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(The FASEB Journal. 2006;20:1561-1563.)
© 2006 FASEB

Melanoma prevention strategy based on using tetrapeptide {alpha}-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity

Zalfa A. Abdel-Malek*,1, Ana Luisa Kadekaro*, Renny J. Kavanagh*, Aleksandar Todorovic{dagger}, Leonid N. Koikov{ddagger}, Joseph C. McNulty§, Pilgrim J. Jackson§, Glenn L. Millhauser§, Sandy Schwemberger||, George Babcock||, Carrie Haskell-Luevano{dagger} and James J. Knittel{ddagger}

* Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA;

{dagger} Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA;

{ddagger} College of Pharmacy, University of Cincinnati, Cincinnati, Ohio, USA;

§ Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA; and

|| Department of Surgery, University of Cincinnati College of Medicine and Shriners’ Burns Hospital, Cincinnati, Ohio, USA

1Correspondence: Department of Dermatology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati 45267-0592, OH, USA. E-mail: abdelmza{at}email.uc.edu

ABSTRACT

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of {alpha}-melanocyte stimulating hormone ({alpha}-MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with {alpha}-MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide {alpha}-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or {alpha}-MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss-of-function MC1R. Analogs, such as 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.—Abdel-Malek, Z. A., Kadekaro, A. L., Kavanagh, R. J., Todorovic, A., Koikov, L. N., McNulty, J. C., Jackson, P. J., Millhauser, G. L., Schwemberger, S., Babcock, G., Haskell-Luevano, C., Knittel, J. J. Melanoma prevention strategy based on using tetrapeptide {alpha}-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity.


Key Words: melanocortins • photoprotection • melanogenesis • DNA repair • melanomagenesis

MELANOMA IS THE DEADLIEST FORM of skin cancer and one of the most challenging human cancers. The incidence of melanoma has reached epidemic levels, and continues to increase at the rate of 4% annually (American Cancer Society, 2005 Facts and Figures (1) ). Melanoma is the fifth and sixth most common cancer among men and women, respectively, and is the most prevalent type of cancer in individuals in their second decade of life (2) . The resistance of advanced melanoma tumors to chemotherapy and their high metastatic potential have underscored the importance of melanoma prevention.

Acute sun exposure has been implicated in melanomagenesis (3) . Exposure of cultured human melanocytes, the precursors of melanoma, to UVR results in dose-dependent induction of DNA photoproducts (4) . Removal of DNA photoproducts by the nucleotide excision repair (NER) pathway is critical for prevention of the malignant transformation of melanocytes to melanoma. Patients with xeroderma pigmentosum, characterized by mutations in the genes for the enzymes involved in the NER pathway, have 1000-fold higher risk for melanoma and nonmelanoma skin cancer than the general population (5) . Melanoma patients seemed to have reduced NER capacity compared to normal control group (6) . Also, mutations in the melanoma susceptibility gene p16 or ARF were found to reduce NER capacity (7) . In addition to induction of DNA photoproducts, exposure of cultured human melanocytes to UVR results in the generation of reactive oxygen species (ROS), such as hydrogen peroxide, which can damage proteins, lipids, and DNA (8) . The genotoxic effects of UVR on human melanocytes are expected to destabilize their genome, increase mutation rate, and ultimately result in melanoma.

Exposure to UVR activates a paracrine/autocrine network in human skin (reviewed in (9) . Among the paracrine factors for melanocytes are the melanocortins {alpha}-MSH and ACTH, both of which are derived from the same precursor peptide, proopiomelanocortin (POMC) (10) . We were among the first to demonstrate that treatment of cultured human melanocytes with {alpha}-MSH or ACTH resulted in stimulation of melanogenesis and proliferation (11) . Others have shown that, as in other mammals, {alpha}-MSH stimulated eumelanin synthesis by human melanocytes (12) . The effects of melanocortins on human melanocytes are mediated by binding and activating the MC1R, a Gs protein-coupled receptor with seven transmembrane domains (13) . The significance of POMC and MC1R genes in the regulation of human pigmentation is supported by the findings that mutations in these genes are associated with red hair/fair skin phenotype, characterized by reduced eumelanin synthesis in follicular and epidermal melanocytes (14) .

