isoobtusilactone-a and Liver-Neoplasms

Hepatoma cells are relatively resistant to TRAIL. We have previously shown that isoobtusilactone A (IOA), a potent anticancer agent isolated from Cinnamomum kotoense, induced mitochondria-mediated apoptosis in hepatoma cells. Here, we report that IOA could potentiate TRAIL-induced apoptosis in Hep G2 cells. The combined treatment with IOA and TRAIL significantly induced caspase-dependent apoptosis. This correlated with the up-regulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5) protein levels. Gene silencing of the DR5 by small interfering RNA abrogated the apoptosis induced by the combined regimen of IOA and TRAIL, suggesting that the sensitization to TRAIL was mediated through DR5. By analyzing the DR5 promoter, we found that IOA induced a CHOP-dependent DR5 transactivation. DR5 expression after IOA treatment was accompanied by provoking intracellular reactive oxygen species (ROS) generation. Pretreatment with N-acetyl-L-cysteine (NAC) attenuated IOA-induced CHOP and DR5 expression and inhibited TRAIL-induced apoptosis. Taken together, our data suggested that ROS-dependent and CHOP-regulated DR5 expression played a pivotal role in the synergistic enhancement of TRAIL-induced apoptosis instigated by IOA in Hep G2 cells.

Topics: Alkanes; Apoptosis; Cinnamomum; Hep G2 Cells; Humans; Lactones; Liver Neoplasms; Plant Extracts; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Up-Regulation

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.

Topics: Administration, Oral; Alkanes; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cinnamomum; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Lactones; Liver Neoplasms; Membrane Potentials; Mitochondria, Liver; NADPH Oxidases; Plant Extracts; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species