CYT387作为IKBKE抑制剂通过激活Hippo通路抑制胶质母细胞瘤恶性进展

发布时间：2018-05-02 10:13

本文选题：IKBKE + Hippo通路　；参考：《天津医科大学》2017年博士论文

【摘要】：最近的研究表明,IKBKE(inhibitor of nuclear factor kappa-B kinase subunit epsilon)已经证实在多种恶性肿瘤中高表达,且沉默IKBKE蛋白表达可以抑制肿瘤进展。在本篇文章中,我们首先使用CCK-8检测方法计算CYT387对2种代表性的胶质母细胞瘤细胞系U87-MG及LN229的半抑制浓度(half maximal inhibitory concentration,IC50)确定胶质母细胞瘤对此种小分子敏感;并且验证CYT387,作为一种潜在的IKBKE抑制剂,能够显著地抑制胶质瘤细胞系细胞的增殖、迁移以及侵袭能力。此外,CYT387还可诱导细胞凋亡和并使细胞周期阻滞在G2期。与此同时我们发现,沉默IKBKE蛋白含量可以增强Hippo通路,降低Hippo通路下游因子的转录水平。另外,我们证实了CYT387不仅仅抑制IKBKE激酶活性同时也降低其在蛋白水平的表达,而这种改变不涉及IKBKE mRNA水平。接下来,通过免疫印迹的办法证实了CYT387从剂量及时间依赖性上促进Hippo信号通路来控制肿瘤的恶性进展。为探查更深一步的机制,我们使用免疫共沉淀(Co-immunoprecipitation,co-IP)的方法及特异性磷酸化抗体证实了IKBKE与YAP1及TEAD2相互作用,促进YAP1及TEAD2入核,调控肿瘤增殖生长。在接下来的体内中,我们发现CYT387能明显抑制裸鼠皮下肿瘤的生长但无法遏制颅内肿瘤的生长,这可能是由于CYT387穿透血脑屏障(blood brain barrier,BBB)差造成的。这些结果表明,CYT387有可能成为新的抗胶质瘤靶向药物但需要寻求通过血脑屏障的方法。方法我们使用CCK-8检测方法设立不同药物浓度,通过检测一定药物浓度梯度下肿瘤细胞成活率,确定胶质母细胞瘤细胞系U87-MG及LN229在不同时间下的半抑制浓度(half maximal inhibitory concentration,IC50)。我们使用CCK-8的检测方法及克隆形成实验测定小分子抑制剂CYT387对肿瘤细胞增殖能力;利用划痕试验及transwell方法检测药物对胶质瘤细胞系迁移及侵袭能力的影响。接下来应用流式细胞仪检测小分子抑制剂CYT387对胶质瘤细胞凋亡及周期的影响;并通过免疫印迹的方法,检测相应周期蛋白及凋亡蛋白的表达;那下面,机制的研究中,我们先使用IKBKE-shRNA沉默IKBKE的蛋白表达,检测Hippo通路核心蛋白LATS2,YAP1,TEAD2,p-YAP1磷酸化127位点及下游效应蛋白Axl,c-myc的表达含量的变化,并通过使用重组质粒过表达IKBKE的蛋白含量,重新检测Hippo通路核心蛋白及下游效应蛋白表达变化,并使用小分子抑制剂CYT387从剂量及时间依赖性上处理胶质瘤细胞系检测这些指标的变化;在接下来对进一步机制的研究中,我们使用免疫共沉淀(Co-immunoprecipitation,co-IP)的方法,从内源性及外源性两个方面说明IKBKE与YAP1,TEAD2存在相互结合的作用;分别提取胞浆及胞核蛋白,分别使用免疫印记的方法说明沉默IKBKE可以抑制YAP1及tead2入核启动转录过程,并应用免疫荧光的办法说明沉默IKBKE可以抑制YAP1的入核;使用real-time RT-PCR证实IKBKE改变YAP1,TEAD2的含量是否牵扯到转录或者翻译水平。使用U87-MG细胞注射4-5周大小裸鼠皮下建立裸鼠皮下肿瘤模型,模型建立一周后使用灌胃针经口灌胃CYT387(100mg/kg/day),饲养30天监测皮下肿瘤大小,随后处死全部裸鼠,获取对照组与实验组全部肿瘤称重,免疫组化的方法检测IKBKE,凋亡,周期,Hippo通路及下游因子指标变化;随后使用感染了luciferase病毒的U87-MG细胞在立体定向仪的辅助下建立裸鼠颅内肿瘤模型,实验组每天灌胃药物CYT387(100mg/kg/day),每7天使用活体荧光成像仪监测颅内肿瘤大小,监测裸鼠体重及裸鼠存活情况,取鼠脑切片做免疫组化染色确定指标变化。结果 1,胶质瘤细胞系U87-MG及LN229对CYT387十分敏感,经CYT387处理后胶质瘤细胞增殖及集落形成能力大大降低。2,CYT387可以明显抑制胶质瘤细胞系U87-MG及LN229迁移及侵袭能力。3,CYT387可以促进胶质瘤细胞系U87-MG及LN229凋亡。4,CYT387促进胶质瘤细胞系U87-MG及LN229周期阻滞,使肿瘤阻滞于G2期。5,CYT387不仅可以抑制IKBKE激酶活性还可以降低IKBKE蛋白表达含量,这种改变主要是翻译后的修饰,在转录水平无明显影响。6,CYT387通过抑制IKBKE表达及活性增强Hippo通路抑制恶性胶质瘤细胞生长。7,IKBKE可以与YAP1及TEAD2直接作用影响Hippo通路,沉默IKBKE可以抑制YAP1及TEAD2向细胞核内转座。8,CYT387可以抑制裸鼠皮下肿瘤生长但对裸鼠颅内原位肿瘤的抑制效果不明显。结论 1,CYT387在体外实验中可以明显抑制胶质母细胞瘤细胞系U87-MG及LN229的生长;促进其凋亡;2,CYT387可以活化Hippo通路抑制肿瘤细胞的生长,而这一作用主要是通过减低IKBKE激酶的活性及表达量实现的。3,IKBKE可与YAP1及TEAD2相互作用,促进YAP1及TEAD2向细胞内转座从而影响Hippo通路从而控制胶质瘤进展;4,CYT387可以抑制皮下肿瘤的生长但对颅内肿瘤影响效果不明显,可能与其透过血脑屏障差有关。
[Abstract]:Recent studies have shown that IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) has been shown to be highly expressed in a variety of malignant tumors, and the expression of silent IKBKE protein can inhibit the progression of tumors. In this article, we first used CCK-8 detection to calculate CYT387 pairs of 2 representative glioblastoma cell lines. -MG and LN229's semi inhibitory concentration (half maximal inhibitory concentration, IC50) determines the sensitivity of glioblastoma to this small molecule, and verifies that CYT387, as a potential IKBKE inhibitor, can significantly inhibit the proliferation, migration and invasiveness of