【作者】 孙岩；

【导师】 邱荣国；

【作者基本信息】 大连理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 变构菌素(Tautomycetin, TMC)作为一种新型的T细胞免疫抑制剂,具有独特的化学结构。目前有研究表明,TMC具有很好的抗肿瘤作用,有望成为极具潜力的抗癌药物,但其抗肿瘤的作用机制还没有被完全阐明。本研究首先通过MTT法测定了变构菌素对不同肿瘤细胞的细胞毒性,结果显示：变构菌素可以抑制乳腺癌细胞MCF-7、子宫颈癌细胞Hela、乳腺癌细胞MCF-7/ADR、肺癌细胞A549、黑色素瘤细胞B16等肿瘤细胞的生长,IC50(μM)分别为0.35、0.67、1.5、5.07、5.32。我们以乳腺癌细胞MCF-7为体外模型,初步探讨了TMC诱导乳腺癌细胞MCF-7凋亡的作用机制。为了进一步证明MTT法所得的结果,我们用流式细胞术对TMC处理的细胞进行了周期和凋亡分析。FCM检测结果表明,经0.5μM TMC处理24h后,G0/G1期细胞数量从43.38%上升到60.14%,S期细胞数量从35.83%降低到17.82%,表明TMC诱导了MCF-7细胞G1/S期阻滞。经0.5、1μM TMC处理24h后,MCF-7细胞早期凋亡率均高于对照组,且随着TMC浓度的升高而升高。Western blot检测凋亡效应因子caspase-7蛋白表达的结果显示,经TMC处理12和24h后,细胞pro-caspase-7蛋白表达明显降低,相应的活化的caspase-7蛋白表达明显升高,而加入caspase抑制剂Z-VAD-FMK后,TMC对MCF-7的细胞毒性受到了明显抑制,表明TMC是通过激活下游效应因子caspase-7诱导细胞凋亡的。Western blot检测凋亡相关蛋白的结果显示,经TMC处理12和24h后,细胞Bcl-2和pro-caspase-9蛋白表达均明显降低,cytochrome-c蛋白表达明显升高,而pro-caspase-8和p53蛋白表达无明显变化。同时,通过细胞转染及报告基因检测技术我们检测了TMC对p53转录活性的影响,结果显示TMC所引起的凋亡反应并不依赖于对p53转录的特异性激活反应。所得结果显示,TMC以时间和剂量依赖的方式诱导体外培养的乳腺癌细胞MCF-7凋亡,其作用通路为线粒体／细胞色素C介导的凋亡通路,凋亡机制为下调Bcl-2蛋白表达,引发cytochrome-c的释放以及caspase-9的活化,从而激活下游效应因子caspase-7,引起细胞凋亡,其作用并不依赖于p53,为进一步研究TMC抗肿瘤作用的机制与靶点,及其临床肿瘤治疗的可行性奠定了一定的理论基础。更多还原

【Abstract】 Tautomycetin (TMC) is a novel T-cell immunosuppressor with unique chemical structure. It has been reported as a potential drug for cancer, and can inhibit the proliferation of cancer cells, but the molecular mechanisms have not been fully elucidated.Our studies showed that TMC could inhibit proliferation of different kinds of cancer cells in vitro including MCF-7, Hela, MCF-7/ADR, A549 and B16, the value of IC50 (μM) was 0.35, 0.35,0.67,1.5,5.07 and 5.32 respectively. We chose MCF-7 to continue our rearch.To confirm the results obtained from MTT assays, we analyzed the effects of TMC on cell cycle progression and apoptosis by flow cytometry assay. The results showed that,0.5μM TMC treatment caused an increase of the percentage of cells in the Gi phase from 43.38 to 60.14% and a decrease in the percentage of those in the S phase from 35.83 to 17.82%, which means TMC caused the G1/S cell cycle arrest in MCF-7 cells. When treated MCF-7 cells with 0.5 and 1μM TMC for 24h, the apoptosis rates were higher than that in control. Then we analyzed the effects of TMC on the expression of caspase-7 by western blot. The result showed that, treating MCF-7 cells with TMC resulted in caspase-7 activation, and the activation could be blocked by an caspase inhibitor, Z-VAD-FMK, indicating the important role of caspase-7 in TMC-induced apoptosis.We studied the expression of apoptosis related proteins by western blot, which showed that, when treated MCF-7 cells with TMC for 12 and 24h, Bcl-2 and pro-caspase-9 expression was down-regulated significantly, cytochrome-c expression was up-regulated, but there was no obvious difference between pro-caspase-8 and p53 expression and that in control. At the same time, we did some research on effects of TMC on p53 transcriptional activity in MCF-7 cells by transfection and transient reporter assay, the results showed that transcriptional activation of p53 is not required for TMC induced apopotosis in MCF-7 cells.The results indicated that TMC could induce apoptosis of MCF-7 cells in a time and dose dependent manner, the key regulators include Bcl-2, caspase-9, cytochrome-c and caspase-7, which are controlled through the intrinsic apoptosis pathway independent of p53.更多还原