To assess the involvement of the Akt pathway in the apoptosis induced by the co treatment with I3C and genistein, the level of phosphorylated Akt protein was investigated by western blotting. As shown in Fig. 3A and 3B, phosphorylated Akt started to decrease 6 h after the co treatment. Twelve hours after the co treat ment caspase 3 started to be activated, suggesting that dephosphorylation of Akt occurs before apoptosis. In addition, we further investigated the expression of phosphorylated caspase 9, a downstream target of Akt, and found that the co treatment significantly reduced the level of phospho caspase 9, resulting in activa tion of caspase 9. Since X chromosome linked inhibitor of apoptosis protein and survivin, inhibitor of apoptosis protein family members, have been recently reported to be activated by Akt, we further investigated the expression of the proteins. As shown in Fig. 3A, both XIAP and survivin expression was markedly downregulated by the combined treatment, consistent with the inhibition of Akt phosphorylation by the treat ment. Since mTOR is another downstream effector of Akt, we further investigated phosphorylated mTOR expression by western blotting.
