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. 2009 Feb 1;69(3):1000-8.
doi: 10.1158/0008-5472.CAN-08-2367. Epub 2009 Jan 27.

Curcumin disrupts the Mammalian target of rapamycin-raptor complex

Christopher S Beevers  1 , Long Chen, Lei Liu, Yan Luo, Nicholas J G Webster, Shile Huang
Affiliations

Curcumin disrupts the Mammalian target of rapamycin-raptor complex

Christopher S Beevers et al. Cancer Res. .

Abstract

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Recently, we have shown that curcumin inhibits phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), two downstream effector molecules of the mammalian target of rapamycin complex 1 (mTORC1) in numerous cancer cell lines. This study was designed to elucidate the underlying mechanism. We observed that curcumin inhibited mTORC1 signaling not by inhibition of the upstream kinases, such as insulin-like growth factor 1 receptor (IGF-IR) and phosphoinositide-dependent kinase 1 (PDK1). Further, we found that curcumin inhibited mTORC1 signaling independently of protein phosphatase 2A (PP2A) or AMP-activated protein kinase AMPK-tuberous sclerosis complex (TSC). This is evidenced by the findings that curcumin was able to inhibit phosphorylation of S6K1 and 4E-BP1 in the cells pretreated with PP2A inhibitor (okadaic acid) or AMPK inhibitor (compound C), or in the cells expressing dominant-negative (dn) PP2A, shRNA to PP2A-A subunit, or dn-AMPKalpha. Curcumin did not alter the TSC1/2 interaction. Knockout of TSC2 did not affect curcumin inhibition of mTOR signaling. Finally, we identified that curcumin was able to dissociate raptor from mTOR, leading to inhibition of mTORC1 activity. Therefore, our data indicate that curcumin may represent a new class of mTOR inhibitor.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Curcumin does not affect phosphorylation of IGF-IR and PDK1. A, Rh1 cells were pretreated with or without curcumin (20 μmol/L) for 2 h and then stimulated with or without IGF-I (10 ng/mL) for 1 h, followed by immunoprecipitation with antibodies to the IGFR β-subunit (IGFRβ) plus protein A/G-agarose, and immunoblotting (IB) with antibodies to phosphotyrosine (p-Tyr) and IGFRβ, respectively. HC, heavy chain of IgG; LC, light chain of IgG. B, Rh1 cells were pretreated with curcumin (0–40 μmol/L) for 12 h and then stimulated with IGF-I (10 ng/mL) for 1 h, followed by Western blot analysis with antibodies against p-PDK1 (Ser241), PDK1, and β-tubulin (loading control), respectively. Top, representative blots; bottom, semiquantitated values of three independent experiments, performed by densitometry using NIH Image J. Statistical analysis was performed by Student’s t test. P < 0.05 was considered to be significant.
Figure 2
Figure 2
Curcumin inhibits mTORC1 signaling in a PP2A-independent manner. A, serum-starved (24 h) Rh1 cells, grown in six-well plates, were treated with 100 nmol/L okadaic acid (OA) for 1 h, followed by treatment with various concentrations of curcumin for 2 h and stimulation with or without IGF-I (10 ng/mL) for 1 h. Whole-cell lysates were subjected to Western blot analysis using the indicated antibodies. B, top, Rh1 cells were stably transfected with vector alone (Rh1/pcDNA) or with a vector expressing HA-tagged dn-PP2Ac (Leu199→Pro), followed by Western blotting using the indicated antibodies. Bottom, Rh1/pcDNA and Rh1/dn-PP2A cell lines were stimulated with or without IGF-I (10 ng/mL) for 10 min, followed by Western blotting using the indicated antibodies. C, Rh1/dn-PP2A cells were exposed to curcumin (0–40 μmol/L) for 2 h followed by stimulation with IGF-I (10 ng/mL) for 1 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies. D, serum-starved (24 h) HT29 cells expressing shRNAs to PP2A-A subunit and GFP (control), grown in six-well plates, were treated with curcumin (0–50 μmol/L) for 2 h, followed by stimulation with or without IGF-I (10 ng/mL) for 1 h. Whole-cell lysates were subjected to Western blot analysis using the indicated antibodies.
Figure 3
Figure 3
Curcumin inhibits mTORC1 signaling independently of AMPK. A, serum-starved (24 h) HT29 cells were exposed to curcumin (0–40 μmol/L) for 2 h followed by stimulation with IGF-I (10 ng/mL) for 1 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies. B, serum-starved (24 h) HT29 cells, grown in six-well plates, were pretreated with compound C (10 μmol/L) for 1 h and then exposed to curcumin (0–50 μmol/L) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies. C, serum-starved Rh30 cells, infected with Ad-dn-AMPKα1 and Ad-GFP (control), were exposed to curcumin (0–50 μmol/L) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies.
Figure 4
Figure 4
Curcumin inhibits mTORC1 signaling independently of TSC. A, Rh1 cells were exposed to curcumin (0–40 μmol/L) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. TSC2 was immunoprecipitated (IP) from the cell lysates, followed by immunoblotting with the indicated antibodies. B, MEF/TSC2−/− and wt cells were exposed to curcumin (0–50 μmol/L) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies.
Figure 5
Figure 5
Curcumin inhibits the kinase activity of mTOR. Serum-starved (24 h) Rh1 cells, grown in 100-mm dishes, were treated with various concentrations of curcumin or rapamycin (100 ng/mL) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. mTOR was immunoprecipitated from whole-cell lysates with antibodies to mTOR and the immunoprecipitates were used in mTORC1 in vitro kinase assay using recombinant 4E-BP1 as a substrate (A), or in mTORC2 in vitro kinase assay using recombinant Akt as a substrate (B), as described in Materials and Methods. The kinase assay products were subjected to Western blot analysis using the indicated antibodies.
Figure 6
Figure 6
Curcumin disrupts mTOR complexes. Serum-starved (24 h) Rh1 cells, grown in 100-mm dishes, were treated with various concentrations of curcumin or rapamycin (100 ng/mL) for 2 h, followed by stimulation with IGF-I (10 ng/mL) for 1 h. mTOR was immunoprecipitated from whole-cell lysates. The cell lysates (A) and the immunoprecipitates (B) were subjected to Western blot analysis using the indicated antibodies.
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