Zha Qing-Bing, Zhang Xiao-Yu, Lin Qiu-Ru, Xu Li-Hui, Zhao Gao-Xiang, Pan Hao, Zhou Dan, Ouyang Dong-Yun, Liu Ze-Huan, He Xian-Hui
College of Life Science and Technology, Jinan University, Guangzhou, China; Department of Fetal Medicine, the First Affiliated Hospital of Jinan University, Guangzhou, China.
College of Life Science and Technology, Jinan University, Guangzhou, China.
PLoS One. 2015 May 13;10(5):e0124355. doi: 10.1371/journal.pone.0124355. eCollection 2015.
Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.
葫芦素是一类具有多种生物活性的天然三萜类化合物，据报道可抑制mTORC1/p70S6K信号通路并诱导自噬。然而，这些活性之间的相关性在很大程度上尚不清楚。在本研究中，我们在人癌细胞中针对葫芦素E（CuE）处理解决了这个问题。我们的结果表明，CuE诱导了自噬，这在HeLa和MCF7细胞中通过LC3-II的形成以及点状LC3与溶酶体标志物LAMP2的共定位得以证明。然而，CuE在ATG5敲低的细胞中诱导的自噬水平要低得多，并且在缺乏功能性ATG5表达的DU145细胞中未能诱导自噬，这表明CuE诱导的自噬依赖于ATG5。与自噬诱导一致，CuE处理抑制了mTORC1活性（以p70S6K和ULK1S758磷酸化反映）。mTOR向溶酶体的募集减少进一步证实了mTORC1活性的抑制，溶酶体是mTORC1的激活位点。相反，CuE迅速激活AMPK，导致其底物磷酸化增加。AMPK激活促成了CuE诱导的mTORC1/p70S6K信号通路抑制和自噬诱导，因为AMPK敲低减弱了这些作用。总体而言，我们的数据表明，CuE在人癌细胞中诱导自噬至少部分是通过下调mTORC1信号通路和上调AMPK活性实现的。
