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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 killing effect of osthole and tumor necrosis factor (TNF) -related apoptosis inducing ligand (TRAIL) on breast cancer stem cells and its mechanism. Methods MCF-7 breast cancer non-stem cells and MCF-7 breast cancer stem cells were treated with TRAIL and osthole in vitro. Cell viability was detected by MTT assay. Flow cytometry was used to detect the apoptosis of breast cancer cells. Western blot assay The activation of caspase-9 and caspase-3, the expression level of apoptotic protease activating facter-1 (Apaf-1) and the release of cytochrome C were detected by immunoprecipitation. The expressions of Apaf-1 and caspase-9 Body interaction. Results The sensitivity of MCF-7 tumor stem cells to TRAIL was significantly lower than that of MCF-7 non-stem cells. The addition of osthole to the culture system of MCF-7 tumor stem cells can significantly increase the cell viability of TRAIL to MCF-7 tumor stem cells. Western blot results show that osthorin can significantly up-regulate the expression of Apaf-1 in MCF-7 tumor stem cells, but does not affect the release of cytochrome C. After transfection of Apaf-1 si RNA in MCF-7 tumor stem cells, the synergistic cytotoxicity of Ostholeclidine and TRAIL to MCF-7 tumor stem cells was significantly inhibited. In addition, co-immunoprecipitation results showed that osthole combined with TRAIL significantly induced the activation of Apaf-1 and caspase-9 precursor in MCF-7 tumor stem cells. Conclusion Osthole could promote the apoptosis of breast cancer stem cells induced by TRAIL by up-regulating the expression of Apaf-1 and promoting the activation of caspase-9 in breast cancer stem cells.