glioma cell lines. In addition, CYT387 can also induce apoptosis and the apoptosis of glioblastoma cells. And the cell cycle was blocked at G2 phase. At the same time, we found that the silencing of IKBKE protein content could enhance the Hippo pathway and reduce the transcriptional level of the downstream factors of the Hippo pathway. In addition, we have confirmed that CYT387 not only inhibits the activity of IKBKE kinase, but also reduces its protein level, but this change does not involve the level of IKBKE mRNA. By immunoblotting, it was demonstrated that CYT387 promotes the malignant progression of the tumor by promoting the dose and time dependence of the Hippo signaling pathway. In order to explore a further mechanism, we use the Co-immunoprecipitation (co-IP) method and specific phosphorylated antibodies to confirm the interaction of IKBKE with YAP1 and TEAD2. In the next body, we found that CYT387 can significantly inhibit the growth of subcutaneous tumors in nude mice but can not inhibit the growth of intracranial tumors in the next body, which may be caused by the poor CYT387 penetration of the blood brain barrier (blood brain barrier, BBB). These results suggest that CYT387 may become a new one. These results suggest that CYT387 may become a new one. Anti glioma targeting drugs need to be sought through the blood brain barrier method. Methods we used CCK-8 detection methods to establish different drug concentrations. By detecting the survival rate of tumor cells under a certain concentration gradient, the U87-MG and LN229 concentration of glioblastoma cell lines at different time (half maximal inhibitory CO) were determined. Ncentration, IC50). We used CCK-8 detection method and clone formation test to determine the proliferation ability of small molecule inhibitor CYT387 to tumor cells; use scratch test and Transwell method to detect the effect of drug on the migration and invasion ability of glioma cell lines. Then, flow cytometry was used to detect small molecule inhibitor CYT387 to glia. The effect of apoptosis and cycle of tumor cells, and the expression of corresponding cyclin and apoptotic protein by immunoblotting, and in the study of mechanism, we first use IKBKE-shRNA silencing IKBKE protein expression to detect the core protein of Hippo pathway LATS2, YAP1, TEAD2, p-YAP1 phosphorylation site 127 and downstream effector protein Axl, c-myc The changes in the expression of the content of IKBKE were redetected by using recombinant plasmids to overexpress the protein content of the Hippo pathway, and the changes in the expression of the core protein and downstream effector protein were redetected, and the changes of the glioma cell lines were treated with the dose and time dependence of the small molecular inhibitor CYT387. In the study, we use the method of Co-immunoprecipitation (co-IP) to explain the interaction between IKBKE and YAP1 and TEAD2 from two aspects of endogenous and exogenous. The cytoplasm and nucleoprotein were extracted respectively. The method of immuno imprinting was used respectively to show that IKBKE could inhibit the transcription