A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

S J Lee,A D Kim,Y H Choi,K A Kang,D H Kim,H S Kim,J W Hyun

doi:10.1038/cddis.2013.273

Abstract

Compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells.

Highlights

Reactive oxygen species (ROS) have an important role in cell death, in cancer cells
HCT-116 colon cancer cells were treated for 24 h with various concentrations of compound K to investigate the cytotoxic activity of compound K against these cells
Transfection of cells with green fluorescent protein (GFP)-light chain 3 (LC3) showed that the fraction of cells with a punctate GFP-LC3 localization was increased following treatment with compound K (20 mg/ml; Figure 2c)

Summary

Introduction

Reactive oxygen species (ROS) have an important role in cell death, in cancer cells. Compound K possesses chemopreventive activity against chemical carcinogens, impairs metastasis in vivo, and constrains tumor growth through the inhibition of 12-O-tetradecanoylphorbol13-acetate-induced cyclooxygenase-2 expression.[10,11,12,13] We recently reported that compound K exhibits cytotoxicity against tumor cells by the induction of apoptosis and cell cycle arrest at G1 phase These actions occur by a caspasedependent pathway via mitochondrial disruption and inhibition of telomerase activity.[14,15] combination treatment with compound K and radiation enhances human lung cancer cell death[16] and compound K induces apoptosis in MCF-7 human breast cancer cells by the generation of ROS and modulation of AMP-activated protein kinase signaling.[17] compound K-mediated generation of ROS leads to apoptosis in HT-29 human colon cancer cells through the modulation of a mitochondriadependent apoptotic pathway and the mitogen-activated protein kinase pathway.[18]

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