一种新硫色满酮衍生物诱导人乳腺癌MCF-7细胞凋亡

初步研究了新合成的(顺)-3-(氯代亚甲基)-5-甲基-硫色满-4-酮(3-chloromethylene-5-fluoro-thiochroman-4-one,CMTK)对人乳腺癌MCF-7细胞增殖和凋亡的影响,分别用不同浓度的CMTK对细胞进行干预,采用改良的MTT法测定CMTK抑制细胞增殖的能力,台盼蓝染色法计数活细胞数,绘制生长曲线,流式细胞术检测细胞对细胞周期的影响,同时采用流式细胞术及TUNEL染色法(terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labelling)检测CMTK诱导凋亡,活性检测法测定细胞中人半胱氨酸蛋白酶8(caspase-8)和人半胱氨酸蛋白酶9(caspase-9)的活性,ELISA法测人死亡受体3(deathreceptor3,DR3)、人肿瘤坏死因子受体1(tumor necrosis factor receptor1,TNFR1)、人肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、人半胱氨酸蛋白酶3(caspase-3)的蛋白表达情况.结果表明,CMTK可明显抑制MCF-7细胞增殖,与顺铂(cisplatin,CDDP)相比,其抗肿瘤活性更明显,IC50为14.24±0.12molL1;生长曲线进一步说明CMTK抑制细胞增殖,其抑制作用呈时间-剂量依赖性;流式细胞仪检测分析显示,加药12和24h后可将细胞阻滞于G0/G1期(**P<0.05);TUNEL法和流式细胞仪分析CMTK可诱导凋亡的结果基本一致;与药物作用后,caspase-8和caspase-9的活性增高,并呈浓度剂量依赖性;ELISA法检测DR3蛋白量和TNF-细胞因子的量无变化,TNFR1和caspase-3的蛋白量显著增高,CMTK诱导肿瘤细胞凋亡的机制可能是:不仅可以通过死亡受体TNFR1介导凋亡途径,激活caspase-8;还可以通过线粒体凋亡途径,激活caspase-9;最终激活caspase-3凋亡通路.本研究为深入研究CMTK诱导肿瘤细胞凋亡的机制提供了依据.

A new methyl-thiochroman-4-ketone derivative induces apoptosis in MCF-7 human breast cancer cells in vitro

To investigate effects of (Z)-3-chloromethylene-5-methyl-thiochroman-4-ketone (CMTK) on apoptosis in human breast cancer MCF-7 cells in vitro, cultured MCF-7 cells were treated with different concentrations of CMTK. The MTT method was used to study inhibition rates; the trypan blue method for cell viability and to construct growth curves; flow cytometry for cell cycles; the TUNEL assay and flow cytometry to assess proportion of apoptotic cells; Ac-IETD-pNA caspase-8/Ac-LEHD-pNA caspase 9 Colorimetric Assay Kit for caspase-8 and caspase-9 activities; and ELISA assays to detect DR3, TNFR1, TNF- and caspase-3 proteins. Results showed that CMTK had dramatic anti-tumor activities at low concentrations (IC 50 : 14.24 ± 0.12 mol L 1 ). Importantly, the IC 50 of CMTK on MCF-7 cells was much higher than that of cisplatin. Growth curves show CMTK can inhibit cell proliferation in time- and dose-dependent manners. In cell-cycle analysis, proportions of MCF-7 cells in G 0 /G 1 phase after 12-h and 24-h treatment with CMTK were significantly different from the control group. In contrast, the proportion of cells in S phase tended to decrease, but not significantly. Use of TUNEL and flow cytometry indicated that CMTK induces apoptosis. In MCF-7 cells, CMTK induces TNFR1 production, activates caspase-8 and caspase-9, and enhances caspase-3 levels in a concentration-dependent way; however, CMTK does not affect production of DR3 and TNF- . These results suggest that the anti-tumor mechanism of CMTK involves inducing apoptosis via caspase-8 activation by death receptor, and caspase-9 activation by the mitochondrial-dependent pathway. Our results provide preliminary data for further investigation of the apoptotic mechanism.