Received for publication April 9, 2004.
Revised August 20, 2004.
Accepted for publication August 27, 2004.

Critical Role of Reactive Oxygen Species and Mitochondrial Membrane Potential in Korean Mistletoe Lectin-induced Apoptosis in Human Hepatocarcinoma Cells

Abstract

Viscum album L. coloratum agglutinin (VCA), isolated from Korean mistletoe, is a strong inducer of apoptosis in a variety of tumor cells; however, the underlying molecular mechanisms responsible are not clear. Here, we show that VCA induces apoptotic killing, as demonstrated by DNA fragmentation, Hoechst 33258 staining, TUNEL assay, and flow cytometry analysis in hepatocarcinoma Hep3B cells. VCA treatment results in a significant increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential (m). Furthermore, treatment with the antioxidant N-acetyl-L-cysteine (NAC) reduces ROS induction by VCA, preventing apoptosis in Hep3B cells, indicating that oxidative-stress is involved in VCA-mediated cell death. Our results also show rapid changes in mitochondrial transition permeability (MTP), Bax translocation, cytochrome c release, caspase-3 activity, and PARP degradation in Hep3B cells occurring in VCA-induced apoptosis. There is much evidence that implicates JNK activation with apoptosis in a variety of cellular and animal models. In this study, we show that VCA induces JNK phosphorylation, which is abolished with pretreatment with a JNK inhibitor. Moreover, Hep3B cells overexpressing JNK1 or SEK1 appear to be more susceptible to cell death from ROS and loss of m induced by VCA, whereas expression of dominant-negative JNK1 or SEK1 in Hep3B cells do not. These data suggest that JNK phosphorylation may be a major regulator involved in VCA-induced apoptosis. Collectively, these results suggest that VCA induces apoptosis by inducing ROS production and a loss of m, in which JNK phosphorylation plays a critical role in these events.

Key words: Jun Kinase, Apoptosis, Mitochondrial toxins, Oxidative stress/antioxidants, Reactive intermediates, Oxidative stress, Mechanisms of cell killing/apoptosis, Tumor suppressors

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