雷公藤红素抑制结直肠癌生长的机制研究

发布时间：2018-06-13 05:25

  本文选题：雷公藤红素 + 结直肠癌　； 参考：《北京协和医学院》2017年博士论文

【摘要】：近年来,中药防治肿瘤受到了人们广泛的关注。雷公藤是我国传统中药材卫矛科雷公藤属植物,它的有效成分之一——雷公藤红素,被证明具有广泛的药理学作用。临床上,雷公藤红素作为传统药物,用于治疗多种炎症疾病和自身免疫性疾病,如过敏性哮喘、肌萎缩侧索硬化症以及类风湿性关节炎等。随着研究的深入,人们逐渐发现,雷公藤红素还可以抑制多种肿瘤的发生发展,如乳腺癌、肝细胞肝癌、膀胱癌、胃癌和前列腺癌等,但在结直肠癌中的报道却非常少见。本研究的目的是系统地探究雷公藤红素对结直肠癌的作用以及其具体的分子机制。MTT和细胞克隆的实验结果表明,雷公藤红素可以显著地抑制结直肠癌细胞系SW480和HCT116细胞的生长。我们用雷公藤红素腹腔注射两种经典的结直肠癌动物模型——-C57BL/6J-ApcMin/+(APCMin/+)小鼠和AOM/DSS小鼠,结果发现雷公藤红素在小鼠体内也有明显的抑瘤作用。同时,Western blot和TUNEL的实验结果证明,雷公藤红素在体内、外可以促进细胞凋亡。此外,我们还发现雷公藤红素可以通过泛素化-蛋白酶体途径降解β-catenin,从而抑制Wnt/β-catenin信号通路的活性。面临床上,大多数患者的结直肠癌组织都存在Wnt/β-catenin信号通路的异常活化。因此,这提示我们,抑制Wnt/β-catenin信号通路可能是雷公藤红素抑制结直肠癌的机制之一。Hippo信号通路与Wnt/β-catenin信号通路有着千丝万缕的联系。Hippo信号通路是一条高度保守的信号通路,参与调控器官大小等。有研究指出,Hippo信号通路的下游主要效应分子——Yes相关蛋白(yes-associated protein,YAP)与β-catenin可以相互作用,从而调节肠道再生以及结直肠癌的发生发展。我们的研究表明,在雷公藤红素降解β-catenin的过程中,YAP存在于β-catenin降解的复合体上,并且YAP的磷酸化对β-catenin的降解至关重要。最近的文献报道,肝脏激酶1(liver kinase B1,LKB1),也被称作丝氨酸-苏氨酸激酶 11(serine-threonine kinase 11,STK11)作为抑癌基因,可以促进YAP的磷酸化,使得YAP从细胞核移至细胞浆中,最后被蛋白酶体降解。我们在SW480和HCT116细胞中利用CRISPR-Cas9体系构建了LKB1缺失的稳定细胞系。在体内,我们利用Loxp-Cre体系构建了在肠道特异性敲除LKB1的小鼠,并且用AOM/DSS诱导其发展为结直肠癌。我们用雷公藤红素处理后,结果发现LKB1的缺失不仅可以在体内外促进结直肠癌细胞的生长,还能拮抗雷公藤红素降解β-catenin,从而削弱雷公藤红素的抑瘤作用。我们进一步还发现了雷公藤红素可以上调热休克因子1(heat shock factor 1,HSF1)的表达,从而增强LKB1的转录活性。LKB1激活其下游分子——AMP活化的蛋白激酶α(AMP-activated protein kinase α,AMPKα),进一步磷酸化 YAP,从而促进了β-catenin泛素化-蛋白酶体途径的降解。综上所述,我们的研究表明,雷公藤红素通过泛素化-蛋白酶体途径降解β-catenin,从而在体内、外明显地抑制结直肠癌细胞的生长,而找到雷公藤红素降解β-catenin的具体途径是关键。我们的研究发现了其潜在的一条通路——雷公藤红素可以通过提高HSF1的蛋白水平,上调LKB1的转录活性,从而激活AMPKα,进一步磷酸化YAP。磷酸化的YAP存在于β-catenin降解的复合体上,促进β-catenin泛素化-蛋白酶体途径的降解。在此过程中,YAP和LKB1在结直肠癌发生发展和雷公藤红素抑瘤过程中起着不可或缺的作用。以上结果为雷公藤红素在临床上用于结直肠癌的治疗提供了很好的理论基础。Hippo信号通路是一条高度保守的信号通路,它参与机体组织和器官的发育过程和维持体内细胞增殖与凋亡的动态平衡,也被证明与肿瘤的发生发展有着密切的联系。Hippo信号通路的主要下游效应分子YAP,是一个转录激活因子,参与调控下游很多靶基因,而这些靶基因多与细胞的增殖与存活相关。作为癌基因,YAP可以促进细胞生长和抑制细胞凋亡。而Hippo信号通路下游的另一个主要效应分子TAZ,与YAP具有将近60%的同源性。因此,我们推测TAZ可能具有与YAP相似的生物学功能。为了验证我们的猜想,我们分别在T-Rex-293细胞和HEK293细胞中构建了 TAZ诱导表达和稳定表达的细胞系。细胞生长曲线、细胞克隆形成以及裸鼠皮下成瘤实验结果证明了,高表达TAZ可以在体内外促进细胞的生长。同时,我们还发现上调TAZ的表达可以部分逆转雷公藤红素诱导的细胞凋亡。接着,我们又找到了 TAZ抵抗细胞凋亡的可能的分子机制——诱导的TAZ高表达可以上调其下游靶基因包括ANKRD,CYR61以及CTGF,并且还可以增加Bcl-2的表达,降低Bax的表达,活化PI3K/Akt信号通路。以上发现揭示了 TAZ作为癌基因可以参与调控细胞增殖和凋亡。
[Abstract]:In recent years, traditional Chinese medicine for the prevention and control of tumor has attracted wide attention. Tripterygium wilfordii is a traditional Chinese medicinal herb of the genus Corey, one of its effective components, tripterin, has been proved to have extensive pharmacological effects. Diseases, such as allergic asthma, amyotrophic lateral sclerosis and rheumatoid arthritis, have gradually been found, with the further research, that tripterin can also inhibit the development of various tumors, such as breast cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, and prostate cancer, but it is rarely reported in colorectal cancer. The purpose of this study was to systematically explore the effect of tripterin on colorectal cancer and its specific molecular mechanism.MTT and cell clones. The results showed that tripterin could significantly inhibit the growth of SW480 and HCT116 cells in the colorectal cancer cell lines. We injected two classic colorectal cancer models with tripterin abdominal cavity. -C57BL/6J-ApcMin/+ (APCMin/+) mice and AOM/DSS mice found that tripterine was also significantly antitumor in mice. Meanwhile, the results of Western blot and TUNEL showed that tripterine could promote apoptosis in the body. In addition, we also found that tripterine could be used as a ubiquitin protease. The body pathway degrades beta -catenin and inhibits the activity of the Wnt/ beta -catenin signaling pathway. In the face of the bed, the colorectal cancer tissues in most patients have abnormal activation of the Wnt/ beta -catenin signaling pathway. Therefore, this suggests that the inhibition of Wnt/ beta -catenin signaling pathway may be one of the.Hippo signals of Lei Gongteng erythropoietin inhibition of colorectal cancer. The pathway is inextricably linked to the Wnt/ beta -catenin signaling pathway. The.Hippo signaling pathway is a highly conservative signal pathway and participates in the regulation of the size of the organ. There is a study that the main effector molecules of the Hippo signaling pathway, the Yes related protein (Yes-associated