ZT-25, a new vacuolar H+-ATPase inhibitor, induces apoptosis and protective autophagy through ROS generation in HepG2 cells

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.