title: Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress.
abstract: Malignant gliomas are the most common primary brain tumors, and novel ways of treating gliomas are urgently needed. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit promising antitumor activity. Here, we evaluated the effects of UA on U87MG cells and explored the underlying molecular mechanisms. The results demonstrated that both G1-phase arrest and autophagy were induced by UA in U87MG cells. Evidence of UA-induced autophagy included the formation of acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. UA was also found to induce ER stress and an increase in intracellular calcium accompanied by ROS production. The increase in free cytosolic calcium induced by UA activated the CaMKK-AMPK-mTOR kinase signaling cascade, which ultimately triggered autophagy. Western blot analysis showed that UA promoted the phosphorylation of PERK and eIF2α; this was followed by the upregulation of the downstream protein CHOP, implying the involvement of the ER stress-mediated PERK/eIF2α/CHOP pathway in glioma cells. Meanwhile, UA activated IRE1α and subsequently increased the levels of phosphorylated JNK and Bcl-2, resulting in the dissociation of Beclin1 from Bcl-2. Furthermore, TUDCA and the silencing of either PERK or IRE1α partially blocked the UA-induced accumulation of LC3-II, suggesting that ER stress precedes the process of autophagy. Additionally, NAC attenuated the UA-induced elevation in cytosolic calcium, ER stress markers and autophagy-related proteins, indicating that UA triggered ER stress and autophagy via a ROS-dependent pathway. Collectively, our findings revealed a novel cellular mechanism triggered by UA and provide a molecular basis for developing UA into a drug candidate.
authors.affiliation: The Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China. Electronic address: shuyingshen19890409@gmail.com.
authors.forename: Shuying
authors.initials: S
authors.lastname: Shen
authors.affiliation: The Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China. Electronic address: zhangyiqiaoqiao@126.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210029, China. Electronic address: zhangrui@njmu.edu.cn.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: The Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xintao
authors.initials: X
authors.lastname: Tu
authors.affiliation: The Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China. Electronic address: gongxg@zju.edu.cn.
authors.forename: Xingguo
authors.initials: X
authors.lastname: Gong
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D015394:Molecular Structure; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014315:Triterpenes / Q000494:pharmacology*; D000097245:Ursolic Acid
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D017382:Reactive Oxygen Species; D014315:Triterpenes; D000097245:Ursolic Acid
keywords: Autophagy; Calcium; ER stress; Ursolic acid