Gypenosides Induce Apoptosis by Ca 2+ Overload Mediated by Endoplasmic-Reticulum and Store-Operated Ca 2+ Channels in Human Hepatoma Cells

Abstract

Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca²⁺-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca²⁺]_i level. Gyp-increased [Ca²⁺]_i level was partly inhibited by removal of extracellular Ca²⁺ by Ca²⁺ chelator EGTA, store-operated Ca²⁺ channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca²⁺-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca²⁺ response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca²⁺-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca²⁺]_i increase and apoptosis in HepG2 cells. Exposure to 300 μg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca²⁺]_i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca²⁺ release from ER stores and increased store-operated Ca²⁺ entry.