Curcumin represses mTORC1 signaling in Caco-2 cells by a two-sided mechanism involving the loss of IRS-1 and activation of AMPK

The mechanistic target of rapamycin complex 1 (mTORC1) is a central modulator of inflammation and tumorigenesis in the gastrointestinal tract. Growth factors upregulate mTORC1 via the PI3K/AKT and/or Ras/MAPK signal pathways. Curcumin (CUR), a polyphenol found in turmeric roots (Curcuma longa) can repress mTORC1 kinase activity in colon cancer cell lines; however, key aspects of CUR mechanism of action remain to be elucidated including its primary cellular target. We investigated the molecular effects of physiologically attainable concentration of CUR (20 μM) in the intestinal lumen on mTORC1 signaling in Caco-2 cells. CUR markedly inhibited mTORC1 kinase activity as determined by the decreased phosphorylation of p70S6K (Thr389, −99%, P < 0.0001) and S6 (Ser235/236, −92%, P < 0.0001). Mechanistically, CUR decreased IRS-1 protein abundance (−80%, P < 0.0001) thereby downregulating AKT phosphorylation (Ser473, −94%, P < 0.0001) and in turn PRAS40 phosphorylation (Thr246, −99%, P < 0.0001) while total PRAS40 abundance was unchanged. The use of proteasome inhibitor MG132 showed that CUR-mediated loss of IRS-1 involved proteasomal degradation. CUR lowered Raptor protein abundance, which combined with PRAS40 hypophosphorylation, suggests CUR repressed mTORC1 activity by inducing compositional changes that hinder the complex assembly. In addition, CUR activated AMPK (Thr172 phosphorylation, P < 0.0001), a recognized repressor of mTORC1, and AMPK upstream regulator LKB1. Although cargo adapter protein p62 was decreased by CUR (−49%, P < 0.004), CUR did not significantly induce autophagy. Inhibition of AKT/mTORC1 signaling by CUR may have lifted the cross-inhibition onto MAPK signaling, which became induced; p-ERK1/2 (+670%, P < 0.0001), p-p38 (+1433%, P < 0.0001). By concomitantly targeting IRS-1 and AMPK, CUR's mechanism of mTORC1 inhibition is distinct from that of rapamycin.