title: ZT-25, a new vacuolar H(+)-ATPase inhibitor, induces apoptosis and protective autophagy through ROS generation in HepG2 cells.
abstract: The vacuolar H(+)-ATPase (V-ATPase) has recently been proposed as a key target for new strategies in cancer treatment. Our previous work has proved that diphyllin glycoside is a novel inhibitor of V-ATPase. Here the cytotoxic effects of ZT-25, the most potent diphyllin glycoside derivatives, were studied and some of the underlying mechanisms were elucidated. ZT-25 displayed strong cytotoxicity on several cancer cell lines and relatively low cytotoxicity on human fetal hepatic cells (WRL-68) at submicromolar concentrations. In human hepatoma cells HepG2, ZT-25 induced G1/G0 phase arrest and apoptosis, as well as mitochondrial membrane potential (MMP) dissipation and ATP depletion. Furthermore, Bcl-2 protein decreased, while Bax protein and cleaved caspase-3 protein increased upon ZT-25 treatment. Benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK), a well-known pan-caspase inhibitor, attenuated ZT-25-induced cell death, suggesting the involvement of caspase-dependent pathway. Intriguingly, ZT-25 induced autophagy in HepG2 cells as characterized by increased the conversion of LC3 I to LC3 II, Beclin-1 expression and autophagosome formation. Meanwhile, p-mTOR expression was decreased which indicated that ZT-25-induced autophagy might be mediated through the suppression of mTOR pathway. Inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) obviously promoted ZT-25-induced cell death, suggesting the protective role of autophagy. Increased intracellular ROS level was found to be the early event in ZT-25-treated HepG2 cells. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) attenuated ZT-25-induced cell death and autophagy. Together, these results provide key insights into the ZT-25-induced cytotoxicity in HepG2 cells, which will have a great impact on the further development of diphyllin derivatives.
authors.affiliation: School of Medicine, Jiangsu University, Zhenjiang 212013, China; Institute of Nautical Medicine, Nantong University, Nantong 226019, China.
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Neurology, Deji Hospital, Shanghai 200331, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Institute of Nautical Medicine, Nantong University, Nantong 226019, China.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Liu
authors.affiliation: Institute of Nautical Medicine, Nantong University, Nantong 226019, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. Electronic address: jinggao@ujs.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: Institute of Nautical Medicine, Nantong University, Nantong 226019, China. Electronic address: zhuli85051796@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D052117:Benzodioxoles / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008938:Mitosis / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
chemical_list: D052117:Benzodioxoles; D004791:Enzyme Inhibitors; D006027:Glycosides; D017382:Reactive Oxygen Species; C000611678:ZT-25 compound; D000255:Adenosine Triphosphate; D020782:Matrix Metalloproteinases; D025262:Vacuolar Proton-Translocating ATPases
keywords: 3-methyladenine (PubChemCID: 1673); Apoptosis; Autophagy; Chloroquine (PubChemCID: 2719); Dichlorodihydrofluorescein diacetate (PubChemCID: 104913); Diphyllin (PubChemCID: 100492); Diphyllin glycoside derivatives; JC-1 (PubChemCID: 5492929); MTT (PubChemCID: 64965); N-acetyl-l-cysteine (PubChemCID: 12035); Propidium iodide (PubChemCID: 104981); Rapamycin (PubChemCID: 5284616); Reactive oxygen species; Vacuolar H(+)-ATPase; benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (PubChemCID: 5497174)