熊果酸通过影响Bax和Bcl-2的表达诱导MCF-7人乳腺癌细胞凋亡

Ursolic acid induces apoptosis through effects on Bax and Bcl-2 expression in human MCF-7 cells

目的:探讨中药成分熊果酸(ursolicacid,UA)诱导MCF7人乳腺癌细胞的凋亡作用,并通过分析UA作用后的MCF7细胞Bax,Bcl2,cytochromec蛋白表达的变化,探讨其诱导MCF7细胞凋亡的分子机制.方法:采用细胞培养技术,用0,10,30和50μmol/L的UA处理MCF7细胞24h,用Ho染细胞,在荧光显微镜下观察凋亡细胞的形态变化.用0,20和50μmol/L的UA处理MCF7细胞24h,用PI染细胞,在流式细胞仪上测定细胞周期的变化.用0和50μmol/L的UA处理MCF7细胞24h,采用荧光免疫细胞化学SABCCy3法检测Bax,Bcl2,cytochromec蛋白表达,在荧光显微镜下拍照,用QWin图像分析软件测定Bax,Bcl2,cytochromec荧光灰度值.结果:用0,10,30和50μmol/L的UA处理MCF7细胞24h,随着UA浓度增加,在荧光显微镜下出现凋亡细胞增多,细胞呈现典型的凋亡形态学特征,核质浓集和凋亡小体.用0,20和50μmol/L的UA处理MCF7细胞24h,流式细胞仪检测亚G1峰比例分别为0.05,0.22,0.43与对照组5mL/LDMSO比较,50μmol/L的UA使MCF7细胞中的Bax灰度(59.3±7.8,213.6±7.4,P<0.01)和cytochromec灰度(88.2±6.9,188.1±15.4,P<0.01)增加,Bax/Bcl2灰度比值升高(1.0±0.1,2.4±0.4,P<0.01),Bcl2灰度(58.1±6.1,92.1±12.4,P<0.01)稍有增加.UA促进线粒体放释cytochromec进入胞质.结论:UA诱导MCF7细胞凋亡,其机制涉及到Bax/Bcl2比值升高引起线粒体释放cytochromec所依赖的凋亡调节信号通路.表明治疗乳腺癌,UA可能是有效的中药成分.

AIM： To study the effects of ursolic acid （UA） on human MCF-7 cell apoptosis and the mechanism probably involved. METHODS： MCF-7 cells were treated with different concentrations of UA （0, 10, 30, 50 μmol/L ） for 24 h, apoptotic cells with condensed or fragmented nuclei were visualized by Ho33258 staining in a fluorescence microscope. After MCF-7cells treated with UA （0, 20, 50 μmol/L ） for 24 h, sub-G1 peak and cell cycle were detected with PI staining by FCM. After MCF-7cells treated with and 50 μmol/L of UA for 24 h, the protein expression of cytochrome c, Bcl-2 and Bax were detected by immunofluo- rescence cell staining （ SABC-Cy3 ） and the grey levels of immunofluorescence on cytochrome c and Bcl-2 and Bax were analyzed with the use of QWin. RESULTS： Twenty- four hours after UA treatment, apoptotic cells increased dose-dependently and the morphological changes of MCF-7 cells displayed many hallmark features of apoptosis, including chromatin aggregation and fragmented nuclei. Sub-G1 rates in 0, 20 and 50 μmol/L of UA detected by FCM were 0.05, 0.22 and 0.43 respectively. Compared with 5 mL/L DMSO, 50 μmol/L of UA significantly increased the grey levels of Bax （59.3 ±7.8, 213.6±7.4, P〈0.01）, cytochrome c （88.2 ±6.9, 188. 1 ± 15.4, P〈0.01） and the grey ratio of Bax/Bcl-2 （1.0±0. 1, 2.4 ±0.4, P〈0.01） and increased noticeably the grey level of Bcl-2 （58. 1 ± 6.1, 92.1 ±12.4, P〈0.01）. Cytochrome c induced by 50 μmol/L of UA released into cytoplasm. CONCLUSION： Apoptosis induced with UA in MCF-7 cells is related to increasing the ratio of Bax/bcl-2 and releasing cytochrome cdependent apoptotic pathway. Our study indicates that UA may be a potential Chinese medicinal component for breast neoplasm.