Ethylacetate extract from Tetrastigma hemsleyanum induces apoptosis via the mitochondrial caspase-dependent intrinsic pathway in HepG₂ cells

Ethylacetate extract of Tetrastigma hemsleyanum (EET) has a potent antitumor activity in vitro and in vivo. However, the molecular mechanism underlying EET-induced apoptosis remains elusive. As part of our continuing studies, we investigated the apoptosis mechanism of HepG₂ cells exposed to different concentrations of EET in vitro. Confocal laser scanning was used to detect the apoptotic morphological changes. Flow cytometer and inverted fluorescence microscope were used to detect the mitochondrial membrane potential and cytosolic Ca²⁺ level. Western blotting analysis was used to evaluate the expression of the apoptosis-related proteins. Annexin V/PI staining was used to investigate cell apoptosis. Spectrophotometry was used to detect the activity of caspase family. The results showed that distinct apoptotic morphological changes occurred in HepG₂ cells treated by EET. EET caused collapse of mitochondrial membrane potential, elevation of cytosolic Ca²⁺ level, and evoked release of cytochrome c from mitochondria in a concentration-dependent manner. The apoptosis was accompanied by a significant activation of caspase-3, caspase-9, and the cleavage of poly (ADP-ribose) polymerase, but there was no significant change in either the activity or the expression level of caspase-8. Furthermore, EET-induced apoptosis could be inhibited by caspase-9 inhibitor Z-LEHD–FMK but not by caspase-8 inhibitor Z-IETD–FMK. Taken together, these overall results demonstrated that EET-induced apoptosis of HepG₂ cells was mediated by the mitochondrial caspase-dependent intrinsic pathway rather than the death receptor/caspase-8-mediated signaling route.