依维莫司对顺铂产生抗人皮肤鳞状细胞癌COLO-16细胞效应的增效机制研究

发布时间：2018-05-06 11:34

本文选题：顺铂 + 细胞凋亡　；参考：《天津医科大学》2017年硕士论文

【摘要】：MTOR是一种细胞增殖、凋亡及生存的主要调节分子。MTOR抑制剂,如雷帕霉素,具有免疫抑制作用及抗肿瘤效应。而依维莫司作为雷帕霉素的一种衍生物,也有着抑制MTOR活性的药物学功能。然而,依维莫司是否有抗肿瘤效应却不明确。我们发现使用依维莫司处理人皮肤鳞状细胞癌系COLO-16细胞后,MTOR磷酸化水平下降,LC3-I向LC3-II的转化增加,但自噬流却并未增强;凋亡标志分子caspase3和PARP也并未发生剪切分裂。同时我们也发现依维莫司单独处理细胞时,细胞死亡的一些关键通路并未受到影响;但是与顺铂共同处理细胞时,增强了顺铂诱导的细胞死亡。依维莫司并没有影响顺铂诱导的细胞凋亡,而Chk1(检测点激酶1)的激活却受到了抑制。我们研究证实,依维莫司通过抑制检测点激酶1的激活增强顺铂诱导COLO-16细胞死亡,而且这种效应不依赖于凋亡和自噬调控,发现了依维莫司新的药物效应作用机制。【目的】探讨依维莫司对顺铂的抗人皮肤鳞状细胞癌COLO-16细胞效应的增效机制。【方法】分别用50、100、200nmol/L的依维莫司和25μmol/L顺铂处理人皮肤鳞状细胞癌系COLO-16细胞12、24小时,用AO标记自噬体囊泡并结合溶酶体酶抑制剂分析自噬和自噬流水平。Western印迹分析自噬标记性分子LC3-Ⅰ→LC3-Ⅱ转化、凋亡和细胞周期相关信号通路,并检测MTOR通路、Chk1磷酸化水平、caspase3和PARP剪切;LDH释放实验分析细胞死亡;AnnexinV-EGFP染色分析细胞凋亡。依维莫司与顺铂联合处理的机制实验通过Western印迹进行MTOR、Akt、DNA损伤相关信号以及Chk通路分析。【结果】50、100、200nmol/L的依维莫司处理COLO-16细胞12、24小时后,MTOR2448位点和2481位点的磷酸化水平降低,Rictor1135位点磷酸化水平也降低。然而,下游信号ULK1蛋白的ser757位点的磷酸化水平、P70S6激酶的thr389位点的磷酸化水平的变化并不显著。12小时LC3-Ⅰ→LC3-Ⅱ转化率分别为3.52±0.21、4.03±0.39、5.05±0.22,两两之间比较,差异无统计学意义(P0.05),与未用药物处理的对照组(2.07±0.05)比较,差异有统计学意义(P0.05)。24小时LC3-Ⅰ→LC3-Ⅱ转化率分别为3.38±0.26、3.29±0.06、6.57±0.16,两两之间比较,差异无统计学意义(P0.05),与未用药物处理的对照组(2.61±0.16)比较,差异有统计学意义(P0.05)。50、100、200nmol/L的依维莫司联合E64d、pepstatin处理COLO-16细胞12小时后,LC3-Ⅱ与β肌动蛋白的比值分别为1.26±0.40、1.16±0.34、1.21±0.39,与E64d、pepstatin单独处理细胞组(1.19±0.27)比较,差异无统计学意义(P0.05)。100nmol/L依维莫司联合E64d、pepstatin自噬体囊泡表达阳性率2.06±0.61,与E64d、pepstatin单独处理细胞组(1.68±0.62)比较,差异无统计学意义(P0.05)。依维莫司处理对Akt的总蛋白水平和磷酸化水平无明显影响。顺铂单独处理COLO-16细胞12、24小时,其中12小时细胞死亡率(%)14.33±3.07,24小时细胞死亡率(%)18.20±1.46。依维莫司单独处理细胞12、24小时时:12小时细胞死亡率(%)0.82±0.47,24小时细胞死亡率(%)8.75±1.17,而顺铂和依维莫司联合处理细胞12、24小时,12小时细胞死亡率(%)28.27±2.12,24小时细胞死亡率(%)42.58±0.93,细胞死亡率明显高于顺铂单独处理时的死亡率。顺铂处理细胞24小时后诱导了caspase3和PARP的剪切分裂,另外,顺铂处理细胞后,膜联蛋白(annexin)和碘化丙啶(PI)标记的细胞数目增多。而且,顺铂单独处理细胞和联合依维莫司处理细胞24小时,caspase3和PARP的剪切分裂没有明显的差异,膜联蛋白(annexin)和碘化丙啶(PI)标记的细胞数目也没有明显差异(P0.05)。而且,顺铂单独处理细胞组与联合依维莫司处理细胞组处理细胞12、24小时LC3-Ⅱ形成、caspase3和PARP剪切、AnnexinV标记细胞数均无统计学差异(P0.05)。顺铂处理COLO-16细胞12、24小时后,Rictor的thr1135位点磷酸化水平和Chk1的ser345位点磷酸化水平明显升高,依维莫司单独处理无类似效应。依维莫司与顺铂联合处理后12和24小时,相对于顺铂单独处理的细胞,上调的Chk1和Rictor磷酸化水平受到明显抑制。【结论】1.依维莫司增强顺铂诱导COLO-16细胞死亡,这种效应不依赖于凋亡和自噬调控。2.COLO-16细胞MTOR通路对依维莫司处理敏感,但其下游信号并非同步产生级联反应。细胞凋亡、Akt通路对依维莫司处理不敏感。3.依维莫司通过抑制检测点激酶1的激活增强顺铂诱导COLO-16细胞死亡,但无加重顺铂所导致的DNA损伤。
[Abstract]:MTOR is a major regulatory molecule.MTOR inhibitor for cell proliferation, apoptosis and survival, such as rapamycin, which has immunosuppressive effects and antitumor effects. As a derivative of rapamycin, iverimus also has a pharmacological function to inhibit MTOR activity. However, it is not clear whether iverimus has an antitumor effect. After the use of imimus to treat human squamous cell carcinoma COLO-16 cells, the level of phosphorylation of