Smac基因在增强乳腺癌细胞株MCF-7对环磷酰胺和多柔比星敏感性中的作用

Smac playing vital role in enhancing the sensitivity of cyclophosphamide and doxorubicin in breast cancer MCF-7 cell line

目的研究Smac基因在乳腺癌细胞株MCF-7中对化疗药物环磷酰胺（CTX）和多柔比星（DOX）化疗敏感性的影响。方法分别用化疗药物CTX、DOX及二者联合处理乳腺癌细胞株MCF-7，用四甲基偶氮唑蓝（MTT）法分析细胞生存率，吖啶橙染色和Ho.33342/PI双染法检测细胞凋亡，RT-PCR和Western blotting检测Smac的mRNA和蛋白水平，并进一步分析促凋亡蛋白活性caspase3和活性caspase9的表达变化。结果CTX、DOX及二者联合应用降低了MCF-7细胞的生存率，并且呈浓度依赖性。4.0 μg/ml CTX、0.2 μg/ml DOX及2.0 μg/ml CTX联合0.1 μg/ml DOX处理细胞48 h后，细胞生存率分别是（52.90±8.78）%、（53.35±6.29）%和（34.19±5.43）%。与4.0 μg/ml CTX、0.2 μg/ml DOX相比，联合用药有更强的抑制细胞生长作用（t=9.051，P=0.014；t=9.074，P=0.014）。2.0 μg/ml CTX联合0.1 μg/ml DOX处理细胞48 h后，Smac的mRNA和蛋白水平分别为7.47±0.82和4.13±0.36，4.0 μg/ml CTX组分别为3.27±0.40（t=-50.120，P=0.000）和2.28±0.27（t=-42.588，P=0.000），0.2 μg/ml DOX组分别为3.34±0.62（t=-46.233，P=0.000）和2.45±0.40（t=-39.541，P=0.000），与单一用药相比，联合用药后Smac在mRNA和蛋白水平上都明显升高。同时发现活性caspase-3和活性caspase-9的蛋白表达水平也明显升高。结论Smac在提高乳腺癌细胞株MCF-7对CTX和DOX化疗敏感性中发挥了重要作用。

ObjectiveTo investigate the influence of Smac to the chemosensitivity of cyclophosphamide （CTX） and doxorubicin （DOX） in MCF-7 cells. MethodsMCF-7 cells were exposed to CTX, DOX and the combination of both. 3-（4,5-dimethyl-2-thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） assay was used to estimate the cell viability. Apoptosis was measured by acridine orange staining and Ho.33342/PI double staining. The mRNA and protein expressions of Smac were determined by RTPCR and Western blotting. The study also analyzed the changes of proapoptotic proteins active caspase3 and active caspase9. ResultsCTX, DOX and the combination of both drugs reduced the cell survival rates in a concentrationdependent manner. The cell viability after being treated with 4.0 μg/ml CTX or 0.2 μg/ml DOX or 2.0 μg/ml CTX and 0.1 μg/ml DOX for 48 hours was （52.90±8.78）%, （53.35±6.29）% and （34.19±5.43）%, respectively. The drug combination developed a stronger inhibitory effect compared to the single drugs （t=9.051, P=0.014; t=9.074, P=0.014）. The Smac mRNA and protein levels in 2.0 μg/ml CTX and 0.1 μg/ml DOX group were 7.47±0.82 and 4.13±0.36, which were higher than those in 4.0 μg/ml CTX group （3.27±0.40 and 2.28±0.27; t=-50.120, P=0.000; t=-42.588,P=0.000） and 0.2 μg/ml DOX group （3.34±0.62 and 2.45±0.40; t=-46.233, P=0.000; t=-39.541, P=0.000）. Furthermore, proapoptotic proteins active caspase-3 and active caspase9 increased activity was confirmed by Western blotting. ConclusionSmac plays a vital role in enhancing the sensitivity of chemotherapeutic drugs CTX and DOX in MCF-7 cell line.