Ann Dermatol.  2014 Apr;26(2):209-213. 10.5021/ad.2014.26.2.209.

Effect of Adenosine on Melanogenesis in B16 Cells and Zebrafish

• Kim MY
• Lee HE
• Im M
• Lee Y
• Kim CD
• Lee JH
• Seo YJ

• KMID: 2171657
• DOI: http://doi.org/10.5021/ad.2014.26.2.209

Abstract

BACKGROUND
Adenosine is a nucleoside, in which an adenine molecule is attached to a ribofuranose sugar moiety. It can be released into the microenvironment by metabolically active cells, and then fulfills a multitude of functions in regulation of cell proliferation, by activating four subtypes of G protein-coupled adenosine receptors.
OBJECTIVE
In this study, we investigated the effect of adenosine on melanogenesis, using B16 melanoma cells.
METHODS
The toxic effects of adenosine on B16 melanoma cells were assessed. To understand the mechanism of the effect of adenosine on melanogenesis in B16 cells, melanin content and tyrosinase activity were measured. Tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase were monitored by Western blotting. Finally, adenosine was applied to zebrafish embryos, and its in vivo effect on pigmentation investigated.
RESULTS
At a low concentration, adenosine increased melanin content and tyrosinase activity, while a high dose of adenosine resulted in inhibition of tyrosinase activity. Western blotting showed that adenosine increased tyrosinase protein levels slightly, while high-dose adenosine decreased the expression of tyrosinase. In zebrafish tests, adenosine slightly inhibited body pigmentation.
CONCLUSION
In this study, we investigated the effect of adenosine on melanogenesis, using the well-established B16 melanoma cell and zebrafish models. The results suggest that adenosine may inhibit pigmentation, through negative regulation of tyrosinase.

Keyword

Adenosine; Melanocytes; Melanogenesis; Pigmentation; Zebrafish

MeSH Terms

Adenine
Adenosine*
Blotting, Western
Cell Proliferation
Embryonic Structures
Melanins
Melanocytes
Melanoma, Experimental
Monophenol Monooxygenase
Pigmentation
Receptors, Purinergic P1
Zebrafish*
Adenine
Adenosine
Melanins
Monophenol Monooxygenase
Receptors, Purinergic P1

Figure

• 

  Fig. 1 Cell viability test. B16 melanoma cells were treated with adenosine at the indicated concentrations for 24 hours. Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results are shown as percentages of control±standard deviation. All experiments were repeated three times.

• 

  Fig. 2 Effect of adenosine on pigmentation in B16 melanoma cells. (A) Pellet color after cell harvesting. After treatment with adenosine, cells were harvested by centrifugation. (B) Melanin content. After treatment with adenosine, cells were dissolved in lysis buffer. After centrifugation, melanin pigments were resuspended in 1 N NaOH, and quantified, using a spectrometer. (C) For measurement of tyrosinase activity, 250 µg of total protein was incubated with L-DOPA, and then quantified, using a spectrometer. The results are shown as percentages of the control.

• 

  Fig. 3 Western blotting of pigmentation-related factors. Total protein (30 µg) was separated by sodium dodecysulfate-polyacrylamide gel electrophoresis, and then incubated with the appropriate antibodies. TYR: tyrosinase, TRP1: tyrosinase-related protein-1, DCT: dopachrome tautomerase.

• 

  Fig. 4 Effect of adenosine on zebrafish pigmentation. (A) Time schedule for zebrafish tests. (B) Synchronized embryos were treated with adenosine at the indicated concentrations. The effects on zebrafish pigmentation were observed under a stereomicroscope. PTU: 1-phenyl-2-thiourea.

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