Department of Cadiovascular Sugery, First Affiliated Hospital of the Medical School, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Department of Urology, First Affiliated Hospital of the Medical School, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Oncol Rep. 2017 Nov;38(5):3137-3143. doi: 10.3892/or.2017.5988. Epub 2017 Sep 21.
Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637 cells in a time- and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzyme A carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compound C partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compound C had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.
从Stephania tetrandra 根部分离得到的双苄基异喹啉生物碱汉防己甲素是一种传统中药，在多种类型的癌症中都具有抗癌能力。先前的研究表明，汉防己甲素通过激活半胱天冬酶级联诱导膀胱癌细胞凋亡。然而，其潜在机制尚未报道。自噬是一种涉及降解破碎蛋白质和衰老细胞器以维持体内平衡的细胞过程。最近的研究表明，自噬与癌症治疗有关。因此，我们专注于汉防己甲素处理人膀胱癌细胞时自噬与凋亡之间的相关性。首先，我们的结果通过透射电子显微镜和共聚焦荧光显微镜观察到，汉防己甲素处理后自噬双膜空泡和红色荧光蛋白-绿色荧光蛋白-LC3（GRP-RFP-LC3）荧光斑点明显增加。其次，汉防己甲素处理 T24 和 5637 细胞时，LC3-II 的表达呈时间和浓度依赖性增加。随后，p62 的下调和 LC3 周转测定进一步证实，汉防己甲素诱导膀胱癌 T24 和 5637 细胞发生自噬流。第三，汉防己甲素处理的细胞中磷酸化-AMP 激活的蛋白激酶（AMPK）和磷酸化乙酰辅酶 A 羧化酶（ACC）的蛋白水平上调，而雷帕霉素（mTOR）相关蛋白水平下调。此外，AICAR，一种常见的 AMPK 激活剂，进一步增加了 LC3-II 的表达，而 AMPK 抑制剂化合物 C 部分逆转了膀胱癌 T24 细胞中 LC3-II 蛋白水平。最后，AICAR 显著增强了 T24 和 5637 细胞中汉防己甲素的生长抑制和凋亡诱导作用，而化合物 C 则产生相反的作用，表明 AMPK 介导的自噬增强了汉防己甲素对人膀胱癌细胞的细胞毒性和促凋亡作用。综上所述，本研究表明，汉防己甲素通过调节 AMPK/mTOR 信号通路诱导人膀胱癌细胞自噬，促进汉防己甲素诱导的细胞凋亡，提示汉防己甲素可能是治疗膀胱癌的潜在抗癌候选药物，自噬可能是癌症治疗的一种可能机制。
