BET表观信号通路蛋白抑制剂JQ1对食管鳞癌细胞增殖抑制活性的研究

发布时间：2018-01-28 19:12

本文关键词： 食管鳞癌 JQ1 治疗小分子抑制剂 BET　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：我国食管癌发病率居世界首位,又是食管癌死亡率最高的国家；其中95%为鳞癌,与欧美国家70%左右为腺癌有明显差异。我国食管鳞癌在城市位于恶性肿瘤发病第4位,死亡第5位；在农村发病和死亡率均为第4位,是危害我国人民健康的主要恶性肿瘤之一。目前,食管鳞癌的治疗以手术和放疗为主,尚缺少有效的化疗药物,急需研究开发新的对食管鳞癌有效的治疗药物。染色质的重构是细胞表型调节的重要机制,在包括食管癌在内的恶性肿瘤的发生发展中发挥重要的作用。组蛋白乙酰化修饰是染色质重构的重要方式之一,组蛋白赖氨酸的乙酰化使染色质结构打开,呈转录激活状态。BET (Bromodomain and extra terminal domain)蛋白家族,是一类含有Bromodomain结构域的分子,是与乙酰化赖氨酸结合的活性区域。BET蛋白通过与乙酰化赖氨酸结合,招募其他转录因子,并组装成转录复合物,激活下游靶基因的转录。JQ1是一种新的BET小分子抑制剂,能够竞争性结合其活性结合区,使转录复合物从染色质区解离,从而抑制其下游分子的转录。研究已发现JQ1对血液系统肿瘤及胰腺癌、卵巢癌有明显的抑制活性。本论文研究了JQ1对食管鳞癌细胞的抑制活性,并初步探讨了其分子机制。我们用不同浓度的JQ1处理了不同的食管鳞癌细胞,在不同的时间点用CCK8检测了细胞的活性,绘制细胞增殖曲线。利用流式细胞分析了JQ1处理细胞的细胞周期分布。利用real time PCR和Western Blot检测了JQ1处理细胞后c-Myc和p21的表达水平。我们首先用20μM的JQ1处理了7株食管鳞癌细胞120h进行了敏感性初筛,发现JQ1能够显著抑制食管鳞癌细胞的增殖,对不同的细胞系表现出不同的抑制活性。随后分别用0.1μM、0.5μM、1μM、2μM、5μM、10μM口20μM的JQ1处理了食管鳞癌细胞系KYSE-30、KYSE-70、KYSE-140、KYSE-150、KYSE-410、KYSE-450和KYSE-510,分别在处理0h、24h、48h、72h、96h和120h检测了细胞活性,增殖曲线表明不同浓度的JQ1对不同的食管鳞癌细胞系具有不同的抑制活性,除了KYSE-140细胞对JQ1敏感性较差以外,其他6株食管鳞癌细胞系对JQ1都具有较高的敏感性。我们进一步分析了JQ1对食管鳞癌细胞增殖抑制活性的剂量效应关系,结果表明,JQ1对KYSE-30、KYSE-70、KYSE-150和KYSE-510的增殖抑制活性具有明显的剂量效应关系；JQ1对KYSE-410和KYSE-450的抑制活性的剂量效应关系并不明显,但JQ1在较低的0.1μM浓度时就有较高的抑制活性,72h抑制率就达到28.67%,对KYSE-450的抑制率达到29.07%。我们进一步分析了JQ1对不同食管鳞癌细胞系增殖抑制活性的IC50值,结果表明JQ1对食管鳞癌细胞增殖抑制的IC50值范围为0.19μM-9.95μM,其中KYSE-410和KYSE-450的IC50值最低,分别为0.92μM和0.19μM。进一步机制研究发现,JQ1处理食管鳞癌细胞后G1期细胞比例明显增高,说明JQ1通过引起食管鳞癌细胞G0/G1期阻滞来发挥其增殖抑制活性。下游蛋白表达分析表明,JQ1抑制了食管鳞癌细胞c-Myc的转录和表达,进而使p21的转录和表达增加,发挥其细胞周期阻滞和增殖抑制活性。综述所述,本论文的研究结果发现新的BET蛋白小分子抑制剂JQ1通过抑制c-Myc和p21的表达诱导了细胞周期G0/G1期阻滞,发挥了其对食管鳞癌细胞的增殖的抑制活性,并具有明显的剂量效应关系。BET蛋白小分子抑制剂JQ1是潜在的食管鳞癌新的治疗药物。
[Abstract]:The incidence of esophageal cancer in China ranks first in the world, and is the country with the highest mortality rate of esophageal cancer; 95% were squamous cell carcinoma, and 70% adenocarcinoma around Europe and the United States have obvious difference. Our city is located in the esophageal squamous cell carcinoma in the incidence of malignant tumor fourth, Fifth deaths in rural areas; the morbidity and mortality rate was fourth. China is one of the major malignant tumors harm people's health. At present, the treatment of esophageal cancer with surgery and radiotherapy, there is a lack of effective chemotherapy drugs, in urgent need of research and development of esophageal squamous cell carcinoma with new treatment. The reconstruction of chromatin is an important mechanism in the regulation of cell types, including esophageal cancer, malignant the occurrence and development of tumor play an important role. Histone acetylation is one of the important ways of chromatin remodeling and histone lysine acetylation to open chromatin structure, a transcription activation state of the.BET (Bro Modomain and extra terminal domain) is a protein family, proteins containing Bromodomain domain, is combined with acetylated lysine with acetyl lysine binding activity of.BET protein, recruit other transcription factors and assembled transcription complexes,.JQ1 activates transcription of downstream target genes is a kind of new small molecule inhibitors of BET, can compete with the activity of the transcription complex binding region from chromatin region dissociation, thereby inhibiting the transcription of the downstream molecules. Studies have found that JQ1 of blood cancer and pancreatic