DNA甲基化酶抑制剂与雷帕霉素对人结直肠癌细胞周期及mTOR信号转导通路的联合作用

DNA methyltransferase inhibitor and rapamycin synergistically modulate cell cycle and mammalian target of rapamycin signaling pathway for human colorectal cancer cells

目的探讨5-氮脱氧胞苷(5-aza-dC)与雷帕霉素(RPM)对人结直肠癌细胞的联合作用及机制。方法应用 MTY 比色法检测5-aza-dC 与 RPM 对3种人结直肠癌细胞株的生长抑制作用;应用流式细胞术检测两者对细胞周期的调控;应用免疫印迹法检测两者对于 mTOR 信号转导通路相关蛋白的影响。结果 5-aza-dC 与 RPM 对人结直肠癌细胞株 SW480、HT29和 HCT116均有生长抑制作用,在相同作用条件下,5-aza-dC 与 RPM 的联合抑制效果明显优于两者单独用药。5-aza-dC 与 RPM单独或联合用药对3种细胞的生长周期均具有调控作用(P<0.05),但作用位点不尽相同。5-aza-dC与 RPM 可下调 mTOR、p70s6K、4E-BP1蛋白的磷酸化水平,对蛋白的总体水平无明显影响,以两者的联合作用最为显著。结论 5-aza-dC 与 RPM 可联合抑制人结直肠癌细胞株 SW480、HT29和 HCT116的生长,并阻滞其细胞周期进程。其作用机制可能与影响 mTOR 信号转导通路蛋白磷酸化水平有关。

Objective To investigate the synergistic effect of 5-aza-2＇-deoxycytidine （5-aza-dC） and rapamycin （RPM） on human colorectal cancer cells and it＇s potential mechanisms. Methods Three human colorectal cancer cell lines SW480, HT29 and HCTI 16 were treated for 48 h with 5-aza-dC, RPM, or 5-aza-dC plus RPM, respectively, and the sensitivity was analysed by an MTT assay. Western blotting analysis was performed to examine the total and phosphorylated levels of mammalian target of rapamycin （mTOR） and its downstream translational signalling intermediates, 70 kDa ribosomal protein $6 kinase （p70s6k） and eukaryotic initiation factor 4E-binding protein 1 （4E-BP1）. The cell cycle progression was evaluated by flow cytometry. Results Both 5-aza-dC and RPM could inhibit viability of three colorectal cancer cell lines, which effect was more significantly in cells treated with 5-aza-dC plus RPM than that 5-aza-dC or RPM only. Treated with 5-aza-dC, RPM, and 5-aza-dC plus RPM all induced blockage of cell cycle progression, although the arrest was confined to different cell cycle phases. In addition to their effect on proliferation and cell cycle, both inhibitors also reduced phosphorylation levels of mTOR, p70s6k, and 4E-BP1, which effect was reinforced when treated cells with 5-aza-dC plus RPM simultaneously, whereas total protein levels remained unchanged. Conclusion 5-aza-dC and RPM inhibit proliferation of colorectal cancer cells synergistically, and induce cell cycle arrest. The potential mechanisms involve modulation of mammalian target of rapamycin signaling pathway.