It is significant that curcumin effectively inhibited mTOR signaling in the noncancerous MEFs, although to a less degree than in PC 3 cells, indicating curcumin mediated inhibition of Akt/mTOR signaling is independent on PTEN status. the phosphorylation of Akt at Thr308 was the first to ever be inhibited. This led to the theory that curcumin can directly inhibit PDK1 mediated phosphorylation of Akt and led to the inhibition of downstream signaling. Phosphorylation Foretinib structure of PDK1 at Ser241 is important because of its activity, though might not be the main regulatory factor. But, curcumin didn’t inhibit the phosphorylation of PDK1 S241. Furthermore, curcumin failed to inhibit the kinase activity of PDK1 to Akt both in vitro and in vivo, suggesting that PDK1 is not the immediate target of curcumin. Similar observations have been noted that Akt/mTOR signaling may be inhibited independent of PI3K/PDK1. Next we examined the position of Akt in curcumin mediated inhibition. Overexpression of Akt or the constitutively activated myr Akt increased the basal level of phosphorylated Akt, mTOR and downstream molecules. Nevertheless, curcumin however effortlessly restricted mTOR and downstream signaling, though to a less degree that will be possibly due to the increased basal phosphorylation mesomerism level. These, particularly curcumin inhibited Akt downstream signaling despite the fact that the phosphorylation of myr Akt was not inhibited in any way, clearly suggest the existence of inhibitory mechanism that will be independent of inhibition of Akt. Coincidentally, AMPK was triggered by curcumin in a time course corresponding to the inhibition of Akt phosphorylation. Overexpression of AMPK in PC 3 cells somewhat potentiated the inhibition of mTOR signaling by curcumin, but neither medicinal inhibitor nor prominent bad overexpression showed important restoration of curcuminmediated inhibition. Although curcumin activated AMPK is not the major reason for curcumin mediated inhibition of Akt/mTOR signaling, how curcumin activates AMPK and its physiological importance deserve further investigation in the future. TSC1/TSC2 complex stops buy Daclatasvir mTOR activity by activating the GTPase activity of Rheb, and both Akt and AMPK converged at TSC1/TSC2 to regulate mTOR activity. Consistent with the incompetence of constitutive activation of Akt or inhibition of AMPK to rescue mTOR signaling, interruption of the event of TSC1/TSC2 complex only marginally saved curcumin mediated inhibition. Knockout of TSC1 in MEFs led to hyperphosphorylation of 4E BP1, mTOR, p70 S6K, and S6, nonetheless, curcumin efficiently inhibited the phosphorylation using a similar concentration dependency to that particular in wild-type MEFs. Moreover, knockdown of TSC2 in PC 3 cells by siRNA slightly increased the basal phosphorylation level of mTOR and 4e-bp1, however the phosphorylation could still be inhibited by curcumin.