cancer, ovarian cancer has obvious inhibitory activity. This paper studied the inhibitory activity of JQ1 for esophageal squamous cell carcinoma, and to explore the molecular mechanism of esophageal squamous cell carcinoma. We treated with different concentrations of JQ1 were different, detection of cell viability by CCK8 at different time points, draw the cell growth curve using. Flow cytometric analysis of cell cycle distribution of the cells treated with JQ1. To detect the expression of JQ1 cells treated with c-Myc and p21 by real time PCR and Western Blot. We first used 20 M JQ1 with 7 strains of esophageal squamous cell carcinoma cell line 120h were sensitive screening, found that JQ1 could significantly inhibit the esophageal squamous cell carcinoma the proliferation of different cell lines showed different inhibition activity. Followed by 0.1 M, 0.5 M, 1 M, 2 M, 5 M, 10 M and 20 M JQ1 treatment KYSE-30, esophageal cancer cell line KYSE-70 KYSE-140, KYSE-150, KYSE-410, and KYSE-450 KYSE-510, 24h, 0h respectively in treatment, 48h, 72h, 96h and 120h to detect cell activity and proliferation curve showed that different concentrations of JQ1 could inhibit the activity of esophageal squamous cell carcinoma cell lines of different, in addition to KYSE-140 cells except for the poor sensitivity of JQ1, other 6 strains of esophageal squamous cell carcinoma cell lines have JQ1 High sensitivity. We further analyze the dose effect relationship, inhibitory activity of JQ1 on the proliferation of esophageal squamous cell carcinoma. The results show that the JQ1 of KYSE-30, KYSE-70, KYSE-150 and KYSE-510 inhibited proliferation activity had obvious dose effect relationship; dose effect relationship of KYSE-410 inhibited the activity of JQ1 and KYSE-450 is not obvious, but at lower JQ1 the 0.1 M concentration had higher inhibitory activity, the inhibition rate of 72h reached 28.67%, the inhibition rate of KYSE-450 reached 29.07%., we analyzed the JQ1 in different esophageal squamous cell carcinoma cell line proliferation inhibitory activity of IC50 values, the results show that inhibition of JQ1 on proliferation of esophageal squamous cell carcinoma of the IC50 range of 0.19 mu M-9.95 mu M among them, KYSE-410 and KYSE-450 of the lowest IC50 value were 0.92 M and 0.19 M. further study mechanism showed that JQ1 treatment of esophageal squamous cell carcinoma after G1 phase cells were increased, indicating JQ1 caused by esophageal squamous cell carcinoma G0/G1 cell cycle arrest to play its anti proliferative activity. Analysis shows that the downstream protein expression, inhibit JQ1 transcription and expression in esophageal squamous cell carcinoma cell line c-Myc, and the transcription and expression of p21 increased, exert its inhibitory activity on cell cycle arrest and proliferation. Review the research results of this paper found that BET the new protein small molecule inhibitors of JQ1 induced G0/G1 cell cycle arrest by inhibiting expression of c-Myc and p21, play the inhibitory activity of esophageal squamous cell carcinoma cell proliferation, and has obvious dose effect relationship between.BET protein small molecule inhibitors of JQ1 therapeutics is a new potential of esophageal squamous cell carcinoma.

【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】：2015
【分类号】：R735.1

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