Cancer cells die by either necrosis or apoptosis. Our laboratory is interested in the understanding of apoptotic pathways and examining factors that upregulate apoptosis in cancer cells. Hypoxia is a hall marker in colon cancers. We thus initially examined hypoxia-induced apoptosis and molecular changes in the apoptotic cells. Three human and one mouse colon cancer cell lines were treated under hypoxic condition. These cells were harvested after various time points and the level of apoptosis was evaluated by JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 - tetraethylbenzimidazolocarbocyanine iodide) staining and Caspase Glo assay. JC-1 staining showed a higher percentage of human cells shifting to green colour (apoptosis) than that of mouse cells after 48 hours. Caspase assay also showed that Caspase 3 level fast increased and reached a peak after 6 hours in HCT116, whereas only a very slow and insignificant increase in Caspase 3 levelwas detected in HT29, SW480 and CT26. These results suggested that human colon cancer cells were more sensitive to hypoxia than mouse colon cancer cells.We therefore examined chemical-induced apoptosis in these cell lines. Cinobufagin, extracted from a classical Chinese medicine Chan-Su, was recently found to reduce cell growth in several cancer cell lines in vitro. Cytotoxicity study (MTT assays) in five human colon cancer cell lines showed that Cinobufagin strongly inhibited SW260 proliferation at the IC50 value as low as 0.04 卬 while the highest value was at 10 占for T84, indicating that the efficiency of Cinobufagin varies significantly from cell to cell. Work is underway to investigate the mechanism that caused such a diference, or whetehr a specific signalling transduction in colon cancer cells is involved. Taken together, our research has established methods to study hypoxic apoptosis in colon cancer cells and current results suggest that Cinobufagin could be an effective pro-apoptotic agent for the therapy of colon cancer.