ZT-25, a New Vacuolar H(+)-ATPase Inhibitor, Induces Apoptosis and Protective Autophagy Through ROS Generation in HepG2 Cells

Eur J Pharmacol. 2016 Jan 15;771:130-8. doi: 10.1016/j.ejphar.2015.12.026. Epub 2015 Dec 12.

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.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Apoptosis / drug effects*
  • Autophagy / drug effects*
  • Benzodioxoles / pharmacology*
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Enzyme Inhibitors / pharmacology*
  • Glycosides / pharmacology*
  • Hep G2 Cells
  • Humans
  • Matrix Metalloproteinases / metabolism
  • Membrane Potential, Mitochondrial / drug effects
  • Mitosis / drug effects
  • Reactive Oxygen Species / metabolism*
  • Vacuolar Proton-Translocating ATPases / antagonists & inhibitors*

Substances

  • Benzodioxoles
  • Enzyme Inhibitors
  • Glycosides
  • Reactive Oxygen Species
  • ZT-25 compound
  • Adenosine Triphosphate
  • Matrix Metalloproteinases
  • Vacuolar Proton-Translocating ATPases