To analyze the mechanisms relevant to IMQ-induced anti-tumoral activity, we first observed the death patterns under light microscopy. As compared with the
RG108 under normal conditions, control, the IMQ treatment cells evidenced enhanced intracellular vesicular structures in the cytoplasm, typical of autophagic vesicles (Fig. 2A). In order to assess IMQ-induced autophagy, LC3 mobility was analyzed after IMQ treatment. Mobilization of LC3, LC3-II, was increased as the result of IMQ treatment and at that time IK-B degradation was detected, which means that IMQ activates NF-kB signaling. This suggests that the anti-tumoral activity we noted in Caco-2 cells as the result of IMQ treatment, was mediated by autophagy-induced cell death (Fig. 2B). In order to clarify the mechanisms relevant to the cell death mechanism of IMQ-induced anti-tumoral activity, we examined other molecules involved in apoptosis. Interestingly, the levels of puma and bcl-xl were uniformly downregulated, which is responsible for pro-apoptotic and anti-apoptotic changes of the bcl-2 family, respectively (Fig. 2B). It appears as if the nonspecific degradation of the bcl-2 family in Caco-2 cells occurred as the result of the activation of autophagy. Importantly, we were unable to detect cleaved PARP and caspase-3 activation in IMQ-treated Caco-2 cells. Collectively, we concluded that there were no signs of apoptotic cell death in IMQ-induced anti-tumoral activity. In order to confirm IMQ-induced autophagy is responsible for cell death in Caco-2 cells, we applied 3-methyl
adenine (3-MA) as an autophagy inhibitor and measured cell viability. The prevention of autophagic cell death was noted in Caco-2 cells treated with 3-MA (Fig. 2C). Therefore, we determined first that IMQ did not induce apoptosis, but rather autophagy and consequent autophagy-induced cell death in Caco-2 cells.
