Nutr Res Pract.  2017 Aug;11(4):275-280. 10.4162/nrp.2017.11.4.275.

Astaxanthin induces migration in human skin keratinocytes via Rac1 activation and RhoA inhibition

Affiliations
  • 1Department of Pharmacology, Faculty of Science, Prince of Songkla University, 15 Hat Yai, Songkhla 90110, Thailand. wanida.su@psu.ac.th
  • 2Department of Anatomy, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand.
  • 3Department of Fishery Product, Faculty of Fishery, Kasetsart University, Bangkok 10900, Thailand.
  • 4Department of Physiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand.
  • 5Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand.

Abstract

BACKGROUND/OBJECTIVES
Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. MATERIAL/METHOD: HaCaT keratinocyte cells were exposed to 0.25-1 µg/mL of ASX. Proliferation of keratinocytes was analyzed by using MTT assays and flow cytometry. Keratinocyte migration was determined by using a scratch wound-healing assay. A mechanism for regulation of migration was explored via immunocytochemistry and western blot analysis.
RESULTS
Our results suggest that ASX produces no significant toxicity in human keratinocyte cells. Cell-cycle analysis on ASX-treated keratinocytes demonstrated a significant increase in keratinocyte cell proliferation at the S phase. In addition, ASX increased keratinocyte motility across the wound space in a time-dependent manner. The mechanism by which ASX increased keratinocyte migration was associated with induction of filopodia and formation of lamellipodia, as well as with increased Cdc42 and Rac1 activation and decreased RhoA activation.
CONCLUSIONS
ASX stimulates the migration of keratinocytes through Cdc42, Rac1 activation and RhoA inhibition. ASX has a positive role in the re-epithelialization of wounds. Our results may encourage further in vivo and clinical study into the development of ASX as a potential agent for wound repair.

Keyword

Carotenoids; cell movement; wound healing; re-epithelialization

MeSH Terms

Blotting, Western
Carotenoids
Cell Movement
Cell Proliferation
Clinical Study
Flow Cytometry
Humans*
Immunohistochemistry
Keratinocytes*
Pseudopodia
Re-Epithelialization
S Phase
Salmon
Skin*
Wound Healing
Wounds and Injuries
Carotenoids

Figure

  • Fig. 1 Cytotoxicity effect of ASX on human skin keratinocyte cells.Cells were exposed to 10-1,000 µg/mL of ASX for 24, 48, and 72 h. Cell viability was determined by MTT assay. The results were presented as a percentage of the control mean. Data are expressed as mean ± SEM values (n = 4). #P < 0.01, *P < 0.001 versus an untreated control.

  • Fig. 2 Proliferative effects of ASX on human skin keratinocyte cells.(A) HaCaT cells were treated with various concentrations (0.125-10 µg/mL) of ASX for 24, 48, and 72 h. Cell proliferation was determined by MTT assay. Cells were incubated with 0.25-1 µg/mL of ASX for 72 h. (B) Percentage values of cell-cycle cell-phase distribution. (C) Distribution of cells in each phase as detected by flow cytometry and PI. Data are expressed as mean ± SEM values (n = 4). †P < 0.05, *P < 0.001 compared to a control group.

  • Fig. 3 Effect of ASX on the migration of skin keratinocyte cells.HaCaT cells were treated with various concentrations (0, 0.25, 0.5 or 1 µg/mL) of ASX for 0, 9, 24, 48, and 72 h. (A) Cell migration was visualized via phase-contrast microscopy. (B) The percentage wound closure area was measured by comparing the change in wound area to that of the control. Data are expressed as mean ± SEM values of four independent experiments. Difference among groups was evaluated by one-way ANOVA. *P < 0.001 compared to time 0, **P < 0.001 compared to a control.

  • Fig. 4 The formation of filopodia and lamellipodia, and the relative blot density of Cdc42, Rac1, and RhoA in response to ASX treatment.(A) HaCaT cells were stained with phalloidin-rhodamine. (B) ASX-treated cells were subjected to western blotting after 9 h of ASX exposure, and expression levels of Cdc42, Rac1 and RhoA were assessed. (C) Relative levels of Cdc42, Rac1 and RhoA in HaCaT cells after treatment with ASX (0.25-1 µg/mL) for 9 h. Densitometric analysis results for the indicated proteins are presented as mean ± SEM values of three independent experiments, #P < 0.01, *P < 0.001 compared to a control.


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