Figure 1.
Resveratrol and verbascoside effects on IL-8 expression in normal human keratinocytes.
Chemical structure of resveratrol (A) and verbascoside (B). (C, D) Quantitative real-time RT-PCR time-dependent changes of IL-8 transcript. Cells were treated with 50 µM resveratrol (Resv) or verbascoside (Verb) for the indicated intervals. Keratinocytes were pre-treated with the polyphenols for 1 h before addition of TNFα (50 ng/ml), and analyzed at the indicated time-points. (E) Profile of IL-8 protein accumulation in cell supernatants measured by specific ELISA. *P<0.05 and §P<0.01 versus controls at the same time-point without polyphenol (−). (F) ELISA quantification of IL-8 in the medium of human keratinocytes treated with escalating concentration of Resv for 24 h. *P<0.05 and §P<0.01 versus controls without polyphenol (0 µM). (G) Quantitative real-time RT-PCR measurement of IL-8 mRNA stability. After 12 h treatment with TNFα, the RNA polymerase II inhibitor DRB (75 µM) was added for the indicated time-points. Data are expressed as the mean ± S.D. of three consecutive determinations in three independent experiments.
Figure 2.
Effects of resveratrol and verbascoside on the phosphorylation status of EGFR, ERK1/2, and the NFκB subunit p65.
(A) Western blot analysis performed in whole-cell lysates of human keratinocytes treated with 50 µM of the indicated polyphenol. Actin was used as a loading control. (B, C, D) Quantification of Western blot bands by densitometry. *P<0.05 versus untreated controls (0 h time-point). (E) Western blot analysis of EGFR and ERK phosphorylation in whole-cell lysates of human keratinocytes treated with 10 and 50 µM Resv for 6 h. (F) Quantification of Western blot bands by densitometry. *P<0.05 versus controls without Resv (0 µM). §P<0.05 versus 10 µM Resv (0 µM). Data are representative of three independent experiments.
Figure 3.
Effects of resveratrol and verbascoside on the TNFα−induced phosphorylation of EGFR, ERK, and the NFκB subunit p65.
(A) Western blot analysis performed in whole-cell lysates of human keratinocytes. Following 1 h pre-incubation with 50 µM polyphenol, cells were treated for further 12 h with TNFα (50 ng/ml). Actin was used as a loading control. (B) Quantification of Western blot bands by densitometry. *P<0.05 versus untreated controls (0 h time-point); §P<0.05 versus TNFα-treated conditions. (C) Binding activity of nuclear cell lysates to NFκB-specific or AP-1-specific DNA consensus sequences. *P<0.05 versus untreated controls; §P<0.05 versus TNFα-treated conditions. Data are representative of three independent experiments.
Figure 4.
Abrogation of both constitutive and resveratrol-dependent EGFR and ERK phosphorylation by specific inhibitor of EGFR kinase.
(A) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD168393 (PD16) for 30 minutes prior to addition of 50 µM resveratrol (Resv) for the indicated time-points. (B) Quantification of Western blot bands by densitometry. *P<0.05 and §P<0.01 versus untreated controls for each time-point. (C) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD16 prior to addition of 50 µM Rv for 1 h. Subsequently, cells were further treated for 12 h with 50 ng/ml TNFα. (D) Quantification of Western blot bands by densitometry. *P<0.05 versus controls treated with TNFα only. Data are representative of three independent experiments.
Figure 5.
Effects of resveratrol on IL-8 expression are sensible to specific inhibitor of EGFR kinase.
(A) Quantitative real-time RT-PCR measurement of IL-8 mRNA. Cells were treated with 2 µM PD168393 (PD16) for 30 min, and then exposed to 50 µM resveratrol (Resv) for 1 h. Cells were cultivated for further 12 h, with or without TNFα (50 ng/ml). *P<0.05 versus untreated controls; §P<0.01 versus TNFα-treated conditions. (B) ELISA of IL-8 protein accumulation in the supernatants of human keratinocytes. Cells were treated with 2 µM PD16 for 30 min, and then, exposed to 50 µM Resv for 1 h. Cells were cultivated for further 24 h, with or without TNFα (100 ng/ml). Data are representative of three independent experiments.
Figure 6.
Resveratrol did not protect phosphorylated EGFR from endogenous phosphatases, led to EGFR accumulation in the keratinocyte membranes, and induced its cytosolic degradation.
Western blot analysis of the plasma membrane fraction, where cadherin was used as a loading control (A) and of the cytosolic fraction (C). After 24h treatment with resveratrol (Resv), keratinocytes were stimulated with TGFα (50 ng/ml) for 10 minutes. Then, 2 µM PD168393 (PD16) were added to the medium for further 10 minutes. (B, D) Quantification of Western blot bands by densitometry. *P<0.05 and §P<0.01 versus untreated controls.
Figure 7.
Resveratrol promoted nuclear accumulation of phosphorylated and non-phosphorylated EGFR alone and in association with TGFα.
(A) Western blot analysis of nuclear levels of phosphorylated (nP-EGFR) and non-phosphorylated (nEGFR), and (B) its densitometric quantification. *P<0.05 and §P<0.01 versus untreated controls.
Figure 8.
Resveratrol effects on 3H-thymidine incorporation and molecular markers of cell proliferation, cell cycle, senescence, and apoptosis.
A. Keratinocyte proliferation was determined by 3H-thymidine incorporation (CPM, counts per minute) 12 h, 24 h, and 36 h after incubation without any agent (C), with DMSO (DMSO), with 50 µM of resveratrol (Resv), with 10 ng/mL of TGFα (TGF-α), or their combination (Resv+TGF-α). *P<0.01 versus untreated controls. B. Characteristic Western blots of p63, PCNA, p53, p21Waf1, caspase 8, Bax, and p16INK4 in NHEK treated with the same agents for 24 h. C. Densitometry of the Western blots obtained in three independent experiments.
Figure 9.
Proposed model of reduction-oxidation reactions of resveratrol in human keratinocytes.
A. Redox cycling of resveratrol, which results in reactive oxygen species scavenging or production. Abbreviations: Resv, parent molecule of resveratrol; Resv-O•, semiquinone of resveratrol; Resv-Q, quinone of resveratrol; Men+, transition metals; ROS, reactive oxygen species; Resv-NOO, nitrated resveratrol; B. Resveratrol hydroxylation by cytochrome P450. Resv-(OH)n, hydroxylated resveratrol; CYP1, cytochrome P450 subfamily 1.