Melanocortins play an important role in the response of human melanocytes to UVR. We reported that {alpha}-MSH, via activation of the cAMP pathway, enables cultured human melanocytes to overcome the UVR-induced G1 arrest and increase melanin sythesis (15) . In fact, we found that in the absence of {alpha}-MSH or any factor that increases cAMP levels, normal human melanocytes fail to respond to UVR with increased melanogenesis, i.e., tanning, which is considered photoprotective against subsequent UVR exposure.

Recently, we discovered a novel role for {alpha}-MSH and ACTH as survival factors that rescue human melanocytes from UVR-induced apoptosis, enhance the repair of DNA photoproducts, and reduce the release of hydrogen peroxide (8) . Cells that undergo apoptosis are usually those with irreparable DNA damage. Therefore, we expect that the survival effect of melanocortins is due to restoration of genomic stability of melanocytes, by diminishing the burden of DNA photoproducts and oxidative DNA damage. These effects of melanocortins precede the increase in melanogenesis. Collectively, the effects of melanocortins are expected to reduce the chance for mutations in human melanocytes and thus prevent the malignant transformation of melanocytes to melanoma.

Based on these novel findings, we have designed a potentially effective strategy for melanoma prevention based on the use of synthetic MC1R agonists. We hereby report the biological effects of three fragment analogs of {alpha}-MSH: 1, the tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 and the N-capped tetrapeptides 2, n-Pentadecanoyl-His-D-Phe-Arg-Trp-NH2, and 3, 4-phenylbutyryl-His-D-Phe-Arg-Trp-NH2 on cultured human melanocytes. Earlier pharmacological studies demonstrated that these analogs are activators of melanocortin receptors, particularly the MC1R (16 , 17) . In the present study, analogs 2 and 3 proved to be potent MC1R agonists that were more effective than {alpha}-MSH in stimulating melanogenesis and promoting human melanocyte survival. Additionally, 2 and 3 mimicked {alpha}-MSH in reducing DNA damage by enhancing the repair of DNA photoproducts and diminishing the generation of hydrogen peroxide in UVR-irradiated human melanocytes. We attribute increased survival of UVR-irradiated melanocytes treated with these analogs to reduction of DNA damage. These results suggest that further modification of these two analogs, particularly 3, which has reversible effects, can potentially lead to the development of a topical agent that reduces the burden of UVR-induced DNA damage and thus prevents melanoma as well as nonmelanoma sun-induced skin cancers.

MATERIALS AND METHODS

Primary human melanocyte cultures were derived from neonatal foreskins and used for experiments before reaching passage 10, as described (11) . In some experiments, we used neonatal melanocyte cultures that naturally express loss-of-function MC1R alleles, as determined by sequencing the MC1R gene, and lack of responsiveness of melanocytes to melanocortins by increasing cAMP formation and tyrosinase activity, as described (18) . Each culture was derived from a single foreskin and was representative of a single donor. For all experiments, melanocytes were deprived of bovine pituitary extract (BPE), known to contain high concentrations of melanocortins (13) , for 24–48 h prior to, and for the entire duration of each experiment. Removing BPE from the culture medium is critical for the response of melanocytes to exogenous melanocortins.

To test the effects of melanocortin analogs on tyrosinase activity and proliferation, human melanocytes were plated into 60 mm dishes at a density of 0.25 x 106 melanocytes/dish (triplicate dishes/group), and 48 h thereafter treated with increasing concentrations of {alpha}-MSH or melanocortin analogs (1 pM–100 nM) for a total of 6 d, as described (11 , 19) . On the fifth day, 3H-tyrosine (2.1 µCi; Perkin Elmer, Boston, MA) was added to each dish, and 24 h thereafter tyrosinase activity was assayed as described (11 , 19) . Tyrosinase activity was determined using the in situ tyrosine hydroxylase activity, based on measuring the 3H-labeled water that is released to the media as melanocytes metabolize 3H-tyrosine to DOPA during the first reaction catalyzed by tyrosinase in the melanin synthetic pathway. Cell number in each dish was counted using a Coulter Particle Counter (Model Z 1 Hialeah, FL).

For measuring the residual effects of melanocortin analogs, melanocytes were plated as described above, and treated for 3 d with 1 nM of {alpha}-MSH, 2, 3, or Ac-[Nle4, D-Phe7]-{alpha}-MSH (NDP-MSH), the known potent tridecapeptide analog (20) . The culture dishes were then washed twice with sterile PBS and maintained in growth medium without any melanocortin peptide for 2 or 4 d. Tyrosinase activity was determined 3 d following treatment with, or 2 and 4 d after removal of, the melanocortin peptides, as described above.

