5-氮杂-2'-脱氧胞苷对食管癌放射敏感性的影响及作用机制

Effect of 5-Aza-CdR on Radiosensitivity of Human Esophageal Carcinoma and Its Mechanism

目的研究5-氮杂-2'-脱氧胞苷(5-aza-CdR)对人食管癌EC9706细胞放射敏感性的影响及可能机制。方法用不同浓度5-aza-CdR(0、0.50、1.00、1.50及3.00μmol/L)和不同射线照射剂量(2、4、6、8及10 Gy)处理EC9706细胞,CCK8法检测EC9706细胞增殖抑制率及放射增敏比(SER);Western blot法检测cleaved caspase-3、Bax、Bcl-2细胞凋亡相关蛋白及DNA损伤相关蛋白ATM/p-ATM的表达;流式细胞术检测5-aza-CdR和放射线对细胞周期的影响;Transwell小室侵袭实验观察EC9706细胞侵袭能力的改变。结果经CCK-8法检测,1.00μmol/L是5-aza-CdR的无毒最高剂量,并以此作为后续研究的干预剂量。5-aza-CdR预处理EC9706细胞,可增强不同射线照射剂量对EC9706细胞增殖的抑制作用(P<0.01),SER为1.335±0.04。5-aza-CdR能升高放射线诱导凋亡相关蛋白cleaved caspase-3、Bax的表达,抗凋亡蛋白Bcl-2表达下降,差异有统计学意义(P<0.01),但p-ATM蛋白表达无明显变化(P>0.05);与单纯照射组相比,射线照射联合5-aza-CdR干预组G0/G1期和S期细胞减少,而G2/M期细胞增多,穿膜细胞数量明显减少,差异有统计学意义(P<0.05或P<0.01)。结论1.00μmol/L 5-aza-CdR可增加人食管癌EC9706细胞放射敏感性,作用机制可能与增加放射线诱导的细胞凋亡、抑制细胞增殖、影响细胞周期及增强放射线抑制细胞侵袭的能力有关。

Objective To study effect of 5-Aza-cytosine deoxyriboside(CdR)on radiosensitivity of human esophageal cancer EC9706 cells and its possible mechanism.Methods EC9706 cells were treated with different concentrations of 5-Aza-CdR(0,0.50,1.00,1.50 and 3.00μmol/L)and different exposure dose of radiation(2,4,6,8 and 10 Gy).CCK8 method was used to detect proliferation inhibition rate and sensitization enhancement ratio(SER)of EC9706 cells,and Western blot method was used to detect expressions of cleaved caspase-3,Bax,bcl-2 apoptosis related proteins and DNA damage related proteins ATM/p-ATM.Effects of 5-Aza-CdR and radiation on cell cycle were detected by flow cytometry,and change of invasion ability of EC9706 cells was observed by Transwell cell migration assay.Results The 1.00μmol/L was the highest non-toxic dose of 5-Aza-CdR by CCK8 method,which was used as the intervention dose in the subsequent study.Pretreatment of EC9706 cells with 5-Aza-CdR enhanced inhibition effects of EC9706 cell proliferation by different exposure doses of radiation(P<0.01),and the SER was(1.335±0.04).The 5-Aza-CdR could increase expressions of protein related to radiation-induced apoptosis including cleaved caspase-3 and Bax,and decrease anti-apoptotic protein Bcl-2(P<0.01),but expression of p-ATM protein had no significant change(P>0.05).Compared with those in simple irradiation group,cells in phase G0/G1 and S were significantly decreased,while cells in phase G2/M were significantly increased,and number of transmembrane cells was significantly decreased(P<0.05 or P<0.01).Conclusion the 1.00μmol/L 5-Aza-CdR may increase the radiosensitivity of human esophageal cancer EC9706 cells,and its mechanism may be related to increasing apoptosis induced by radiation,inhibiting cell proliferation,affecting cell cycle and enhancing the ability of inhibiting cell invasion.