MTOR decreased and the transformation of LC3-I to LC3-II increased, but the autophagic flow did not increase; the apoptotic markers, Caspase3 and PARP, did not occur in shear division. The pathway was not affected, but the cell death induced by cisplatin was enhanced when cisplatin was treated together with cisplatin. Imus did not affect cisplatin induced apoptosis, but the activation of Chk1 (detection point kinase 1) was inhibited. Our study confirmed that the activation of the Imus through inhibitory detection point kinase 1 enhanced cisplatin induced COLO-1 6 cell death, and this effect is not dependent on apoptosis and autophagy regulation, and a new mechanism of effector effect of Imus is found. [Objective] to explore the synergistic mechanism of imimols on cisplatin against human skin squamous cell carcinoma COLO-16 cell effect. [Methods] the treatment was treated with 50100200nmol/L's imimus and 25 mol/L cisplatin respectively. Human skin squamous cell carcinoma cell line COLO-16 cells are 12,24 hours, using AO to mark autophagosome vesicles and combine with lysosomal enzyme inhibitors to analyze autophagic and autophagic flow level.Western blot analysis of autophagic markers LC3- I to LC3- II transformation, apoptosis and cell cycle related signal pathways, and detect MTOR pathway, Chk1 phosphorylation level, Caspase3 and PARP. Shear; LDH release assay was used to analyze cell death; AnnexinV-EGFP staining was used to analyze cell apoptosis. The mechanism of combined treatment with imimus and cisplatin was carried out by Western blot for MTOR, Akt, DNA damage related signals and Chk pathway analysis. [results] 50100200nmol/L's everimus treated COLO-16 cells for 12,24 hours, MTOR2448 site and 2 The phosphorylation level of the 481 loci was reduced and the phosphorylation level of the Rictor1135 site decreased. However, the level of phosphorylation of the ser757 loci of the downstream signal ULK1 protein, the change in the phosphorylation level of the thr389 site of the P70S6 kinase was not significantly.12 hours LC3- I to LC3- II conversion rate was 3.52 + 0.21,4.03 + 0.39,5.05 + 0.22, respectively, 22, respectively. The difference was not statistically significant (P0.05), compared with the control group (2.07 + 0.05) without drug treatment (2.07 + 0.05), the difference was statistically significant (P0.05).24 hours LC3- I to LC3- II conversion rate was 3.38 + 0.26,3.29 + 0.16 respectively, the difference was not statistically significant (P0.05), compared with the control group (2.61 + 0.16), which was not treated with drug treatment (2.61 + 0.16). The ratio of LC3- II to COLO-16 cells was 1.26 + 0.40,1.16 + 0.34,1.21 + 0.39 respectively after 12 hours of COLO-16 cells