Gypenosides Induce Apoptosis in Human Hepatoma Huh-7 Cells through a Calcium/Reactive Oxygen Species-Dependent Mitochondrial Pathway

 

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Abstract

We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca²⁺) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca²⁺ increase with a Ca²⁺ chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca²⁺ concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.

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Shih-Lan Hsu, PhD

Department of Education and Research

Taichung Veterans General Hospital

No. 160, Section 3, Chung-Gang Road

40705 Taichung

Taiwan

Republic of China

Phone: +886-4-23592525 ext. 4037

Fax: +886-4-23592705

Email: h2326@mail.vghtc.gov.tw

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