Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent
cancer. In this study, the effects of
isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human
hepatoma Hep G2 cells were studied. Under our experimental conditions,
isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by
isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of
cytochrome c, followed by activation of
caspase 3 and PARP cleavage, and finally cell death.
Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (
N-acetyl-L-cysteine) and an inhibitor of
NADPH oxidase (
diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of
isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to
oral administration of 350, or 700 mg/kg bw
isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the
body weight gain, the
body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of
NADPH oxidase plays an essential role in apoptosis induced by
isoobtusilactone A, and the dosages of
isoobtusilactone A tested in this study did not cause animal toxicity.