We performed a colony formation assay to research the result of the combined treatment with OBP 801/YM753 and LY294002.On another hand, LY294002 at 6. 3 uM or more inhibited cell growth with a decrease of phosphorylated Akt. LY294002 at 1-2. 5 uM did not somewhat decrease the number of colony E3 ubiquitin ligase inhibitor formation, while OBP 801/YM753 at 4 nM reduced it by 60-inch. Interestingly, LY294002 increased the inhibitoryeffect of OBP 801/YM753 o-n colony formation. Under the conditions above, an increase of decrease and acetylated histone H4 of phosphorylated Akt were seen. We investigated the aftereffect of LY294002 and OBP 801/YM753 on the cell cycle progression of HEC 1A cells by flow cytometric analysis. G2/M phase arrest was caused by obp 801/YM753, whereas LY294002 caused G1 arrest for 24?72 h. On-the other hand, the combined therapy for 48 and 72 h markedly induced apoptosis. More over, the combination index valueswere b1. 0, suggesting synergistic apoptosis inducing effectiveness. SAHA may be the most clinically used HDAC inhibitor. To compare SAHA and OBP801/YM753 in conjunction with LY294002, we reviewed sub G1 by flow cytometry. As shown in Fig. 3D, OBP 801/YM753 or SAHA alone almost equally induced Endosymbiotic theory apoptosis, but denver treatmentwith OBP LY294002 and 801/ YM753 more effectively induced apoptosis than that with LY294002 and SAHA in HEC 1A cells. These results indicate that OBP 801/ YM753 is a lot more potent than SAHA in conjunction with LY294002 in HEC 1A cells. To investigatewhether the apoptosis is caspase dependent,we examined the effect of a caspase inhibitor. As shown in Fig. 3E, the apoptosis induced by the combination was very nearly com-pletely inhibited by the overall caspase inhibitor zVAD fmk. More over, the combination obviously improved the cleavage of caspases and increased the expression of Bim. These results suggest that the combined treatment with LY294002 and OBP 801/YM753 induces caspase dependent apoptosis via an intrinsic process such as the regulation of Bim. To research whether ROS are related to the apoptosis induced by the combined therapy with OBP 801/YM753 and LY294002, we examined the aftereffect of the accumulation of intracellular ROS in the cells selective c-Met inhibitor subjected to OBP 801/YM753 and/or LY294002 utilising the ROS signal CM H2DCFDA. The mixture notably increased the accumulation of intracellular ROS, that was blocked by N acetylcysteine. More over, the apoptosis induced by the combinationwas very nearly com-pletely inhibited by NAC. At the molecular level, NAC inhibited the activation of caspases and induction of Bim from the mixture. These results suggest that the apoptosis induced by the mixture is mediated by the regulation of Bim through the accumulation of the intracellular ROS.