The transfected cells were treated with different concentrations of curcumin, and then cell proliferation and the phosphorylated protein levels were examined by Western blotting and 3H thymidine incorporation assay. Over-expression of Akt somewhat renewed curcumin mediated inhibition of Akt phosphorylation, but showed less influence on the inhibition of the phosphorylation Tipifarnib clinical trial of 4E BP1, mTOR and S6. Overexpression of myr HA Akt, which is anchored at the cell membrane by the myr party and hence constitutively activated by PDK1, triggered highly phosphorylated Akt which could not be inhibited by curcumin, and augmented the basal phosphorylation of mTOR, 4E BP1, and S6, but interestingly, the phosphorylation of mTOR, 4E BP1 and S6 was still significantly inhibited by curcumin. Similarly, overexpression of HA Akt or myr HA Akt partially but considerably restored cyclin D1 stage and the proliferation of PC 3 cells treated with curcumin. These suggest Metastasis the inhibition of Akt phosphorylation partially led to curcuminmediated inhibition of mTOR signaling and cell proliferation, but is unlikely to become the primary mechanism targeted by curcumin. AMPK and MAPKs were triggered by curcumin although not responsible for curcumin mediated inhibition of Akt/mTOR signaling AMPK is just a negative upstream regulator of mTOR. Indeed, we discovered that curcumin induced a prompt and sturdy phosphorylation of AMPK at Thr172, which will be necessary for AMPK activation. Simultaneously, ACC, a substrate of AMPK, was also phosphorylated upon curcumin treatment. We firstly tested the effect of an AMPK inhibitor, compound C, to gauge the effort of AMPK in curcumin mediated inhibition of mTOR signaling. As shown in Fig. 4A, pretreating the cells with Compound buy Lapatinib D inhibited the phosphorylation of AMPK and ACC, but, it showed no influence on curcumin mediated inhibition of mTOR signaling. Then the Thr172 of AMPK1 was mutated to Ala to make a dominant negative form of AMPK, and the inhibition of cellular AMPK action by overexpression of the AMPK1/T172A in PC 3 cells was confirmed by inhibition of the phosphorylation of ACC. Overexpression of AMPK1 somewhat potentiated the inhibitory effect of curcumin on mTOR signaling, as indicated by phosphorylation of 4e-bp1, mTOR and S6. None the less, curcumin mediated inhibition remained unchanged. These indicate that activation of AMPK by curcumin is not the primary reason for curcumin mediated inhibition of mTOR signaling. Curcumin also triggered Erk1/2, JNK, and p38 in PC 3 cells. All over again, specific inhibitors from the activated MAPK paths had no impact on curcumin mediated inhibition of mTOR signaling. Interruption of TSC1/TSC2 complex only partially renewed curcumin mediated inhibition of mTOR signaling Both AMPK control mTOR and Akt signaling through TSC1 TSC2 complex. Here we tested the possible role of TSC1 TSC2 in curcumin mediated inhibition by utilizing TSC1 knockout MEFs or siRNA against TSC2/tuberin.