Calyculin A is a potent protein serine/threonine phosphatase inhibitor which inhibits both PP1 and PP2A, whereas okadaic acid potently inhibits PP2A but have much less effect on PP1, and tautomycin preferentially inhibits PP1 activity. Treatment method of PC-3 cells with calyculin A or okadaic acid induced a slight increase of basal phosphorylation level. Notably, pretreatment with calyculin A concentration-dependently reversed curcumin-mediated inhibition on the phosphorylation of Akt, mTOR, S6, and 4E-BP1, with 100 nM of calyculin A thoroughly blocked curcumin-mediated inhibition. Okadaic acid showed a very similar but very much weaker result when compared to calyculin A. To the other hand, tautomycin had no result on curcumin-mediated inhibition of Akt/mTOR signaling even at a concentration of one ?M .
The effects of calyculin A on curcumin-mediated inhibition of cyclin D1 and cell proliferation have been also established. As shown in Inhibitors 6B, calyculin A thoroughly reversed the inhibition of cyclin D1 expression by curcumin. 3H-leucine incorporation assay was put to use for proliferation assay considering the fact that MTS or 3H-TdR assays require longer treatment method but prolonged XL765 incubation with calyculin A result in cell detachment and death. Even though a hundred nM of calyculin A itself somewhat inhibited 3H-leucine incorporation, pretreatment with calyculin A remarkably reversed curcumin-mediated inhibition . The data propose that curcumin inhibits Akt/mTOR signaling by calyculin A-sensitive protein phosphatase , and restoration of Akt/mTOR phosphorylation by calyculin A reversed curcumin?s anti-proliferative effects.
PP2A core enzyme consists of catalytic C and regulatory A subunits, as well as C subunits is targeted to reversible methylation that regulates PP2A exercise . Having said that, incubation of PC-3 cells with curcumin modified neither the protein level nor the methylation state Fostamatinib of PP2A C subunit . Subsequent the cellular protein phosphatase action upon curcumin remedy was determined by Malachite Green Phosphatase assay. As proven in Inhibitors 6D, incubation of PC-3 cells with curcumin for ten min concentration-dependently increased the protein phosphatase exercise during the cell extract, and this curcumin-stimulated exercise could be inhibited by calyculin A. Taken together, these data indicate that incubation with curcumin activated PP2A and/or unspecified calyculin A-sensitive protein phosphatase , and led to dephosphorylation of Akt, mTOR, and their downstream substrates.
Discussion Curcumin has been shown to inhibit the phosphorylation and activation of Akt in PC-3 cells ; even so, the effects of curcumin to the downstream signaling of Akt haven’t been explored.