| Trichothecin is a four-ring 12,13-epoxy skeleton of the toxic metabolite, which produced by Fungi Imperfecti and other toxin-producing fungi under certain conditions. It is very broad in nature, largely hazards to animals'and humans'healthy. Trichothecin have a wide range of biological effects to tissues and organs of the body; it is thought the mechanism of action to inhibit the protein and DNA synthesis. Recent studies have shown that trichothecin could induce tumor cells apoptosis.Mitochondria plays a central role in the regulation of apoptosis, many important events are closely related with it, including the release of caspase activators, the changes in electron transport chain, the loss of membrane potential, the reactive oxygen species changes, Bcl-2 family proteins participation in promoting and inhibition of apoptosis, and so on.The present study was to investigate the effects of trichothecin on cellular proliferation and apoptosis of Hep G2 cells. Cell viability of Hep G2 cells were evaluated by MTT. LDH activity of cell supermatant was determined to confirm whether necrosis or apoptosis was induced. The effect of trichothecin on apoptotic morphology was observed by DAPI fluorochrome staining. DNA ladder was observed to evaluate the role of trichothecin on DNA damage. Finally, the apoptotic rate of Hep G2 cells which treated with trichothecin was detected by flow cytometry with Annexin V-EGFP/PI double-staining. Cell apoptosis was verified by morphology, cytology and molecular biology of multiple means. In order to underly the mechanism of trichothecin induced apoptosis, the expression change of Bax/Bcl-2 and FAS/FASL was detected by RT-PCR, and then affirmed by Real-Time PCR. The change of the reactive oxygen species (ROS) level was determined by using fluorescent probe DCFH-DA and ELISA. Intracellular Ca2+ concentration was determined by using fluorescent probe Fura 2-AM, while mitochondria membrane potential was tested by flow cytometry with rhodamine 123 staining. Finally, the pro-caspase-9 and Caspase-3 relative activity were investigated by Western blot analysis and absorption spectroscopy assay.Detection with MTT method showed trichothecin inhibited Hep G2 cells proliferation in dose and time-dependent manner. LDH activity of Hep G2 cells treated with trichothecin was similar to that of control, which means necrosis was not appeared. Typically apoptotic morphological changes, including chromatin condensation, chromatin crescent formation and nucleus fragmentation could be observed by fluorescence microscope after trichothecin treated Hep G2 cells for 24 hours and 48 hours. The apoptotic ratio of 10μg/ml trichothecin treated 24 hours was markedly increased 37.97% compared with the control groups in dose and time-dependent manner. Meanwhile with the time prolonged, early apoptosis changed to late apoptosis gradually. Hep G2 cells treated with 5μg/mL trichothecin for 48 hours demonstrated obvious apoptotic DNA ladder by Agarose gel electrophoresis analysis. Experiments above have proved that trichothecin can induce apoptosis of Hep G2 cells. After treated with 1,5,10μg/ml trichothecin for 6 hours, the RNA of Hep G2 cells were purified, then RT-PCR showed that FAS and FASL have no significant changes; while Bax significantly increased to 1.18±0.03; 1.22±0.03; 1.69±0.05 of control, and Bcl-2 decreased significantly to 0.85±0.01; 0.67±0.06; 0.50±0.05 of control, which imply the activation of the mitochondrial apoptosis pathway. The level of ROS in trichothecin-treated Hep G2 cells was increased in a short time, When 3 h, ROS levels reached the maximum, compared with the control 1.38±0.21,1.45±0.13 and 1.75±0.19-fold respectively,; then decreased with the time prolonged. The concentration of intracellular Ca2+ in 1,5,10μg/ml trichothecin-treated Hep G2 cells after 12h increased to the control of 1.295±0.02,1.32±0.06,1.32±0.07 and 1.43±0.03 times, which increased continuously and quickly. The mitochondrial membrane potential of trichothecin-treated Hep G2 cells was decreased in a short time. Trichothecin induced proteolytic cleavage of pro-caspase-9 into the active form in treated Hep G2 cells by western blotting. The caspase-3 activity of Hep G2 cells treated by 1,5,10μg/ml trichothecin for 24 hours was increased 1.46±0.11,1.85±0.24 and 2.47±0.29-fold of control.We may conclude that trichothecin induced ROS production and Ca2+ concentration increase in the cytoplasm in Hep G2 cells, leading to the loss of MMP and release of cytochrome c from mitochondria to cytosol, then facilitated the cell apoptosis through the activation of caspase cascade, which lead to cell apoptosis.
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