新型HDACi N25抗肿瘤作用及其通过抑制HDAC3诱导胶质瘤细胞自噬研究

发布时间：2019-06-17 19:56

【摘要】：胶质瘤是一种常见且高致死率的原发性脑肿瘤,在小孩和成人中皆可发生。胶质瘤患者经过标准治疗后,中位生存期只有13-16个月,并且超过70%的胶质瘤患者在确诊后的2年内死亡。为了提高胶质瘤治疗的有效性及改善胶质瘤患者的存活状况,开发出新的安全有效的治疗药物显得尤为重要。N25(注:N25化学名:N-(2,5-二甲氧基苯基)-N`-羟基辛二酰胺,专利授权号:ZL201310087797.9),一种新型的组蛋白去乙酰化酶抑制剂,我们在SAHA的基础上进行构效修饰而获得。细胞自噬,又称Ⅱ型细胞死亡,是指细胞将自生胞浆蛋白或细胞器包裹形成囊泡,并与溶酶体融合形成自噬溶酶体进而降解自身成分的过程。细胞自噬在肿瘤中发挥着重要作用,其与肿瘤的关系十分复杂,目前尚未完全阐明。为了研究N25的抗肿瘤作用及其诱导胶质瘤细胞自噬的分子机制,我们进行了系列相关实验。首先,我们使用CCK-8法检测了N25的体外抗增殖活性,并通过建立裸鼠胶质瘤移植模型评价N25的体内抗肿瘤作用;然后,我们通过透射电子显微镜观察了N25是否诱导胶质瘤细胞自噬;此外,我们随机收集了20例临床胶质瘤样本及3例正常脑组织,通过western blot分析了临床胶质瘤样本中LC3,HDAC3,Tip60等蛋白的表达情况;最后,我们用western blot分析了N25对胶质瘤细胞中HDAC3,Tip60,ULK1(Atg1),Beclin-1(Atg6)等蛋白的影响,并检测了选择性HDAC3抑制剂RGFP966是否诱导胶质瘤细胞自噬。我们的研究结果显示,N25的体外抗增殖活性略强于SAHA,通过计算得出N25与SAHA的IC50分别为2.55μM和3.58μM,并且从绘制的生长抑制曲线可以发现,在相对较低浓度范围内,N25较SAHA能够更显著地抑制胶质瘤U87细胞的生长;在胶质瘤移植模型中也发现,N25具有显著的体内抗肿瘤活性且略强于SAHA,其中,N25高剂量组(96 mg/kg)对胶质瘤移植模型的抑瘤率约为68%。在透射电子显微镜的观察中,我们发现N25(5μM)可以明显诱导胶质瘤细胞自噬体的产生。在对临床胶质瘤样本的分析中,我们发现,与正常脑组织相比,HDAC3是明显上调的,而LC3与Tip60是明显下调的。当我们用N25处理胶质瘤细胞后,发现HDAC3明显受到抑制,而LC3与Tip60明显增加。此外,我们也发现N25明显诱导胶质瘤细胞中ULK1(Atg1),Beclin-1(Atg6)等自噬相关蛋白的表达,并且发现选择性HDAC3抑制剂RGFP966也可明显诱导胶质瘤细胞自噬。总之,这些数据表明N25具有显著的抗肿瘤活性,且部分原因可能与HDAC3的抑制有关;此外,N25可能通过一条新的通路诱导胶质瘤细胞自噬,即N25通过下调HDAC3,激活Tip60,进而刺激ULK1和Beclin-1而诱导胶质瘤细胞自噬。
[Abstract]:Glioma is a common and lethal primary brain tumor, which can occur in both children and adults. After standard treatment, the median survival time of glioma patients was only 13 脳 16 months, and more than 70% of glioma patients died within 2 years after diagnosis. In order to improve the efficacy of glioma treatment and improve the survival of glioma patients, it is particularly important to develop new safe and effective therapeutic drugs. N25 (N25 chemical name: N25 (2, 5-dimethoxyphenyl)-N`hydroxyoctanediamide, patent authorization number: ZL201310087797.9), a new histone deacetylase inhibitor, we obtained on the basis of SAHA structure-activity modification. Autophagy, also known as type II cell death, refers to the process in which cells encapsulate authigenic cytoplasmic proteins or organelle to form vesicles and fuse with lysosomes to form autophagy lysosomes and then degrade their own components. Autophagy plays an important role in tumor, and the relationship between autophagy and tumor is very complex, which has not been fully clarified at present. In order to study the antitumor effect of N25 and the molecular mechanism of inducing autophagy of glioma cells, we carried out a series of related experiments. First, we detected the anti-proliferation activity of N25 in vitro by CCK-8 assay, and evaluated the antitumor effect of N25 in vivo by establishing a nude mice glioma transplantation model. Then, we observed whether N25 induced autophagy of glioma cells by transmission electron microscope. In addition, 20 clinical glioma samples and 3 normal brain tissues were randomly collected, and the expression of LC3,HDAC3,Tip60 and other proteins in clinical glioma samples was analyzed by western blot. Finally, we analyzed the effect of N25 on HDAC3,Tip60,ULK1 (Atg1), Beclin-1 (Atg6) and other proteins in glioma cells by western blot, and detected whether RGFP966, a selective HDAC3 inhibitor, induced autophagy of glioma cells. Our results show that the anti-proliferation activity of N25 in vitro is slightly stronger than that of SAHA,. The IC50 of N25 and SAHA are 2.55 渭 M and 3.58 渭 M, respectively. From the drawn growth inhibition curve, it can be found that N25 can inhibit the growth of glioma U87 cells more significantly than SAHA in a relatively low concentration range. It was also found that N25 had significant antitumor activity in vivo and was slightly stronger than SAHA, in glioma transplantation model. The tumor inhibition rate of N25 high dose group (96 mg/kg) was about 68%. In the observation of transmission electron microscope, we found that N25 (5 渭 M) could obviously induce the production of autophagy in glioma cells. In the analysis of clinical glioma samples, we found that compared with normal brain tissue, HDAC3 was significantly up-regulated, while LC3 and Tip60 were significantly down-regulated. When we treated glioma cells with N25, it was found that HDAC3 was significantly inhibited, while LC3 and Tip60 were significantly increased. In addition, we also found that N25 significantly induced the expression of ULK1 (Atg1), Beclin-1 (Atg6) and other autophagy related proteins in glioma cells, and found that selective HDAC3 inhibitor RGFP966 could also induce autophagy in glioma cells. In conclusion, these data suggest that N25 has significant antitumor activity, and some of the reasons may be related to the inhibition of HDAC3. In addition, N25 may induce autophagy of glioma cells through a new pathway, that is, N25 can induce autophagy of glioma cells by down-regulating HDAC3, to activate Tip60, and stimulating ULK1 and Beclin-1.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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