Procaspase-8的异常表达抑制TRAIL诱导肿瘤细胞凋亡

发布时间：2018-05-29 05:00

本文选题：procaspase-8 + TRAIL　；参考：《昆明理工大学》2015年硕士论文

【摘要】：TRAIL ((Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand,肿瘤坏死因子相关凋亡诱导配体)属于TNF-α相关细胞因子家族成员,并因其选择性抗肿瘤且对正常细胞无毒性的特点,使其成为肿瘤治疗的潜力药物。AT-406是凋亡抑制蛋白(Inhibitor of Apoptosis Proteins, IAPs)的一种泛拮抗剂。Rocaglamide是从楝科米仔兰属中分离得到的天然化合产物,并被证明它能从翻译水平抑制c-FLIP(Cellular Fadd-Like (Flice)-Inhibitory Protein)的表达,而c-FLIP被证实能够竞争性阻断TRAIL凋亡通路。在我们的研究中,发现大部分实体瘤细胞系对ART(AT-406(A), Rocaglamide (R)和TRAIL (T))联合用药方式敏感。我们发现,ART联合用药方式能够有效地诱导大部分实体瘤细胞系凋亡,但对一株乳腺癌细胞系MCF-7,几株神经胶质瘤细胞系(U251, U373, U87)以及人体正常牙髓细胞(Pulp)没有诱导凋亡的作用。在本篇研究中,我们进—步证实在这些对ART联合用药极为耐受的细胞中,除了c-FL IP和IAPs这些抑凋亡蛋白有很高的表达量,procaspase-8的表达水平非常低,而其是激活TRAIL凋亡通路的上游蛋白。上调这些细胞中的procaspase-8蛋白表达量能够提高这些细胞对ART联合用药方式的敏感性。在此研究中,我们利用MTT细胞活性检测技术以及Western Blots技术分析TRAIL所诱导的外源性凋亡通路中的各个蛋白表达水平。通过这些方法来研究ART联合用药方式所诱导的凋亡效率。实验结果显示乳腺癌细胞系MCF-7,几株神经胶质瘤细胞及正常牙髓细胞pulp都对ART联合用药方式表现出极强的耐性。通过、Vestern Blots蛋白分析发现,这些细胞中procaspase-8表达水平非常低,但凋亡通路中的其他蛋白的表达水平却很高,包括死亡受体DR4/5,Fas相关的死亡结构域FADD, c-FLIP和XIAP。我们通过利用Dup-697(一种COX-2的抑制剂),丙戊酸钠(一种去乙酰化酶抑制剂)以及5-氮杂-2’-脱氧胞苷(一种甲基化酶抑制剂)来预处理这些细胞,提高procaspase-8蛋白在这些细胞中的表达水平,提高了ART联合用药方式对这些细胞的促凋亡率。总体来说,我们的实验数据证明了ART联药方式有效地诱导大部分实体瘤细胞系的凋亡。但有部分的实体瘤细胞和神经胶质瘤细胞仍对ART联药方式有很高的耐性。上调procaspase-8的表达量可以提高凋亡率。同时,人正常牙髓细胞也表现出对ART联合用药很高的耐性,提高牙髓细胞中procaspase-8的表达量可以提高联合用药对其凋亡率。提示我们的ART联合用药方式对人正常细胞没有毒性,而其原因可能就是procaspase-8的低表达。因此,我们认为这些肿瘤细胞系和人正常牙髓细胞能够逃逸凋亡的机制在于procaspase-8的低表达水平,procaspase-8在TRAIL凋亡过程必不可少。探究procaspase-8的主要异常情况,为未来以TIAIL为基础的癌症治疗方法提供进一步的治疗策略。
[Abstract]:TRAIL Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (tumor necrosis factor-related apoptosis-inducing ligand) is a member of TNF- 伪 associated cytokine family. AT-406 is a pan-antagonist of apoptosis inhibitor of Apoptosis Proteins, IAPs). Rocaglamide is a natural compound isolated from the genus Melia, and has been shown to inhibit the expression of c-FLIP(Cellular Fadd-Like Flicesis-Inhibitory protein at translation level. C-FLIP has been proved to be able to competitively block TRAIL apoptosis pathway. In our study, it was found that most solid tumor cell lines were sensitive to the combination of ARTI-AT-406A (Rocaglamide) and TRAIL (TX). We found that the combined use of art can effectively induce apoptosis in most solid tumor cell lines, but it has no effect on one breast cancer cell line MCF-7, several glioma cell lines U251, U373, U87) and pulp cells of human normal dental pulp. In this study, we further demonstrated that in these cells that are extremely resistant to ART combination, the expression of proc caspase-8, in addition to c-FL IP and IAPs, is very low. It is the upstream protein that activates the apoptosis pathway of TRAIL. Upregulating the expression of procaspase-8 protein in these cells increased the sensitivity of these cells to the combination of ART. In this study, we analyzed the expression levels of various proteins in exogenous apoptotic pathway induced by TRAIL by MTT assay and Western Blots technique. These methods were used to study the apoptotic efficiency induced by the combination of ART. The results showed that the breast cancer cell line MCF-7, several glioma cells and normal dental pulp cell line pulp all showed strong tolerance to the combination of ART. Vestern Blots protein analysis showed that the expression of procaspase-8 was very low in these cells, but the expression of other proteins in apoptosis pathway was very high, including FADDD, c-FLIP and XIAPs associated with death receptor DR4 / 5Fas. We pretreated these cells with Dup-697, an inhibitor of COX-2, sodium valproate (a deacetylase inhibitor) and 5-aza-2-deoxycytidine (a methylase inhibitor). The expression of procaspase-8 protein in these cells was increased, and the apoptotic rate of these cells was increased by the combination of ART. In general, our experimental data demonstrate that ART-combined drugs can effectively induce apoptosis in most solid tumor cell lines. However, some solid tumor cells and glioma cells still have high tolerance to ART combination. Upregulation of procaspase-8 expression can increase apoptosis rate. At the same time, human normal dental pulp cells also showed a high tolerance to combined use of ART. Increasing the expression of procaspase-8 in dental pulp cells could increase the apoptosis rate of combined treatment. The results suggest that our combined use of ART has no toxicity to human normal cells, which may be due to the low expression of procaspase-8. Therefore, we believe that the mechanism that these tumor cell lines and human normal dental pulp cells can escape apoptosis lies in the low expression level of procaspase-8 in the process of TRAIL apoptosis. Explore the main abnormalities of procaspase-8 and provide further strategies for future TIAIL-based cancer treatment.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-36

【相似文献】