Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells.
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, terminal deoxynucleotidyl transferase dUTP nick-end labeling 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 (DeltaPsim). Furthermore, treatment with the antioxidant N-acetyl-L-cysteine 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, Bax translocation, cytochrome c release, caspase-3 activity, and poly(ADP-ribose) polymerase degradation in Hep3B cells occurring in VCA-induced apoptosis. There is much evidence that implicates c-Jun NH2-terminal kinase (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 stress-activated protein kinase kinase (SEK1) seem to be more susceptible to cell death from ROS and loss of DeltaPsim 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. Together, these results suggest that VCA induces apoptosis by inducing ROS production and a loss of DeltaPsim, in which JNK phosphorylation plays a critical role in these events.
Kim
Won-Ho
WH
Division of Metabolic Disease, Department of Biomedical Science, National Institutes of Health, Seoul, South Korea.
Park
Won Bong
WB
Gao
Bin
B
Jung
Myeong Ho
MH
D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D010936:Plant Extracts / Q000494:pharmacology; D028321:Plant Preparations / Q000494:pharmacology*; D010940:Plant Proteins / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological / Q000494:pharmacology*
D010936:Plant Extracts; D028321:Plant Preparations; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C488862:mistletoe lectin I; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases