To determine if the inhibition of cell viability by snake venom toxin was due to the induction of apoptosis, we evaluated the changes in the chromatin morphology of cells by applying DAPI staining followed by TUNEL staining assays, and then a double order Avagacestat labeled cells were analyzed by fluorescence microscope. The cells were treated with different concentrations of snake venom toxin for 24 h. DAPI stained TUNELpositive cells were concentration dependently improved and greatest concentration of snake venom toxin caused nearly all of cells TUNEL positive, and the apoptosis rates were 51. 25 2. Six months in HCT116 cells and 50.. 43 1. 4% in HT 29 cells.. These demonstrated that snake venom toxin therapy clearly induced apoptosis in cancer of the colon cells. Effect of snake venom toxin Metastasis on the ROS generation in human colon cancer cells Several chemotherapeutic agents induce apoptosis by increase of ROS. . We investigated whether snake venom toxin also induced ROS in colon cancer cell lines, since we had unearthed that ROS is implicated in the snake venom toxin induced neuroblastoma cell death. Hence, we identified the part of ROS in mediating SVTinduced apoptosis of HT and HCT116 29 cells by measuring ROS levels after-treatment of varying levels of snake venom toxin for 30 min. As shown in Figure 2A, snake venom toxin improved ROS levels in a dose dependent fashion in both HCT116 and HT 29 cells. Effect of snake venom toxin on the expression of death receptors in human colon cancer cells Several studies demonstrated the ROS generation is involved in DR4 and DR5 up-regulation by treatment of chemotherapeutic agents including curcumin, baicalein and ursolic acid. We investigated the possible involvement of ROS within the expression of death receptors after treatment of snake venom toxin. We considered changes in expression of many death receptors and their ligands in HCT116 and HT 29 cancer of the colon cells using RT PCR. Consistent with the increase of apoptosis, Hedgehog antagonist the words of DR4 and DR5 was notably improved by treatment of snake venom toxin in a dose-dependent fashion in HCT116 and HT 29 cells. But expression of other death receptors such as TNF R1, TNFR2, DR3, DR6 and Fas and death receptor ligands such as FasL and TRAIL was not improved by treatment of snake venom toxin. The increased expression of DR4 and DR5 was also confirmed by western blotting. Taken together, these indicated that snake venom toxin induced apoptosis by up regulation of DR5 and DR4 in colon cancer cells. Effect of snake venom toxin on the expression of caspase and bax in human colon cancer cells To elucidate the relationship between apoptosis and the expression of apoptosis regulatory protein by snake venom toxin, expression of caspase Bax and cytochrome C was examined since these are DR relevant down signal cell death proteins. Cells were treated with snake venom toxin, and whole cell extract was afflicted by Western blotting.