Resveratrol induces autophagy through mTOR inhibition

In order to understand resveratrol-induced autophagy, we examined the effect of resveratrol on autophagy in GFP-LC3 expressing HeLa cells. Resveratrol treatment induces autophagy, as evidenced by the accumulation of LC3B-II and LC3 puncta formation14 (supplementary Fig. S1). Along with this result, treatment with mTOR kinase inhibitor pp242 demonstrated that mTORC1 activity is inversely correlated with the level of autophagy (supplementary Fig. S1A). This result implicates the possible involvement of mTORC1 in resveratrol-induced autophagy. To test the position of mTOR in resveratrol-induced autophagy, we used various drugs that induce autophagy. We found that combinatory treatment with resveratrol and PP242 did not show any additive effect (Fig. 1A,B), indicating that resveratrol acts via the same pathway as PP242. Decreased cyclic-AMP (cAMP) levels induce autophagy in an mTOR-independent manner17. Because reduction of cAMP induces autophagy through inhibition of PKA18, we utilized H-89, a PKA inhibitor, to induce mTOR-independent autophagy. As expected, treatment with H-89 increased autophagy without reduction of mTORC1 activity. Additionally, unlike the resveratrol-PP242 combination, co-treatment with H-89 and resveratrol or H-89 and PP242 had an additive effect on the accumulation of LC3B-II (Fig. 1A). This result indicates that H-89 induces autophagy through an mTOR-independent pathway, and resveratrol and H-89 use different pathways to induce autophagy. Taken together, these combinatory drug treatment experiments indicate that resveratrol induces autophagy at the level as same as mTOR inhibition.

Figure 1: Resveratrol induces autophagy through mTOR inhibition. (A) Autophagy induction was analyzed by measuring accumulation level of LC3-II. Resveratrol (100 μM), PP242 (1.25 μM), H-89 (10 μM), or a combination of two different chemicals was administered to HEK293 cells for 4 h. *P < 0.05 n = 3. (B) LC3 puncta formation induced by resveratrol (50 μM), PP242 (1.25 μM), or a combination of these chemicals was examined in HeLa cells stably expressing GFP-LC3 for 2 h. Scale bars in fluorescent pictures represent 20 μm Full size image

ULK1 is required for resveratrol-induced autophagy

mTOR regulates autophagy through inhibitory phosphorylation of ULK1 and inhibition of mTOR decreases the inhibitory phosphorylation level of ULK1 and increases autophagy13,14. Therefore, to confirm the involvement of mTOR in resveratrol-induced autophagy and to examine the ULK1 dependency, we analyzed the level of autophagy induced by resveratrol in the presence or absence of ULK1. ULK1 knockdown using shRNA abolished resveratrol-induced autophagy, indicating that ULK1 is required for resveratrol-induced autophagy (Fig. 2A,B). However, ULK1 knockdown did not abolish autophagy induced by H-89 treatment (Fig. 2C), again confirming that the mTOR-regulated autophagy is independent of the cAMP-PKA pathway. Together, we concluded that resveratrol induces autophagy through the mTOR-ULK1 pathway, which includes inhibition of mTOR.

Figure 2: ULK1 is required for resveratrol-induced autophagy. (A) Autophagy induction by resveratrol (50 μM, 2 h) in the presence or absence of ULK1. Lenti virus encoding vector or shULK1 was introduced into GFP-LC3 expressing HeLa cells. (B) Autophagy induction by resveratrol (50 μM, 2 h)in the presence or absence of ULK1. cDNA encoding vector or shULK1 was transfected into HEK293 cells. Scale bars in fluorescent pictures represent 20 μm. (C) Autophagy induction by H-89 (10 μM, 2 h) in the presence or absence of ULK1. cDNA encoding vector or shULK1 was transfected into HEK293 cells. Full size image

Resveratrol reduces viability of mTOR inhibition sensitive cancer cells in ULK1 dependent manner

