吡唑并[4,3-d]嘧啶衍生物的抗肿瘤筛选及其机制研究

Study on antitumor screening and mechanism of pyrazolo[4,3-d]pyrimidine derivatives

目的对一系列吡唑并[4,3-d]嘧啶衍生物进行抗肿瘤活性评价,选取显著抑制神经胶质瘤U87-MG细胞增殖的化合物9,研究其体外对U87-MG细胞的抗肿瘤活性及机制。方法采用MTT比色法观察化合物1-10对四种肿瘤细胞(人肝癌SMMC-7721细胞、人胃癌SGC-7901细胞、人胃癌MGC-803细胞和人神经胶质瘤U87-MG细胞)增殖的影响。采用流式细胞仪检测化合物9诱导的U87-MG细胞凋亡率,以及Western blot法检测细胞凋亡相关蛋白Bcl-2和Bax的表达和相关通路蛋白p-Akt、Akt、p-mTOR和mTOR的表达情况。结果 MTT结果显示,化合物4、5、8和9对四种肿瘤细胞株的增殖均具有一定抑制作用,其中,化合物9抑制神经胶质瘤U87-MG细胞增殖的能力最强;化合物9体外抑制U87-MG细胞增殖的作用具有时间-剂量依赖性;流式细胞仪检测结果显示,U87-MG细胞凋亡率随着化合物9浓度增加而升高,呈剂量依赖性。Western blot结果显示,随着化合物9浓度增加,Bcl-2蛋白表达下调,Bax蛋白表达上调;同时,Akt和mTOR蛋白表达量基本不变,p-Akt和p-mTOR蛋白表达均下调。结论吡唑并[4,3-d]嘧啶衍生物9明显抑制神经胶质瘤U87-MG细胞增殖,并且诱导细胞凋亡,其诱导凋亡可能机制为通过抑制PI3K/Akt/mTOR通路达到的。

Objective To evaluate the in vitro anti-proliferative activity of a series of pyrazolo[4,3-d]pyrimidine derivatives,and to study the preliminary antitumor mechanism of compound 9,which exhibits the strongest activities among synthesized compounds in glioma U87-MG cells.Methods MTT assay was used to observe the effect of compound 1-10 on the proliferation of four tumor cells(human liver cancer SMMC-7721 cells,human gastric cancer SGC-7901 cells,human gastric cancer MGC-803 cells and human glioma U87-MG cells).Annexin-V/PI double staining was used to detect the cell apoptosis induced by compound 9,and the expressions of apoptosis-related proteins Bcl-2 and Bax and the related pathway proteins p-Akt,Akt,p-mTOR and mTOR were detected by Western blot.Results MTT results showed that compounds 4,5,8 and 9 had inhibitory effects on the proliferation of four tumor cell lines.Among them,compound 9 had the strongest ability to inhibit the proliferation of glioma U87-MG cells in a time-and dose-dependent manner;Flow cytometry results showed that the apoptosis rate of U87-MG cells increased with the increase of compound 9 concentration,showing a dose-dependent manner.Western blot results showed that as the concentration of compound 9 increased,the expression of Bcl-2 protein was down-regulated and the expression of Bax protein was up-regulated.Mearwhile,the expression of Akt and mTOR protein remained basically unchanged,and the expression of p-Akt and p-mTOR protein was down-regulated.Conclusion Pyrazolo[4,3-d]pyrimidine derivative 9 significantly inhibits the proliferation of glioma U87-MG cells and inducs apoptosis,and its possible mechanism of inducing apoptosis is achieved through inhibition of PI3 K/Akt/mTOR pathway.