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Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1.

Oi N, Yuan J, Malakhova M, Luo K, Li Y, Ryu J, Zhang L, Bode AM, Xu Z, Li Y, Lou Z, Dong Z - Oncogene (2014)

Bottom Line: Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis.We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53.Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Resveratrol (trans-3,5,4'-truhydroxystilbene) possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to the suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1.

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The p53-dependent effect of resveratrol relies mainly on G3BP1(a) The effect of resveratrol was diminished in HCT116 p53−/− cells compared with p53+/+ cells. Both cell types were treated with resveratrol (0–40 µM) for 72 h and then cellular apoptosis was determined by flow cytometry. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (b) HCT116 p53−/− cells are less sensitive to resveratrol’s effect on proliferation. Both cell types were treated with resveratrol for 72 h and then formazan production was determined by MTS assay. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (c) The p53-dependent effect of resveratrol mainly relies on G3BP1. HCT116 p53−/− cells expressing the indicated shRNAs were treated with resveratrol for 72 h and then early apoptosis and formazan production were determined by flow cytometry or MTS assay, respectively. Data are shown as means ± S.D. from 3 independent experiments and no significant difference was observed compared with shCtrl cells (*p < 0.05). (d) G3BP1 is overexpressed in human melanoma skin tissue. G3BP1 levels were analyzed by immunohistochemistry using 40 cases of human melanoma skin tissue and 8 cases of normal skin tissue, and then the density score from each sample was determined. Representative cases are shown (right panels). (e) A working model of G3BP1-mediated p53 regulation by resveratrol.
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Figure 7: The p53-dependent effect of resveratrol relies mainly on G3BP1(a) The effect of resveratrol was diminished in HCT116 p53−/− cells compared with p53+/+ cells. Both cell types were treated with resveratrol (0–40 µM) for 72 h and then cellular apoptosis was determined by flow cytometry. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (b) HCT116 p53−/− cells are less sensitive to resveratrol’s effect on proliferation. Both cell types were treated with resveratrol for 72 h and then formazan production was determined by MTS assay. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (c) The p53-dependent effect of resveratrol mainly relies on G3BP1. HCT116 p53−/− cells expressing the indicated shRNAs were treated with resveratrol for 72 h and then early apoptosis and formazan production were determined by flow cytometry or MTS assay, respectively. Data are shown as means ± S.D. from 3 independent experiments and no significant difference was observed compared with shCtrl cells (*p < 0.05). (d) G3BP1 is overexpressed in human melanoma skin tissue. G3BP1 levels were analyzed by immunohistochemistry using 40 cases of human melanoma skin tissue and 8 cases of normal skin tissue, and then the density score from each sample was determined. Representative cases are shown (right panels). (e) A working model of G3BP1-mediated p53 regulation by resveratrol.

Mentions: Because resveratrol has been reported to induce p53-dependent as well as p53-independent apoptosis in a certain types of human cancer cells (30), we compared the effect of resveratrol in HCT116 p53+/+ and p53−/− cells. The effects of resveratrol in inducing apoptosis and inhibiting cell proliferation were significantly reduced in HCT116 p53−/− cells compared with HCT 116 p53+/+ cells (Fig. 7a,b). A similar phenomenon of inhibition of proliferation was observed in SK-MEL-5 cells by depletion of p53 (Supplemental Fig. 6a). Although the effect of resveratrol is diminished in p53 depleted or deficient cells, resveratrol still induced apoptosis and inhibited cell proliferation, indicating resveratrol exerts both p53-dependnt and -independent effects. To examine the consequence of G3BP1 in p53-dependent effect of resveratrol, G3BP1 was depleted in HCT116 p53−/− or RPMI7951 (a p53- human skin malignant melanoma) cells and then cells were treated with resveratrol. Interestingly, the effect of resveratrol on either inducing apoptosis or inhibiting proliferation was not affected by depletion of G3BP1 in HCT116 p53−/− (Fig. 7c) and RPMI7951 (Supplemental Fig. 6b) cells. We therefore concluded that the p53-dependent effect of resveratrol mainly relies on G3BP1. Furthermore, human skin melanoma tissue array analysis showed that the protein levels of G3BP1 were significantly higher in skin melanoma tissue compared with normal skin tissue (Fig. 7d), which is consistent with a role of G3BP1 in promoting cancer cell proliferation. Overall, our study demonstrates that resveratrol directly targets G3BP1, which in turn prevents the G3BP1/USP10 interaction and consequently increases USP10-regulated deubiquitination of p53 (Fig. 7e).


Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1.

