Cannabinoid inhibition of melanin formation in human melanocytes

Rasha Abdel-Aziz1,2, Tag El din Anbar1, Hamza Abdel-Raouf1, Steve Alexander2, Michael Garle2, David Kendall2. 1Dermatology Department, Al-Minya University, Al-Minya, Egypt, 2School of Biomedical Sciences and Institute of Neuroscience, University of Nottingham, Nottingham, NG7 2UH, United Kingdom.

Melanin is synthesized in specialized skin cells named melanocytes; it is not only responsible for skin colour but also plays a crucial role in protecting keratinocytes from the mutagenic effects of ultraviolet radiation. The factors controlling pigment formation are imperfectly understood and there is a need to develop improved therapies for hyper- and hypo-pigmentation diseases such as vitiligo.

The aim of the present study was to examine the effects of cannabinoids on melanin production in cultured human melanocytes and to determine the involvement of cyclic AMP (cAMP) signalling in any cannabinoid effect.

The non-tumorigenic human melanocytic cell line Hermes-2B, immortalized by telomerase reverse transcriptase expression (Sviderskaya, et al. 2002) were grown to approximately 80% confluence, serum-starved for 24 hours and then exposed to histamine (10uM), a known activator of melanin synthesis, in the absence and presence of the cannabinoid CB1/2 receptor agonist HU210 (1uM) and the CB1 receptor antagonist AM251 (1uM) for 5 days, with two changes of media during the stimulation period. On the day of assay, the cells were solubilised with 0.2 M NaOH and the concentration of melanin measured by monitoring its absorbance at a wavelength of 475nm with reference to a synthetic melanin calibration curve (10-100ug/ml).

Histamine produced a significant increase in melanin content from 53.0 ± 1.5µg/106 cells, n = 3) in control cells to 67.3 ± 0.9µg/106 cells, n = 3). HU210 alone induced a significant decrease in melanin content (29.7 ± 2.0µg/106 cells, n = 3) when compared with the basal level (P<0.001, one way ANOVA) and pre-simulation with HU210 significantly decreased the stimulatory effect of histamine on melanin synthesis (36.3 ± 1.9µg/106 cells, n = 3) (P<0.001). The CB1 antagonist AM251 failed significantly to reverse the effect of HU210.

Since cAMP is known to be involved in melanin synthesis, the acute effects of the drugs on cAMP formation in the Hermes-2B cells were assessed using a [3H]-adenine pre-labelling assay (Alvarez and Daniels, 1992). Histamine (10uM, 15 mins) increased the accumulation of [3H]-cAMP (expressed as % conversion from [3H]-adenine) from 1.9 ± 1.3 %to 8.7 ± 1.3% (n = 3) and this was inhibited by cimetidine (10uM) (5.9 ± 1.9%, n = 3; P<0.01) indicating mediation by H2 receptors. However, HU210 alone had no significant effect on accumulation of [3H]-cAMP (1.61 ± 0.3%, n = 3) when compared with basal and failed to affect [3H]-cAMP accumulation in response to histamine (7.6 ± 5.2%, n = 3) (P>0.05). Immunohistochemical analysis indicated that the cells do not express CB2 receptors.

In conclusion, the cannabinoid receptor agonist HU210 inhibits melanin formation in human melanocytes, although the receptor mediating the effect and its mechanism of action are uncertain at the present time. The data do, however, support the proposal that cannabinoid drugs might be of value in treating hyper-pigmentation disorders.

Sviderskaya E. V, Hill S. P, Evans-Whipp T. J, Chin L et al (2002). P16Ink4a in melanocyte senescence and differentiation, J Natl Cancer Inst 94, 446–54.

Alvarez R. and Daniels D.V (1992): A separation method for the assay of adenylyl cyclase using tritium-labelled substrate. Anal. Biochem, 203, 76-82.

This work was sponsored by the Egyptian Government.