ABT737通过延迟宫颈癌细胞放射后DNA损伤修复及诱导凋亡而增敏放疗

摘要

[目的]探讨类似BH-3的小分子物质ABT737诱导增敏放疗的作用及其分子机制.[方法]噻唑蓝法(MTT)检测不同浓度ABT737对HeLa细胞的生长抑制作用；细胞克隆形成实验检测ABT737联合放疗对放疗的增敏作用；免疫荧光法检测γ-H2AX观察DNA损伤修复情况；流式细胞术检测细胞凋亡；免疫印迹法检测Caspase-3、PARP的表达观察凋亡.[结果]与对照组相比,ABT737能显著抑制宫颈癌HeLa细胞的增殖(P＜0.05),并呈浓度依赖性,其IC50为15.7 μmol/L.体外培养克隆形成实验结果显示放疗+ABT737不同用药时间组的DEF值均大于1,放疗+8 μmol/L ABT737持续用药组为1.88,放疗+12 μmol/L ABT737用药72 h组为1.13,细胞存活分数(SF值)持续用药组为0.84,用药72 h组SF值为0.82；免疫荧光结果显示ABT737联合放疗处理HeLa细胞1h后,放射线导致的γ-H2AX聚焦点数量及有γ-H2AX聚焦点生成的细胞数量均明显增加,上述处理24 h后,单纯放疗组γ-H2AX焦点消失,而联用ABT737处理组仍可观察到γ-H2AX焦点聚集.流式细胞术结果显示,单纯放疗组早期凋亡率(Annexin V+,PI-)为23.3％,ABT737联合放疗可以明显提高放射线诱导的细胞凋亡,早期凋亡率最高达50.3％.免疫印迹结果显示10 μmol/L ABT737与2 Gy放射联合作用于Hela细胞后,凋亡蛋白cleaved Caspase-3与cleayed PARP的表达较单纯放疗组增加.[结论]ABT737对宫颈癌Hela细胞具有放疗增敏作用,其机制与ABT737可延迟宫颈癌细胞放疗后DNA损伤修复及诱导凋亡有关.%[Objective] To explore the pivotal role of ABT737, a BH3 mimetic small molecule inhibitor, in enhancing radiosensitivity and its mechanism. [Methods] MTT assay was conducted to determine the inhibition rate of ABT737 in HeLa cells. The radiosensitizing effect of ABT737 in HeLa cells was detected by cell colony assay. The DNA damage of HeLa cells was examined by detection of histone variant H2AX (γ-H2AX) foci using indirect immunofluorescence. Flow cytometry with Annexin V-PI staining was used to assess the apoptosis. The expression of apoptotic proteins caspase-3 and PARP were detected using Western blot analysis. [Results] ABT737 significantly inhibited cell growth of HeLa cells with IC50 value of 15.7 μmol/L. Result of the colony formation assay indicated that the clonogenic survival of HeLa cells was decreased after combining the radiation. The DEF values for both combination groups were over one; 1.88 for the group treated with 8 μmol/L ABT737and 1.13 for the group treated with 12 μmol/L ABT-737. The survival fraction (SF) values for the group treated with 8 μmol/L ABT737 and the group treated with 12 μmol/L were 0.84 and 0.82, respectively. ABT737 increased the number of γ-H2AX focis and the cells that detected γ-H2AX foci in HeLa cells exposed radiation. After 24 h, γ-H2AX focis disappeared in radiation group, but the accumulation of γ-H2AX focis was also observed after combining ABT737. The expression levels of both cleaved-Caspase-3 and cleaved-PARP and the apoptotic rate of HeLa cells were increased in the combination group comparing to those in the groups that were treated with only ABT-737 or radiation. [Conclusion] Our data imply that ABT-737 can enhance radiosensitizing effects in human cervical cancer HeLa cells via delaying DNA damage repair after radiation and inducing apoptosis.