Resveratrol is a class IA phosphoinositide 3-kinase inhibitor

Abstract

Resveratrol, a polyphenol found in fruits, possesses chemopreventive and chemotherapeutic properties and has been shown to increase lifespan in yeast and metazoans, including mice. Genetic evidence and in vitro enzymatic measurements indicate that the deacetylase Sir2/SIRT1, an enzyme promoting stress resistance and aging, is the target of resveratrol. Similarly, down-regulation of insulin-like pathways, of which PI3K (phosphoinositide 3-kinase) is a key mediator, promotes longevity and is an attractive strategy to fight cancer. We show here that resveratrol inhibits, in vitro and in cultured muscle cell lines, class IA PI3K and its downstream signalling at the same concentration range at which it activates sirtuins. Our observations define class IA PI3K as a target of resveratrol that may contribute to the longevity-promoting and anticancer properties and identify resveratrol as a natural class-specific PI3K inhibitor.

Figure 1. Resveratrol inhibits the insulin-activated PI3K–PKB pathway and IRS1-associated PI3K activity in human primary myotubes

(A) Fully differentiated myotubes were serum-starved overnight and subsequently stimulated for 20 min with 100 nM insulin. Resveratrol (Res.) (100 μM) was added to the cells 30 min prior to insulin stimulation. Separated proteins were immunoblotted with antibodies to PKB phospho-Ser-473 (pS473), PKB phospho-Thr-308 (pT308), total PKB – to monitor equal loading – and pFoxO4. (B) Upper panel: IRS-1-associated PI3K activity was measured in IRS1 immunoprecipitates obtained from starving (–) or insulin-stimulated (+, 100 nM insulin, 20 min) cells. Immune complexes were pretreated with the indicated concentrations of resveratrol. ³²P-labelled PtdIns3P was separated by TLC (‘O’, origin). Lower panel: phosphoimager quantification of PtdIns3P. PI3K activity was set at 100 for the insulin-stimulated condition. Results are means±S.E.M. (n=4). The significance of resveratrol inhibition was compared with the insulin-stimulated condition using Student's t test. *P<0.01, **P<0.001.

Figure 2. Inhibition of the PI3K–PKB pathway, but not of the MAPK pathway, by resveratrol in L6 and CCL cells

(A) L6 myoblasts were serum-starved overnight, pretreated for 30 min with the indicated concentrations of resveratrol and subsequently stimulated for 10 min with 1000 nM insulin. Separated total proteins were immunoblotted with antibodies to PKB pS473, PKB and pMAPK. Furthermore, equal amounts of lysates were immunoprecipitated with antibodies to IRS1 and IRS2, and the associated PI3K was visualized by immunoblotting with anti-p85α antibodies. I.P., immunoprecipitation. (B) IRS1-associated PI3K activity was measured in IRS1 immunoprecipitates obtained from starving (–) or insulin-stimulated (+, 1000 nM insulin, 10 min) L6 cells. Immune complexes were pretreated with the indicated concentrations of resveratrol. ³²P-Labelled PtdIns3P was separated by TLC as in Figure 1. (C) Resveratrol dose–response inhibition of insulin-induced PKB Ser-473 and Thr-308 phosphorylations in CCL cells. Equal loading was verified by immunoblotting of total PKB.

Figure 3. Resveratrol inhibits PKB Ser-473 phosphorylation induced by various agonists

(A) L6 cells were infected for 48 h with an adenovirus expressing a constitutively active p110α (Adp110αCAAX) as indicated. After overnight serum starvation, cells were pretreated for 30 min with 100 μM resveratrol (Res.) as indicated and subsequently stimulated with 10% FCS. Cell lysates were immunoblotted with antibodies to PKB pS473 and pMAPK. (B) CCL cells were starved overnight, pretreated for 30 min with the indicated concentrations of resveratrol and subsequently stimulated with 10 μM LPA. Cell lysates were immunoblotted with antibodies to PKB pS473 and PKB.

Figure 4. Resveratrol inhibits recombinant class IA, but not class IB, PI3K lipid kinase activity

(A) Relative dose–response inhibition of class IA p85α/p110α, p85α/p110β and class IB GST–p110γ in lipid kinase assays using PtdIns as substrate. Representative TLCs for each PI3K isoform are shown at the bottom. Assays for each experimental condition were performed at least four times. Activity in the absence of resveratrol was normalized at 100%. (B) Relative dose–response inhibition of class IA p85α/GST–p110α in a lipid kinase assay using PtdIns(4,5)P₂ as substrate and representative TLC. Assays for each experimental condition were performed in triplicate. (C) Inhibition of class IA p85α/GST–p110α by 100 μM resveratrol by using PtdIns(4,5)P₂ or PIs as substrates. Results are means±S.D. for at least three independent assays; activity in the absence of resveratrol was normalized at 100%. The significance of inhibition was compared with the assays without resveratrol using Student's t test. *P<0.05, **P<0.01, ***P<0.001.

Figure 5. Resveratrol inhibits recombinant class IA, but not class IB, PI3K protein kinase activity

Relative dose–response inhibition of class IA p85α/GST–p110α and class IB GST-p110γ autophosphorylation activities. ‘Relative autophosphorylation’ refers to the radioactivity incorporated into p85α and GST–p110γ bands relative to the autophosphorylation reaction without resveratrol. Lower panel: quantification of incorporated radioactivity from three independent experiments. Results are means±S.D. The significance of inhibition was compared with the assays without resveratrol by using Student's t test. *P<0.1, **P<0.05. p85α/GST–p110α and GST–p110γ expression levels were visualized by Coomassie Blue staining (see Supplementary Figure 2A).

Figure 6. Resveratrol targets the PI3K ATP-binding site in a non-covalent fashion

(A) The lipid kinase activity of class IA p85α/p110α was assayed in the absence (–) or in the presence (+) of 100 μM resveratrol (Res.) and with increasing ATP concentrations as indicated. The experiment shown is representative of two independent ones in which each condition was tested in duplicate. ‘% activity’ is the mean for four independent reactions. (B) Serum-starved L6 cells were pretreated with 50 μM LY294002 (LY), 100 μM resveratrol (R) or 100 nM wortmannin (W) as indicated for 30 min. Prior to a 10 min stimulation with 1000 nM insulin, the medium was replaced in the lanes indicated (washout), thus allowing the elimination of non-covalently bound inhibitor. Separated proteins were immunoblotted with antibodies to PKB pS473, PKB pT308 and total PKB to monitor equal loading.

Figure 7. SIRT1 is not found in IRS1 immune complexes and resveratrol inhibits the insulin-activated PKB independently of sirtuin expression

(A) L6 myoblasts were serum-starved overnight and subsequently stimulated for 10 min with 1000 nM insulin. Separated total proteins were immunoblotted with antibodies to p85α (that also recognizes the short-spliced forms p50 and p55α) and SIRT1. In parallel, equal amounts of lysates were immunoprecipitated with antibodies to IRS1 and immunoblotted with anti-p85α and α-SIRT1 antibodies. IP, immunoprecipitation. (B) L6 myoblasts were transfected with siRNAs directed to SIRT1. After an overnight starving and a 30 min pretreatment with 100 μM resveratrol as indicated, cells were stimulated with 1000 nM insulin for 10 min. Total lysates were immunoblotted with antibodies to PKB pS473 and SIRT1.