Figure 1.
DCE and CS decrease intracellular GSH level in human keratinocytes.
(A) Reaction scheme of GSH and CS or DCE interaction. DCE and CS, as well as other natural sesquiterpenes, have in common a reactive α-β-unsaturated carbonyl group that may react by a Michael-type addition with nucleophiles, such as cysteine sulfhydryl groups. DCE (B) and CS (C) time- and dose-dependently induce the drop in cellular GSH content measured by spectrophotometric analysis. Treatment of cultures with culture medium alone do not vary GSH release. Data are presented as means of GSH content ± SD of results of four independent experiments. Terpene-treated groups were compared to untreated groups (time 0). * p≤0.01 and ** p≤0.05.
Figure 2.
DCE and CS inhibit STAT3 and STAT1 signalings whereas increase EGFR/ERK1/2 phosphorylation.
Total STAT3, Tyr705- or Ser727-phosphorylated STAT3, Lys685-acetylated STAT3, total STAT1, and Tyr701- or Ser727-phosphorylated STAT1 were detected by Western blotting of lysates from cultured human keratinocytes (A). STAT3 and STAT1 were analyzed in IL-22 and IFN-γ-activated keratinocyte cultures, respectively, treated or not with DCE, HCS or CS (all at 12.5 µM with 1-h pre-treatment). Graphs represent densitometric analyses of phospho-STAT3, acetylated STAT3, and phospho-STAT1 (D. U., Densitometric Units; F. I., Fold Induction). Effects of terpenes on the STAT3 or STAT1 activities were evaluated by assaying luciferase activity of STAT3 or STAT1 responsive plasmids (B). Data are expressed as means ± SD of Firefly luciferase values normalized to Renilla luciferase and µg of total proteins. * p≤0.01 and ** p≤0.05. Phospho-ERK1/2 (C), phospho-EGFR (D) and phospho-AKT (E) were evaluated by Western blotting on keratinocyte cultures treated with DCE, HCS or CS for 30 min. Graphs represent densitometric analyses. * p≤0.01 and ** p≤0.05. Immunoblot stainings are representative of 3 independent experiments performed on 3 different keratinocyte strains obtained from biopsies of different healthy donors.
Figure 3.
Modulation of the IL-22- and IFN-γ-induced gene expression by DCE and CS.
The IL-22-induced SOCS3, CCL2, HBD-2, S100A7, IL-20 mRNA (A) and the IFN-γ-induced SOCS1, CCL2, CXCL10, ICAM-1, IRF-1 mRNA (B) expression were evaluated by real-time PCR analysis of RNA from cultured keratinocytes stimulated with the specific cytokines in presence or absence of DCE, HCS or CS, and normalized to HPRT-1 mRNA. * p≤0.01 and ** p≤0.05.
Figure 4.
Inhibition of proliferation and cell-cycle progression of keratinocytes by DCE and CS.
Proliferation of keratinocytes treated with DCE, CS or HCS either in presence or absence of IL-22 was proportional to crystal violet incorporation (A), which was measured with an ELISA reader after 2 d of culture. Data are expressed as fold induction of treated vs. untreated samples, which were given a value of 1. * p≤0.01 and ** p≤0.05. B) PCNA, cyclin D1, and pRB were analyzed by Western blotting. Graphs show densitometric values of PCNA, cyclin D1, and pRB staining on blots (D. U., Densitometric Units; F. I., Fold Induction). Western blots are representative of 3 independent experiments performed on 3 different keratinocyte strains obtained from biopsies of different healthy donors. * p≤0.01. C) Cell-cycle distribution analysis of cultured keratinocytes treated or not with DCE, CS, or HCS, for 16 h. The percentage of keratinocytes in G0-G1, S, and G2/M phases are indicated in each histograms.
Figure 5.
Apoptosis is enhanced in DCE- or CS-treated keratinocytes.
Apoptosis of cultured keratinocytes treated with DCE, CS, or HCS in presence or absence of the pro-apoptotic stimulus TNF-α (50 ng/ml for 48 h) was examined by measuring Annexin/PI fluorescence through FACS analysis (A) or DNA fragmentation by ELISA (B). In A), a representative experiment of four performed is shown, with numbers indicating the percentage of PI+ (upper left), Ann V+ (lower right), PI/Ann V+ (upper right), or negative (lower left) cells. In B), * p≤0.05.
Figure 6.
DCE and CS accelerates in vitro wound healing by enhancing keratinocyte migration.
Scratch assays were carried out on scratched keratinocyte cultures incubated or not with 30 ng/ml IL-22 for 18 h, in presence of DCE, CS or HCS (A). Cultures were also treated with 10 µM mitomycin and stimulated as indicated in B). Residual gap between migrating keratinocytes is expressed as percentage of initial scratched area. * p≤0.01 and ** p≤0.05 vs untreated samples.