Addition of exogenous PIP3 successfully rescued the inhibitory effects of particular PI3K inhibitor LY294002 on the downstream signaling, but, it had no influence on the curcumin induced inhibition. curcumin inhibited the phosphorylation of Akt, FoxO1, GSK3B, tuberin/TSC2, mTOR, p70 S6K, S6, 4e-bp1, eIF4G in an identical concentrationdependent manner. At the same time, curcumin induced the phosphorylation of AMPK and certainly one of its substrates, Acetyl-coa FK866 1198425-96-5 Carboxylase, suggesting that AMPK was activated. MAPKs, including ERK1/2, JNK, and p38MAPK, were also activated by curcumin treatment. But, the phosphorylation state of PDK1 and PKC remained unchanged. In the following studies we dedicated to the Akt/mTOR signaling axis. When PC 3 cells were treated with 40 uM of curcumin, the phosphorylation of Akt at Thr308 was instantly restricted within 5 min, followed closely by inhibition of phosphorylation of mTOR, Akt at Ser473, and then the other downstream targets including 4e-bp1, eIF4G, p70 S6K and S6. In most experiments the total Akt, mTOR, 4e-bp1, p70 S6K, and S6 were also blotted and showed no significant change. More over, the expression of cyclin D1 was also inhibited after 1 hr of curcumin therapy, similar as reported in. Curcumin acted at downstream Cellular differentiation of PI3K/PDK1 PI3K catalyzes the generation of PIP3, hence activates downstream signaling including Akt/ mTOR. The activity of PI3K is managed by the binding of regulatory subunits to catalytic subunits and a series of phosphorylation events. In our experiments the phosphorylated p85/p55 was barely noticeable and no change in its phosphorylation state upon curcumin therapy was seen. The phosphorylation of PDK1 at Ser241 about the activation loop, which can be required for PDK1 activity, was also not altered by curcumin treatment at the tested concentrations and time points. We further tested the result of PIP3 on curcumin mediated inhibition. We suspected that curcumin might directly inhibit PDK1 task towards Akt, because the phosphorylation of Akt at T308, which is Celecoxib catalyzed by PDK1, was the very first one to become inhibited. To try this hypothesis, the effect of curcumin on activity was evaluated using purified His tagged Akt1 as substrate. Pure active PDK1 minus the first 52 amino acids or endogenous PDK1 immuno precipitated from curcumin addressed PC 3 cells was employed for in vitro kinase assay. Nevertheless, curcumin did not prevent PDK1 activity both in vivo and in vitro. More over, the phosphorylation of PKC, which can be catalyzed by PDK1, was not significantly improved by curcumin treatment, indicating that PDK1 isn’t the immediate target of curcumin. Overexpression of Akt or constitutively activated Akt only partly repaired curcuminmediated inhibition To measure the function of Akt in curcumin mediated inhibition of mTOR signaling and cell proliferation, PC 3 cells were transiently transfected with plasmids encoding HA Akt, myr HA Akt or empty vector.