The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner

Abstract

Background: The isothiocyanate sulforaphane (SFN) has multiple protein targets in mammalian cells, affecting processes of fundamental importance for the maintenance of cellular homeostasis, among which are those regulated by the stress response transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) and the serine/threonine protein kinase mechanistic target of rapamycin (mTOR). Whereas the way by which SFN activates NRF2 is well established, the molecular mechanism(s) of how SFN inhibits mTOR is not understood.

Hypothesis/purpose: The aim of this study was to investigate the mechanism(s) by which SFN inhibits mTOR STUDY DESIGN AND METHODS: We used the human osteosarcoma cell line U2OS and its CRISPR/Cas9-generated NRF2-knockout counterpart to test the requirement for NRF2 and the involvement of mTOR regulators in the SFN-mediated inhibition of mTOR.

Results: SFN inhibits mTOR in a concentration- and time-dependent manner, and this inhibition occurs in the presence or in the absence of NRF2. The phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B (PKB) is a positive regulator of mTOR, and treatment with SFN caused an increase in the phosphorylation of AKT at T308 and S473, two phosphorylation sites associated with AKT activation. Interestingly however, the levels of pS552 β-catenin, an AKT phosphorylation site, were decreased, suggesting that the catalytic activity of AKT was inhibited. In addition, SFN inhibited the activity of the cytoplasmic histone deacetylase 6 (HDAC6), the inhibition of which has been reported to promote the acetylation and decreases the kinase activity of AKT.

Conclusion: SFN inhibits HDAC6 and decreases the catalytic activity of AKT, and this partially explains the mechanism by which SFN inhibits mTOR.

Keywords: HDAC6; NRF2; PI3K-AKT; Sulforaphane; mTOR.

Graphical abstract

Figure 1

Sulforaphane (SFN) inhibits mTOR in an NRF2-independent manner. (A) Chemical structures of the NRF2 activators used in this study. (B,C,E,F) Immunoblotting analysis of phosphorylated p70S6K (T389), p70S6K, phosphorylated S6 (S235/236), and S6 in lysates from U2OS cells, which had been treated with vehicle (0.1% DMSO), rapamycin (20 nM), SFN (20 μM, unless otherwise indicated) (B,C,F), S-4 (E), or BITC (E) for the specified periods of time. The levels of β-actin served as a loading control. (D) Cell proliferation in the presence of increasing concentrations of SFN.

Figure 2

Sulforaphane (SFN) causes transient inhibition of HDAC6 and AKT. (A,B) U2OS cells were treated with vehicle (0.1% DMSO), SFN (20 μM) or rapamycin (20 nM) for the indicated periods of time. Immunoblotting analysis of whole-cell lysates was used to determine the levels of: phosphorylated AKT (S473 and T308), AKT, acetylated α-tubulin (K40), and α-tubulin (A); phosphorylated AMPK (T172) and AMPK (A); phosphorylated β-catenin (S552) and β-catenin (B). (C) Cells were transfected with an empty vector or a plasmid encoding FLAG-HDAC6. After 48 h, they were treated with SFN (20 μM) for a further 4 h. The levels of HDAC6, acetylated α-tubulin (K40), α-tubulin, phosphorylated p70S6K (T389), p70S6K, phosphorylated S6 (S235/236), and S6 were determined in whole-cell lysates by immunoblotting. (D) Following pre-treatment with tubastatin (5 μM) for the indicated periods of time, cells were treated with SFN (20 μM) for 4 h in the presence of tubastatin. The levels of acetylated α-tubulin (K40), α-tubulin, phosphorylated p70S6K (T389), p70S6K, phosphorylated S6 (S235/236), and S6 were determined in whole-cell lysates by immunoblotting. (E) Cells were pre-treated with the indicated concentrations of MS-275 or CBHA for 12 h, and subsequently treated with SFN (20 μM) for 4 h in the presence of the HDAC inhibitors. The levels of acetylated histone H3 (K9 and K27), histone H3, acetylated α-tubulin (K40), α-tubulin, phosphorylated p70S6K (T389), p70S6K, phosphorylated S6 (S235/236), and S6 were determined in whole-cell lysates by immunoblotting. In all cases, the levels of β-actin served as a loading control.