2-(3-methoxyphenyl)-6,7-methylenedioxoquinolin-4-one (MMEQ) is a novel synthesized compound, and this study investigated the effects of MMEQ on molecular signal pathways of the induction of apoptosis in TSGH8301 human bladder cancer cells. The studies included examining the effects of morphological changes by contrast-phase microscope, the percentage of viable cells, cell cycle distribution mitochondria membrane potential (ΔΨm), ROS and caspase activities were examined by flow cytometry, apoptotic cells were examined by DAPI staining and the changes of associated apoptosis proteins levels were examined by Western blotting. Release of apoptotic factors from mitochondria was examined by confocal laser microscope. Our results showed that MMEQ caused morphological changes and inhibited the cell growth of TSGH8301 cells in a time- and dose-dependent manner. MMEQ induced G2/M arrest through the promotion of chk1, chk2 and cdc25c in TSGH8301 cells. MMEQ caused a marked increase in the percentage of DNA damage and apoptosis as characterized by DAPI and DNA fragmentation. The specific inhibitors of caspase-8, -9, and -3 blocked MMEQ-induced growth inhibition action. A remarkable loss of ΔΨm and increase in ROS production were observed after a 24-h treatment. MMEQ promoted the levels of caspase-3, caspase-8, caspase-9, Bax, Bcl-xs, decreased the levels of Bcl-2 and Bid and then led to dysfunction of ΔΨm, following the releases of cytochrome c, AIF and Endo G from mitochondria to cytosol and nuclei, and finally caused cell apoptosis. In conclusions, these molecular mechanisms provide insight into MMEQ-caused growth inhibition, G2/M arrest and apoptotic cell death in TSGH8301 cells.

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