Oi N, Yuan J, Malakhova M, Luo K, Li Y, Ryu J, Zhang L, Bode AM, Xu Z, Li Y, Lou Z, Dong Z - Oncogene (2014)

The p53-dependent effect of resveratrol relies mainly on G3BP1(a) The effect of resveratrol was diminished in HCT116 p53−/− cells compared with p53+/+ cells. Both cell types were treated with resveratrol (0–40 µM) for 72 h and then cellular apoptosis was determined by flow cytometry. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (b) HCT116 p53−/− cells are less sensitive to resveratrol’s effect on proliferation. Both cell types were treated with resveratrol for 72 h and then formazan production was determined by MTS assay. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (c) The p53-dependent effect of resveratrol mainly relies on G3BP1. HCT116 p53−/− cells expressing the indicated shRNAs were treated with resveratrol for 72 h and then early apoptosis and formazan production were determined by flow cytometry or MTS assay, respectively. Data are shown as means ± S.D. from 3 independent experiments and no significant difference was observed compared with shCtrl cells (*p < 0.05). (d) G3BP1 is overexpressed in human melanoma skin tissue. G3BP1 levels were analyzed by immunohistochemistry using 40 cases of human melanoma skin tissue and 8 cases of normal skin tissue, and then the density score from each sample was determined. Representative cases are shown (right panels). (e) A working model of G3BP1-mediated p53 regulation by resveratrol.
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Related In: Results  -  Collection

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Figure 7: The p53-dependent effect of resveratrol relies mainly on G3BP1(a) The effect of resveratrol was diminished in HCT116 p53−/− cells compared with p53+/+ cells. Both cell types were treated with resveratrol (0–40 µM) for 72 h and then cellular apoptosis was determined by flow cytometry. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (b) HCT116 p53−/− cells are less sensitive to resveratrol’s effect on proliferation. Both cell types were treated with resveratrol for 72 h and then formazan production was determined by MTS assay. Data are shown as means ± S.D. from 3 independent experiments (*p < 0.05). (c) The p53-dependent effect of resveratrol mainly relies on G3BP1. HCT116 p53−/− cells expressing the indicated shRNAs were treated with resveratrol for 72 h and then early apoptosis and formazan production were determined by flow cytometry or MTS assay, respectively. Data are shown as means ± S.D. from 3 independent experiments and no significant difference was observed compared with shCtrl cells (*p < 0.05). (d) G3BP1 is overexpressed in human melanoma skin tissue. G3BP1 levels were analyzed by immunohistochemistry using 40 cases of human melanoma skin tissue and 8 cases of normal skin tissue, and then the density score from each sample was determined. Representative cases are shown (right panels). (e) A working model of G3BP1-mediated p53 regulation by resveratrol.
Mentions: Because resveratrol has been reported to induce p53-dependent as well as p53-independent apoptosis in a certain types of human cancer cells (30), we compared the effect of resveratrol in HCT116 p53+/+ and p53−/− cells. The effects of resveratrol in inducing apoptosis and inhibiting cell proliferation were significantly reduced in HCT116 p53−/− cells compared with HCT 116 p53+/+ cells (Fig. 7a,b). A similar phenomenon of inhibition of proliferation was observed in SK-MEL-5 cells by depletion of p53 (Supplemental Fig. 6a). Although the effect of resveratrol is diminished in p53 depleted or deficient cells, resveratrol still induced apoptosis and inhibited cell proliferation, indicating resveratrol exerts both p53-dependnt and -independent effects. To examine the consequence of G3BP1 in p53-dependent effect of resveratrol, G3BP1 was depleted in HCT116 p53−/− or RPMI7951 (a p53- human skin malignant melanoma) cells and then cells were treated with resveratrol. Interestingly, the effect of resveratrol on either inducing apoptosis or inhibiting proliferation was not affected by depletion of G3BP1 in HCT116 p53−/− (Fig. 7c) and RPMI7951 (Supplemental Fig. 6b) cells. We therefore concluded that the p53-dependent effect of resveratrol mainly relies on G3BP1. Furthermore, human skin melanoma tissue array analysis showed that the protein levels of G3BP1 were significantly higher in skin melanoma tissue compared with normal skin tissue (Fig. 7d), which is consistent with a role of G3BP1 in promoting cancer cell proliferation. Overall, our study demonstrates that resveratrol directly targets G3BP1, which in turn prevents the G3BP1/USP10 interaction and consequently increases USP10-regulated deubiquitination of p53 (Fig. 7e).

Bottom Line: Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis.We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53.Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Resveratrol (trans-3,5,4'-truhydroxystilbene) possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to the suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1.

Show MeSH
Related in: MedlinePlus