As previous studies showed, resveratrol-induced autophagy suppresses cancer in vitro and in vivo4,6,19. Therefore, we examined whether resveratrol-induced suppression of cancer progression occurred through mTOR and inhibition of ULK1. To examine mTOR dependency on resveratrol induced-cancer cell suppression, we utilized two different cell lines that were previously reported mTOR inhibition sensitive and insensitive, respectively20. MCF7, a breast cancer cell that has constitutive PI3K activation and higher mTOR activity so that it is sensitive to mTOR inhibitor, showed very sensitive response to resveratrol treatment (Fig. 3A). However, neither PP242 nor resveratrol reduced the viability of Ras transformed colorectal cancer cells, SW620 (Fig. 3B). This result indicates that resveratrol effect on cancer cell viability is largely dependent on impact of mTOR, which varies in cancer cell types. To examine whether the effect of resveratrol on cellular viability is reflected in intra-cellular signaling, we measured mTOR activity in MCF7 and SW620 cells upon resveratrol and PP242 treatment. The basal level of mTOR activity and responsiveness to resveratrol and PP242 were significantly lower in SW620 cells compared to MCF7 cells, further demonstrating that the modulation of mTOR activity is involved in the physiological effects of resveratrol (Fig. 3C). Next, we examined whether ULK1 is involved in resveratrol-induced suppression of cancer cell viability. Like knockdown of ULK1 blunted the autophagy induction by resveratrol, reduction of viability of MCF7 cells was partially restored by ULK1 knockdown (Fig. 3D). This restoration of viability was also observed in PP242 induced cancer cell suppression (Fig. 3D). These cancer cell specific and ULK1 dependent effect of resveratrol further supports the idea that resveratrol-induced cellular behavior alterations occurred through mTOR-ULK1 pathway.

Figure 3: Resveratrol reduces viability of mTOR inhibition sensitive cancer cells. (A) MCF7 Cells were treated with resveratrol (50 or 100 μM) or PP242 (500 nM) or CCCP (10 μM) for 48 h followed by cell viability measurement by MTT assay. (B) Chemical treatment and viability measurement for SW620 cells were conducted same as MCF7 cells. (C) Intra-cellular signaling was examined in MCF7 cells and SW620 cells after 4 h treatment of resveratrol (50 μM) or PP242 (500 nM). n = 3. (D) MCF7 cells were infected with lenti virus containing vector or shULK1. After 48 h infection chemical treatment was conducted for another 48 h followed by viability measurement. The Y-axis of the graph indicates the levels relative to those of untreated negative control samples. Full size image

mTOR-associated DEPTOR level is not changed by resveratrol

DEPTOR is a negative regulator of both mTORC1 and mTORC2. Liu et al. showed that resveratrol increased the interaction affinity between mTOR and DEPTOR and suggested that DEPTOR is required for the suppression of mTOR activity upon resveratrol treatment21. To examine the involvement of DEPTOR, we conducted a pull-down assay of FLAG-tagged mTOR under various conditions, including resveratrol treatment. Rapamycin was used as a positive control for chemically-induced complex dissociation. Unexpectedly, in this condition, both the 10 min and 60 min resveratrol treatments did not increase the affinity between mTOR and DEPTOR although resveratrol clearly decreased mTOR activity. Treatment with rapamycin led to the detachment of Raptor from mTOR (supplementary Fig. S2).

Resveratrol inhibits mTOR kinase activity in cell-free systems

Next, we examined whether resveratrol directly suppresses mTOR activity. To address this question, we performed an in vitro kinase assay. We observed that resveratrol dose-dependently decreased the phosphorylation level of 4E-BP1 (Fig. 4A). In addition to the experiments with full-length mTOR, we used a recombinant fragment mTOR that some regulatory domains are deleted, but still contains the intact kinase domain for a kinase assay. Notably, resveratrol could decrease the phosphorylation of S6K, a substrate of mTOR (Fig. 4B), similar to the results of immunoprecipitated full-length mTOR (Fig. 4A). Additionally, we found that resveratrol has an IC 50 value of ~10 μM against mTOR kinase activity (Fig. 4C). Taken together, resveratrol inhibits mTOR kinase activity in cell-free systems.

Figure 4: Resveratrol inhibits mTOR kinase activity in cell-free systems. (A) An in vitro mTOR kinase assay was performed with purified mTOR from HEK293 cells. HA-tagged mTOR was introduced into cells and immunoprecipitated with the anti-HA antibody. (B) An in vitro mTOR kinase assay was performed using GST-tagged recombinant mTOR. (C) Inhibition curve of mTOR activity with the indicated concentration of resveratrol. An in vitro kinase assay was performed as in B. n = 3. Full size image

Computational simulation of the interaction between mTOR and resveratrol

To assess the mode of action for inhibiting the kinase activity of mTOR, we carried out docking simulations of resveratrol in the ATP-binding site of mTOR for determining the lowest-energy binding mode of resveratrol (Fig. 5A). Resveratrol appears to be stabilized in the binding pocket formed by the Gly loop, hinge region of the ATP-binding site, and the interface residues of N- and C-terminal domains. To examine the possibility of allosteric inhibition of mTOR by resveratrol, we performed additional docking simulations with extended 3D grid maps that include the whole kinase domain. However, no peripheral binding site was found in which resveratrol could be stabilized with negative free energy of binding. It is thus expected that the micromolar-level inhibitory activity of resveratrol against mTOR stems from the specific binding in the ATP binding site.