protein, YAP), can interact with the beta -catenin, thereby regulating the interaction between the Hippo signaling pathway and the beta -catenin. Intestinal regeneration and the development of colorectal cancer. Our study showed that in the course of tripterine degradation of beta -catenin, YAP exists on the complex of beta -catenin degradation, and the phosphorylation of YAP is essential for the degradation of beta -catenin. Recent literature reports that liver irritable enzyme 1 (liver kinase B1, LKB1), also known as serine - Su. As a tumor suppressor gene, ammonia kinase 11 (serine-threonine kinase 11, STK11) can promote the phosphorylation of YAP, causing YAP to move from the nucleus to the cytoplasm and eventually degrade the proteasome. We construct a LKB1 deletion stable cell line in SW480 and HCT116 cells. In vivo, we use the Loxp-Cre system to construct the cell. The mice in the intestinal specific knockout of LKB1 were induced to develop colorectal cancer with AOM/DSS. After treatment with tripterine, we found that the absence of LKB1 could not only promote the growth of colorectal cancer cells in vivo and in vivo, but also antagonize the degradation of beta -catenin by tripterine and weaken the tumor suppressor effect of tripterin. It is also found that tripterin can increase the expression of heat shock factor 1 (heat shock factor 1, HSF1), thereby enhancing the transcriptional activity of LKB1 to activate its downstream molecules - AMP activated protein kinase alpha (AMP-activated protein kinase A, AMPK alpha), and further phosphorylation YAP, thus promoting beta ubiquitination - protease. In summary, our study shows that tripterin degrades beta -catenin through the ubiquitin proteasome pathway and thus significantly inhibits the growth of colorectal cancer cells in the body, and the specific pathway for the discovery of tripterin to degrade beta -catenin is the key. Our study found a potential pathway - Lei Gongteng erythropoietin can activate the transcriptional activity of LKB1 by raising the protein level of HSF1 and activating the AMPK alpha, and further phosphorylated YAP. phosphorylation YAP exists on the complex of beta -catenin degradation, promoting the degradation of beta -catenin ubiquitination proteasome pathway. In this process, YAP and LKB1 are developed in colorectal cancer and in the development of colorectal cancer and Lei Gongteng Erythropoietin plays an indispensable role in tumor suppression. The above results provide a good theoretical basis for the clinical application of tripterin in the treatment of colorectal cancer. The.Hippo signal pathway is a highly conservative signal pathway. It participates in the development of tissues and organs and maintains the dynamic balance of cell proliferation and apoptosis in the body. It has also been shown that the main downstream effector, YAP, which is closely related to the development of the.Hippo signaling pathway, is a transcriptional activator that participates in the regulation of many downstream target genes, which are related to cell proliferation and survival. As a oncogene, YAP can promote cell growth and inhibit apoptosis. Hippo TAZ, another major effector downstream of the signal pathway, has nearly 60% homology with YAP. Therefore, we speculate that TAZ may have a biological function similar to that of YAP. In order to verify our conjecture, we constructed the cell lines of TAZ induced and stable expression in T-Rex-293 and HEK293 cells. The cell growth curve, The results of cell cloning and subcutaneous tumor formation in nude mice showed that high expression of TAZ could promote cell growth in vitro and in vivo. Meanwhile, we also found that up regulation of TAZ could partly reverse the apoptosis induced by tripterin. Then, we found the possible molecular mechanism of TAZ resistance to apoptosis - induced TA The high expression of Z can increase its downstream target genes including ANKRD, CYR61 and CTGF, and also increase the expression of Bcl-2, reduce the expression of Bax and activate the PI3K/Akt signaling pathway. These findings reveal that TAZ as a oncogene can participate in the regulation of cell proliferation and apoptosis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285
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本文编号：2012885