Addition of exogenous PIP3 successfully rescued the inhibitory effects of specific PI3K inhibitor LY294002 on the downstream signaling, nevertheless, it’d 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 a similar concentrationdependent manner. At the same time, curcumin induced the phosphorylation of AMPK and one of its substrates, Acetyl CoA purchase IPA-3 Carboxylase, suggesting that AMPK was triggered. MAPKs, including ERK1/2, JNK, and p38MAPK, were also triggered by curcumin treatment. However, 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 rapidly inhibited within 5 min, followed closely by inhibition of phosphorylation of mTOR, Akt at Ser473, and then your other downstream targets including 4e-bp1, eIF4G, p70 S6K and S6. In all experiments the S6, mTOR, 4E BP1, p70 S6K, and total Akt were also blotted and showed no significant change. Furthermore, the expression of cyclin D1 was also inhibited after 1 hr of curcumin treatment, similar as reported in. Curcumin served at downstream Ribonucleotide 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 a series of phosphorylation events and regulatory subunits to catalytic subunits. In our experiments the phosphorylated p85/p55 was barely detectable and no change in its phosphorylation state upon curcumin treatment was observed. The phosphorylation of PDK1 at Ser241 to the activation loop, which will be essential for PDK1 action, was also not altered by curcumin therapy at the tested concentrations and time points. We further tested the effect of PIP3 on curcumin mediated inhibition. We thought that curcumin may possibly directly inhibit PDK1 action towards Akt, because the phosphorylation of Akt at T308, which is Imatinib price catalyzed by PDK1, was the first one to become restricted. To try this hypothesis, the effect of curcumin on activity was analyzed using pure His branded Akt1 as substrate. Purified effective PDK1 with no first 52 proteins or endogenous PDK1 immuno precipitated from curcumin treated PC 3 cells was employed for in vitro kinase assay. Nevertheless, curcumin failed to inhibit PDK1 activity both in vivo and in vitro. Moreover, the phosphorylation of PKC, which is catalyzed by PDK1, was not significantly changed by curcumin treatment, indicating that PDK1 is not the direct target of curcumin. Over-expression of Akt or constitutively activated Akt only partially 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.