Figure 5: Resveratrol inhibits mTOR through ATP competition. (A) Docking pose of resveratrol in the ATP-binding site of mTOR. (B) Detailed binding mode of resveratrol in the ATP-binding site of mTOR. The carbon atoms of resveratrol and mTOR are shown in green and cyan, respectively. Hydrogen bonds are indicated with dotted lines. (C) Suppression of activity by resveratrol was dependent on the level of ATP. An in vitro kinase assay was performed with the indicated amounts of ATP and resveratrol. mTOR kinase inhibitor PP242 was used as a positive control. (D) In vitro kinase was performed with increased amount of ATP. (E) In vitro kinase was performed with WT mTOR and D2195A mTOR *P < 0.05 n = 3. (F) WT mTOR or D2195A mTOR, and HA-Raptor were transfected into HEK293T cells. After 24 h post transfection, Resveratrol (50 μM) was administered to HEK293 cells for 4 h to measure the accumulation of LC3B-II. *P < 0.05 n = 3. Full size image

We now address the detailed interactions responsible for stabilization of resveratrol in the ATP-binding pocket. The binding mode of resveratrol calculated from docking simulations is shown in Fig. 5B. We observed that the terminal phenolic group of resveratrol receives and donates a hydrogen bond from the backbone amidic nitrogen to the aminocarbonyl oxygen of V2240, respectively. This hydrogen bond seems to be important for the biochemical potency of resveratrol because the formation of hydrogen bonds with same region was also observed in the structures of mTOR complex with potent inhibitors22. In the assessed mTOR-resveratrol complex, two additional hydrogen bonds are established between the benzene-1,3-diol moiety and the side-chain carboxylate groups of E2190 and D2195. On the basis from docking simulations, it can be argued that the inhibitory activity of resveratrol can be attributed to the multiple hydrogen bonds.

Resveratrol inhibits mTOR through ATP competition

To verify the simulation results, we performed an in vitro kinase assay with various concentrations of ATP. PP242, a kinase inhibitor of mTOR, was used as a positive control for mTOR inhibition. We found that resveratrol-induced mTOR inhibition was restored by the addition of ATP (Fig. 5C,D). To clarify whether resveratrol inhibits mTOR through direct interaction, we generated a resveratrol-resistant mTOR mutant. Based on the resolved mTOR-ATP structure22 and our computational simulation (Fig. 5A,B), Asp 2195 was deemed to be a resveratrol-binding residue that does not affect the mTOR-ATP interaction. Thereafter, we generated an mTOR mutant in which Asp 2195 was substituted with alanine (hereafter, referred to as D2195A) and performed an in vitro kinase assay. As expected, resveratrol inhibited wild-type (WT) mTOR but not D2195A mTOR. The basal activity level of the D2195A mutant was lower than that of WT mTOR, possibly due to the fact that the mutated residue resides in the kinase domain of the protein. We observed that PP242 also inhibited DA mTOR, albeit the inhibitory potency was less than WT mTOR (Fig. 5E). Although Asp 2195 was suggested as a residue for the interaction between mTOR and PP242, other residues also participate the interaction between them and not all residues are shared between PP242 and resveratrol for mTOR binding. We speculate that other residues but not D2105 may be essential for the PP242 binding to mTOR and that is why D2195A mTOR was not completely resistant to PP242. To examine whether the resveratrol resistant mTOR blocks the resveratrol-induced autophagy, we measured the autophagy levels upon resveratrol treatment in the WT or D2195A mTOR along with Raptor transfected cells. We found that resveratrol induced autophagy in the WT mTOR transfected cells, but not in the D2109A mTOR transfected cells (Fig. 5F). Taken together, the results from the experiments using resveratrol resistant mTOR suggest that resveratrol induces autophagy by directly inhibits mTOR through ATP competition.