的亲和力而抑制DNA损伤修复,进而促进结肠癌HT29细胞发生周期阻滞而发挥抗结肠癌细胞增殖并促进其周期阻止的作用,为以细胞周期调控为靶点的肿瘤治疗策略提供理论依据和实验基础。

关键词：结肠癌; TSA; Ku70乙酰化; 周期阻止

Abstract：

Objective  To investigate the effect and mechanism of histone deacetylase inhibitor TSA on the proliferation of colon cancer HT29 cells. Methods  Colon cancer HT29 cells were used as the research object. The IC50 values of the cells were calculated by concentration gradient TSA, and the effects of different concentrations of TSA on the proliferation of colon cancer HT29 cells were observed by MTT assay. The effects of different concentrations of TSA on the cell cycle of colon cancer HT29 were observed by flow cytometry. The levels of Ku70 protein acetylation and CyclinB1, CDC25C and cyclin P21 in colon cancer HT29 cells were observed by Western blot. Its upstream regulation of the expression of protein P53. Results  MTT assay and flow cytometry results showed that TSA could inhibit the proliferation of colon cancer HT29 cells and promote G2/M phase arrest in a concentration-dependent manner. In addition, western blot analysis results showed that TSA could significantly up-regulate the acetylation level of Ku70 protein in colon cancer HT29 cells, and up-regulate the expression of cyclic inhibitory proteins P21 and P53, and inhibit the expression of cell cycle-associated proteins CyclinB1 and CDC25C. The difference was statistically significant (P<0.05). Conclusion Histone deacetylase inhibitor (TSA)may inhibit the DNA damage by promoting the acetylation of Ku70 protein, inhibiting its affinity for DNA binding, and then promoting cell cycle arrest of colon cancer HT29 cells. Furthermore, exerting anti-colon cancer cell proliferation and promoting its cycle arrest. Which provides a theoretical basis and experimental basis for tumor therapy strategies targeting at cell cycle regulation.

Key Words:  Colon cancer; TSA; Ku70 acetylation; Cycle arrest