treated with.50100200nmol/L and E64d, and LC3- II and beta actin were respectively compared with E64d and pepstatin alone (1.19 + 0.27). There was no statistical difference (P0.05) with.100nmol/L imimols. E64d, pepstatin autophagic vesicle positive rate was 2.06 + 0.61, compared with E64d, pepstatin alone treated cell group (1.68 + 0.62), the difference was not statistically significant (P0.05). The total protein level and phosphorylation level of Akt were not significantly affected by the imimolus treatment. The 12 hour cell death rate of 12 hours (%) was 1 of cisplatin alone at 12,24 hours. 4.33 + 3.07,24 hours cell death rate (%) 18.20 + 1.46. imimus treated cells for 12,24 hours alone: 12 hours cell mortality (%) 0.82 + 0.47,24 hours cell mortality (%) 8.75 + 1.17, while cisplatin and everimus combined with 12,24 hours, 12 hours cell mortality (%) 28.27 + 2.12,24 hours cell mortality (%) 42.58 + 0.93, The cell death rate was significantly higher than that of cisplatin alone. 24 hours after cisplatin treated cells, the shear division of Caspase3 and PARP was induced. In addition, after cisplatin treated cells, the number of cells marked by annexin and PI was increased. Moreover, cisplatin treated cells alone and combined with ivimols for 24 hours. There was no significant difference in the shear division between Caspase3 and PARP, and there was no significant difference in the number of cells labeled by annexin (annexin) and propidium iodide (PI). Moreover, the cells treated by cisplatin alone and in the combined Imus treated cell group were treated with 12,24 hours LC3- II formation, Caspase3 and PARP shear, and AnnexinV labeled cells. No statistical difference (P0.05). After COLO-16 cells were treated with cisplatin for 12,24 hours, the level of phosphorylation of Rictor at thr1135 site and the phosphorylation level of ser345 loci of Chk1 were significantly increased. There was no similar effect by imimus alone. 12 and 24 hours after the combination of imimus and cisplatin, the up regulation of Chk1 and Ric relative to the cells treated by cisplatin alone The level of tor phosphorylation was significantly inhibited. [Conclusion] 1. imimoll enhanced cisplatin induced COLO-16 cell death, which is not dependent on apoptosis and autophagy regulating the sensitivity of the MTOR pathway to the imimus treatment, but the downstream signal is not synchronized with the cascade reaction. Cell apoptosis, Akt pathway is not sensitive to imimus treatment. Sensory.3. everolimus enhanced cisplatin induced COLO-16 cell death by inhibiting the activation of checkpoint kinase 1, but did not aggravate cisplatin induced DNA damage.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

【参考文献】