The Second Hospital of Lanzhou University, Lanzhou, Gansu 730030, P.R. China.
Int J Oncol. 2013 Apr;42(4):1417-26. doi: 10.3892/ijo.2013.1817. Epub 2013 Feb 8.
Matrine has potent antitumor activity against a broad variety of cancer cells and our previous study showed that both autophagy and apoptosis were activated during matrine-induced gastric cancer cell death. The aim of the present study was to determine the significance of autophagy in antineoplastic effects of matrine and the molecular mechanism by which matrine induces autophagy in gastric cancer cells. Western blot analysis showed that exposure of gastric cancer cells to matrine resulted in the extent of autophagy increasing in a dose- and time-dependent manner by detecting micro-tubule-associated protein 1 light chain 3 (LC3). This induction was due to activation of autophagic flux, as supported using the lysosome inhibitor, bafilomycin A1, which produced an accumulation of LC3-II. Propidium iodide staining demonstrated that matrine induced cell death in a dose-dependent manner and the autophagy inhibitor 3-methyladenine (3-MA) or bafilomycin A1 enhanced lethality of matrine against gastric cancer cells. Moreover, after pretreatment with 3-MA, some of the gastric cancer cells treated with matrine exhibited prototypical characteristics of apoptosis by transmission electron microscopy. The ability of 3-MA to increase matrine-induced apoptosis was further confirmed by Annexin V-FITC/PI staining. Also, the combination of matrine and 3-MA was more potent than matrine alone in inhibiting the proliferation of SGC-7901 cells assessed by sulphorhodamine B assay. Furthermore, administration of the pan-caspase inhibitor zVAD-fmk or autophagy inducer rapamycin decreased the matrine-induced cell death. In addition, matrine treatment did not inhibit the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin (mTOR) as well as p70 ribosomal protein S6 kinase (p70S6K), although the levels of the total Akt and mTOR were decreased. These results suggest that autophagy was activated as a protective mechanism against matrine-induced apoptosis and inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of matrine in gastric cancer.
苦参碱对多种癌细胞具有强大的抗肿瘤活性，我们之前的研究表明，苦参碱诱导胃癌细胞死亡过程中同时激活了自噬和细胞凋亡。本研究旨在确定自噬在苦参碱抗肿瘤作用中的意义，以及苦参碱诱导胃癌细胞自噬的分子机制。Western blot 分析显示，苦参碱处理胃癌细胞后，微管相关蛋白 1 轻链 3（LC3）的量和时间依赖性增加，表明自噬程度增加。这种诱导是由于自噬流的激活，因为使用溶酶体抑制剂巴弗洛霉素 A1 后，LC3-II 积累。碘化丙啶染色表明，苦参碱呈剂量依赖性诱导细胞死亡，自噬抑制剂 3-甲基腺嘌呤（3-MA）或巴弗洛霉素 A1 增强苦参碱对胃癌细胞的致死作用。此外，在用 3-MA 预处理后，一些用苦参碱处理的胃癌细胞通过透射电子显微镜表现出典型的凋亡特征。用 Annexin V-FITC/PI 染色进一步证实了 3-MA 增加苦参碱诱导的凋亡的能力。同样，苦参碱与 3-MA 联合使用比单独使用苦参碱更能抑制 SGC-7901 细胞的增殖，这通过磺酰罗丹明 B 测定法评估。此外，使用泛半胱天冬酶抑制剂 zVAD-fmk 或自噬诱导剂雷帕霉素处理可降低苦参碱诱导的细胞死亡。此外，苦参碱处理不会抑制 Akt 及其下游效应子哺乳动物雷帕霉素靶蛋白（mTOR）以及 p70 核糖体蛋白 S6 激酶（p70S6K）的磷酸化，尽管 Akt 和 mTOR 的总水平降低。这些结果表明，自噬被激活作为一种对抗苦参碱诱导的细胞凋亡的保护机制，抑制自噬可能是增强苦参碱在胃癌中抗肿瘤潜力的一种有吸引力的策略。
