阿柔比星与米托蒽醌联合应用对HeLa细胞凋亡及拓扑异构酶Ⅱ表达的影响

Effects of mitoxantrone combined with aclarubicin on the apoptosis and topoisomeraseⅡ expression in HeLa cells

目的 探讨拓扑异构酶Ⅱ (TopoⅡ )催化抑制剂阿柔比星 (ACR)对TopoⅡ毒性抑制剂米托蒽醌(MIT)杀伤HeLa细胞的影响。方法 应用MTT方法检测药物单用和联合应用对HeLa细胞的抑制作用 ;采用单细胞凝胶电泳方法检测药物对DNA链断裂的影响 ;双荧光方法检测细胞凋亡率 ;免疫细胞化学方法观察TopoⅡ表达变化。结果 MTT法检测表明 ,ACR能够减低MIT对HeLa细胞的抑制作用。单细胞凝胶电泳显示 ,MIT在 1,10 ,10 0nmol·L- 1浓度作用下 ,细胞DNA断裂长度分别为 (3.0± 0 .3) ,(6 .8± 0 .4 ) ,(10 .8± 1.0 ) μm。上述浓度MIT与 0 .2μmol·L- 1的ACR共同作用后 ,DNA断裂长度依次减低为 (1.0± 0 .3) ,(3.9± 0 .4 ) ,(6 .1± 0 .3) μm。两者相比差别明显 (P <0 .0 1)。细胞凋亡检测表明 ,MIT在 1,10 ,10 0nmol·L- 1浓度作用下 ,细胞凋亡分别为(9 .9± 1.7) % ,(5 5 .4± 3.9) % ,(98.0± 7.1) %。上述浓度MIT与 0 .2 μmol·L- 1的ACR共同作用后细胞凋亡依次降低为 (3.9± 0 .3) % ,(2 5 .5± 3.5 ) % ,(79.2± 5 .3) %。两者相比差别明显 (P <0 .0 1)。进一步采用免疫组织化学方法检测表明 ,ACR与MIT合用组较MIT单用组TopoⅡ的表达显著减少。结论 ACR可拮抗MIT对HeLa细胞的杀伤作用。

AIM To investigate the influence of topoisomeraseⅡ(TopoⅡ), a catalytic inhibitor, aclarubicin(ACR) on the effect of mitoxantrone(MIT), a toxic inhibitor of TopoⅡ on HeLa cells. METHODS The MTT assay was used to measure the cell growth. DNA damages were detected by the single cell gel electrophoresis assay . The acridine orange/ethidium bromide staining technique was used to observed apoptotic cells. Immunocytochemical analysis was performed to revealed TopoⅡ expressions of the cells. RESULTS ACR could inhibit the effect of MIT on HeLa cells. The length of DNA breaks was (3.0±0.3), (6.8±0.4) and (10.8± 1.0)μm, respectively following treatment with 1, 10, 100 nmol·L -1 of MIT. When combined with 0.2 μmol·L -1 ACR, the DNA breakage level reduced to (1.0±0.3) , (3.9±0.4) and (6.1±0.3)μm, respectively. The apoptotic rates were (9.9± 1.7)% , (55.36±3.9)%, (98.04± 7.1)% when treated with 1, 10, 100 n mol·L -1 of MIT alone and decreased to (3.9± 0.3)% , (25.5±3.5) and (79.2±5.3)%, respectively when combined with 0.2 μmol·L -1 ACR. It was also noted that the TopoⅡ expression was reduced obviously in ACR+MIT group compared to that of MIT alone. CONCLUSION ACR can antagonize MIT in killing HeLa cells.