Keratinocytes Migration Promotion, Proliferation Induction, and Free Radical Injury Prevention by 3-Hydroxytirosol

Abstract

3-hydroxytyrosol (HT) is the main phenolic compound found in olive oil with known antioxidant, anti-inflammatory, and antimicrobial properties in several dermatological conditions, both when taken in the form of olive oil or pure in cosmeceutical formulations. To date, its direct effect on the wound healing process and the molecular mechanisms involved have not yet been elucidated. Thus, in the present study, we aimed to explore its effects in vitro in epidermal keratinocyte cultures focusing on the molecular mechanism implied. HT was able to induce keratinocyte proliferation in the low micromolar range, increasing the expression of cyclin dependent kinases fundamental for cell cycle progression such as CDK2 and CDK6. Furthermore, it increased cell migration through the activation of tissue remodeling factors such as matrix metalloproteinase-9 (MMP-9) protein. Then, we evaluated whether HT also showed antioxidant activity at this concentration range, protecting from H₂O₂-induced cytotoxicity. The HT prevented the activation of ATM serine/threonine kinase (ATM), Checkpoint kinase 1 (Chk1), Checkpoint kinase 2 (Chk2), and p53, reducing the number of apoptotic cells. Our study highlighted novel pharmacological properties of HT, providing the first evidence of its capability to induce keratinocyte migration and proliferation required for healing processes and re-epithelialization.

Keywords: 3-hydroxytyrosol; cosmeceuticals; oxidative stress; wound healing.

Figure 1

Evaluation of 3-hydroxytyrosol (HT) effect in HaCaT keratinocytes. Cells were cultured in the presence of the indicated HT concentrations (0–100 µM) for 24 or 48 h before the MTT assay (A) or BrdU incorporation (B). Results are expressed as mean (± SD) and are representative of four independent experiments carried out in triplicate. Data are reported as percentage vs. control (untreated cells at 24 h or 48 h) (2-way ANOVA, * p < 0.05, ** p < 0.01 vs. control).

Figure 2

HT induced expression of cell cycle progression proteins. (A) Western blot analysis of cyclin D1, CDK6, CDK2, and cyclin D3 in whole cell extracts from HaCaT cells cultured for 24 h with HT 1–10 µM. α-Tubulin was used as a control for protein loading. The panel shows a representative western blot of three different experiments with similar results. (B) Histograms represent mean (±SD) in densitometry units of scanned immunoblots from three different experiments (2-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 3

Improvement of the migratory capacity of HaCaT cells exposed to HT. (A) Wound healing assay was carried out in HaCaT cells treated for 18 h with HT (1–10 µM) in complete medium. Light microscope images shown are representative of four independent experiments. Dotted white lines indicate the wounded area from the initial scratch. Magnification ×200; (B) Bar columns correspond to the mean scratch area expressed as percentage respect to basal time point area. The measurement was carried out in four different experiments. Results are shown as mean (± SD) (2-way ANOVA, *** p < 0.001).

Figure 4

HT induces migration protein expression in HaCaT cells. (A) Western blot analysis of MMP-9, Phospho Erk1/2, Erk1/2, Phospho-Akt, Akt, PI3 Kinase, RhoA, Rac1, and Ras in whole cell extracts from HaCaT cells treated for 24 h with HT 1–10 µM. β-Actin and α-Tubulin were used as the control of protein loading. The panel shows a representative western blot of four different experiments with similar results. (B) Histograms represent mean (± SD) in densitometry units of scanned immunoblots from four different experiments (2-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 5

HT ameliorated cytotoxicity and apoptosis induced by H₂O₂ in keratinocytes. (A) HaCaT cells were cultured for 6 h in the presence of H₂O₂ (25–800 µM) before the MTT assay. The results are expressed as means ± SD of three independent experiments and reported as percentage vs. the untreated control (2-way ANOVA, * p < 0.05, ** p < 0.01, and *** p < 0.001). (B) HaCaT cells were cultured for 18 h in the presence of HT (1–10 µM) before treatment with H₂O₂ for 6 h. The results are expressed as means ± SD of three independent experiments reported as percentage vs. the untreated control (2-way ANOVA, * p < 0.05 and ** p < 0.01 vs. control). (C) Flow cytometric analysis of annexin V and propidium iodide (PI) double staining in HT-pretreated HaCaT cells for 18 h after H₂O₂ exposure for 6 h. Bars indicate the total percentage of early (annexin V-positive cells/PI-negative cells) and late apoptotic events (annexin V/PI-double positive cells) as well as necrotic cells (annexin V-negative cells/PI-positive cells). The results are representative of three independent experiments and expressed as mean ± SD (ANOVA, * p < 0.05 and ** p < 0.01).

Figure 6

HT prevented the activation of cell death molecular pathways. (A) Western blot analysis of Phospho-ATM, Phospho-Chk1, Phospho-Chk2, and p53 (total and phosphorylated) in whole cell extracts from HaCaTs for 18 h in the presence of HT (1–10 µM) and H₂O₂ (200 µM) for 6 h. α-Tubulin was used as a control for protein loading. The panel shows a representative western blot of three different experiments performed with similar results. (B) Histograms represent mean (±SD) in densitometry units of scanned immunoblots of three different experiments (2-way ANOVA, * p < 0.05, ** p < 0.01, and *** p < 0.001).