The mechanisms of isoobtusilactone A-induced apoptosis in human hepatoblastoma cell line (Hep G2)

by Wu, Mei-jen

Chemoprevention using naturally occurring substances has now been considered a promising strategy for the prevention of 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 and the underlying mechanisms in isoobtusilactone A-induced apoptosis are thoroughly evaluated. Under the experimental conditions adapted by this study, isoobtusilactone A was found to exhibit a concentration-dependent growth impediment (IC50 = 37.5 £gM). The demise of the cells induced by isoobtusilactone A was apoptotic in nature, showing progressive sub-G1 fraction and DNA fragmentation when the concentration of the substrate was increased. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by the 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 ( m). The presence of a ROS scavenger (N-acetyl-L-cysteine,NAC) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride,DPI) blocked ROS production and the subsequent apoptotic cell death. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A. To clarify whether caspases were the sole mediators for eliciting the observed apoptotic process, the effects of a broad caspases inhibitor, Z-VAD.fmk, was studied. Interestingly, Z-VAD.fmk was found to completely inhibit the isoobtusilactone A-induced oligonucleosomal DNA fragmentation, yet it could only prevent limited amount of cells from becoming apoptosis-prone. These data implied that other mechanism(s) might also be important factors and led us to study the possible involvement of apoptosis-inducing factor (AIF), a mediator arbitrating caspase-independent apoptosis, in isoobtusilactone A-induced apoptotic process. Our data indicated that isoobtusilactone A could elicit the nuclear translocation of AIF observed along with the occurrence of large-scale DNA fragmentation. Reduction of AIF expression by AIF-siRNA transfection suppressed large-scale DNA fragmentation. Interestingly, inhibition of AIF expression by AIF-siRNA did not prevent isoobtusilactone A-induced oligonucleosomal DNA fragmentation. When the cells were simultaneously treated with AIF-siRNA and Z-VAD.fmk, both large-scale DNA and oligonucleosomal DNA fragmentations were almost completely prevented. In conclusion, our data suggest that isoobtusilactone A induced apoptotic cell death was caused by the increase of ROS, followed by the disruption of mitochondrial transmembrane potential ( m), further mediated by both caspase-dependent and caspase-independent pathways.