of YAP1 and tead2, and the transcription process of YAP1 and tead2 was inhibited. The immunofluorescence method was used to show that silent IKBKE could inhibit the nucleation of YAP1; real-time RT-PCR was used to confirm that IKBKE changed YAP1, whether the content of TEAD2 was involved in the transcription or translation level. The subcutaneous tumor model of nude mice was established under the subcutaneous injection of U87-MG cells for 4-5 weeks in nude mice, and the model was established by intragastric intragastric perfusion a week later. 87 (100mg/kg/day), the size of the subcutaneous tumor was monitored for 30 days, then all nude mice were killed and all the tumor weighed in the control group and the experimental group. The immunohistochemical method was used to detect the changes of IKBKE, apoptosis, cycle, Hippo pathway and downstream factor, and then the U87-MG cells infected with luciferase virus were established with the aid of stereotactic instrument. The model of nude mice intracranial tumor, the experimental group was intragastric CYT387 (100mg/kg/day) every day, the size of intracranial tumor was monitored every 7 days by living fluorescent imager, the weight of nude mice and the survival of nude mice were monitored. The rat brain slices were determined by immunohistochemical staining. Results 1, the U87-MG and LN229 of glioma cell line were very sensitive to CYT387, and CYT387 After treatment, the proliferation and colony formation ability of glioma cells greatly reduced.2. CYT387 could obviously inhibit the migration and invasion of glioma cell lines U87-MG and LN229.3. CYT387 can promote the apoptosis of glioma cell lines U87-MG and LN229, and CYT387 promotes the U87-MG and LN229 cycle arrest of glioma cell lines. Only inhibition of IKBKE kinase activity can also reduce the expression of IKBKE protein, which is mainly translated after translation, and there is no obvious effect on.6 at transcriptional level. CYT387 inhibits the growth of malignant glioma cells by inhibiting the expression of IKBKE and the activity of Hippo to inhibit the growth of malignant glioma cells. IKBKE can affect the Hippo pathway directly with YAP1 and TEAD2, silent IK. BKE can inhibit the transposition of YAP1 and TEAD2 into the nucleus.8, CYT387 can inhibit the growth of nude mice, but the inhibitory effect on nude mouse intracranial tumor in situ is not obvious. Conclusion 1, CYT387 can obviously inhibit the growth of U87-MG and LN229 in the cell line of glioblastoma in vitro; promote its apoptosis; 2, CYT387 can activate the Hippo pathway. Inhibit the growth of tumor cells, and this effect is mainly by reducing the activity and expression of IKBKE kinase.3. IKBKE can interact with YAP1 and TEAD2 to promote YAP1 and TEAD2 transposing to the Hippo pathway to control the progression of glioma; 4, CYT387 can inhibit the growth of subcutaneous tumors but affect the effect of intracranial tumors. The fruit is not obvious. It may be related to poor blood brain barrier.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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