蛇床子素联合肿瘤坏死因子诱导凋亡配体对乳腺癌干细胞的杀伤效应

Anti-tumor Effect of Osthol Combined with Tumor Necrosis Factor-Related Apoptosis Inducing Ligand on Breast Cancer Stem Cells

目的探讨蛇床子素联合肿瘤坏死因子诱导凋亡配体[tumor necrosis factor(TNF)-related apoptosis inducing ligand,TRAIL]对乳腺癌干细胞的杀伤效应及机制。方法用TRAIL及蛇床子素体外处理MCF-7乳腺癌非干细胞及MCF-7乳腺癌干细胞,MTT法检测乳腺癌细胞的细胞活力;流式细胞术检测乳腺癌细胞的凋亡;Western blot试验检测caspase-9和caspase-3的活化,凋亡酶激活因子(apoptotic protease activating facter-1,Apaf-1)的表达水平,细胞色素C的释放;免疫共沉淀法检测Apaf-1和caspase-9前体的相互作用。结果 MCF-7肿瘤干细胞对TRAIL的敏感性显著低于MCF-7非干细胞。在MCF-7肿瘤干细胞的培养体系中加入蛇床子素能显著提高TRAIL对MCF-7肿瘤干细胞的细胞活力抑制率。Western blot实验结果表明,蛇床子素能显著上调MCF-7肿瘤干细胞中Apaf-1的表达水平,但不影响细胞色素C的释放。在MCF-7肿瘤干细胞中转染Apaf-1 si RNA后,蛇床子素联合TRAIL对MCF-7肿瘤干细胞的协同杀伤活性受到显著抑制。另外,免疫共沉淀实验结果表明,蛇床子素联合TRAIL能显著诱导MCF-7肿瘤干细胞中Apaf-1与caspase-9前体的相互作用,并使之发生活化。结论蛇床子素通过上调Apaf-1的表达促进TRAIL对乳腺癌干细胞caspase-9的活化,从而增强TRAIL对乳腺癌干细胞的凋亡诱导效应。

OBJECTIVE To investigate the synergistic effect and mechanism of osthole and tumor necrosis factor （TNF）-related apoptosis inducing ligand （TRAIL） in breast cancer stem cells. METHODS After treatment of osthole and TRAIL in the MCF-7 cancer stem cells （MCF-7-CSCs） as well as the MCF-7-non-cancer stem cells （MCF-7-non-CSCs）, MTT assay was performed to detect the cell viability; flow cytometry analysis was performed to measure the cell apoptosis; Western blot analysis was performed to evaluate the activation of caspase-9 and caspase-3, expression of Apaf-1, and release of cytochrome C; co-immunoprecipitation was performed to detect the interaction with Apaf-1 and pro-caspase-9, respectively. RESULTS The sensitivity of MCF-7-CSCs to TRAIL was significantly lower than the MCF-7-non-CSCs. Addition of osthole significantly increased the inhibitory rate of MCF-7-CSCs treated with TRAIL. The results of Western blot indicated that the treatment of osthole could significantly upregulate the expression of Apaf-1 without changing the release of cytochrome C. Transfection with Apaf-1 siRNA abolished the synergistic effect of osthole and TRAIL in MCF-7-CSCs. In addition, the results of co-immunoprecipitation indicated that the combination of osthole and TRAIL significantly induced the interaction with Apaf-1 and pro-caspase-9, which led to the activation of caspase-9 in MCF-7-CSCs. CONCLUSION Osthole promotes the TRAIL-induced activation of caspase-9 and apoptosis by upregulating the expression ofApaf-1 in the breast cancer stem cells.