To demonstrate that the effects of 2 and 3 on human melanocytes are mediated by binding to the MC1R, we tested the ability of ASIP-YY to counteract the stimulatory effects of these analogs. ASIP-YY is a synthetic analog with improved folding properties of agouti signaling protein (ASIP (21) , the physiological MC1R antagonist (22) . Melanocytes were plated and treated with 0, or 1 nM 2 or 3, in the absence or presence of 50 nM ASIP-YY for a total of 6 d, and tyrosinase activity was then determined, as in the experiments described above.

The effects of 2 and 3 on apoptosis of UVB-irradiated melanocytes were determined by Annexin V staining. Melanocytes were plated and treated with 0; or 1 nM {alpha}-MSH, 2 or 3, or 1 µM forskolin for a total of 4 d, then irradiated with a single dose of 105 or 120 mJ/cm2 UVB emitted by FS 20 lamps with the UVC component filtered by Kodacel filter. This procedure was followed by incubation in medium lacking BPE and containing the appropriate melanocortin peptide for 24 h. Thereafter, attached as well as detached melanocytes were collected and stained with APC-Annexin V (BD Pharmingen, San Diego, CA) and propidium iodide following the manufacturer’s instructions. Melanocytes were then immediately analyzed by flow cytometry on BD LSR (BD Pharmingen), and the data were analyzed by CELLQuest software.

To evaluate the effects of melanocortin analogs on the release of hydrogen peroxide by UVB-irradiated human melanocytes, we plated melanocytes into 100 mM dishes at a density of 1 x 106 cells/dish and irradiated them 48 h thereafter with a single dose of 105 mJ/cm2 UVB from filtered FS 20 lamps. Immediately after irradiation, melanocytes were treated with 0, or 1 nM {alpha}-MSH, 1, 2, or 3. Triplicate dishes were included in each group. The release of hydrogen peroxide was measured immediately and 15, 45, and 60 min after irradiation, by determining the luminescence of luminol, as described (8) .

The effects of melanocortin peptides on the repair of cyclobutane pyrimidine dimers (CPD) in UVR-irradiated human melanocytes were assessed as follows. Melanocytes were plated at a density of 1 x 106 cells/100 mm dish, treated for 4 d with 0, 1 nM 3, then irradiated with 21 mJ/cm2 from unfiltered FS 20 lamps, as UVC rays emitted by the lamps contribute markedly to the induction of DNA photoproducts (unpublished results). DNA was extracted immediately (T0) to measure induction of CPD, or 12 h (T12) post-irradiation to measure their rate of repair, and Southwestern blot analysis was performed to detect CPD as described (8) . Triplicate lanes were included for each group, and the resulting bands were analyzed by densitometry using Alpha-Innotech Imaging System and the AlphaEase Stand Alone Software (San Leandro, CA).

RESULTS

Treatment of human melanocytes with increasing doses of {alpha}-MSH, 1, 2, or 3 resulted in dose-dependent stimulation of tyrosinase activity (Figs. 1 and 2 A. The EC50 value of 1 was equivalent to 10 nM (6.5–15 nM, 95% confidence interval), compared with EC50 value of 0.9 nM (0.43–1.8 nM, 95% confidence interval) for {alpha}-MSH, and maximal stimulation of tyrosinase activity was achieved upon treatment with 1 at a dose of 50 nM and with {alpha}-MSH at a dose of 5 nM (Fig. 2A ). Analogs 2 and 3 had similar EC50 values, equivalent to 0.16 (0.06–0.37 nM, 95% confidence interval) and 0.18 nM (0.06–0.5 nM, 95% confidence interval), respectively. Treatment with 2 or 3 resulted in stimulation of tyrosinase activity beginning at 1 pM, and maximal activity was achieved in response to 10 nM of 2 (420% of control) and 5 nM of 3 (470% of control) (Fig. 2A ). Direct comparison of the effects of 1 and 2 in one experiment (data not shown), and of 1 and 3 in another experiment on tyrosinase activity (Fig. 2A ) revealed that the maximal achievable tyrosinase activity was higher in melanocytes treated with 2 or 3 than in melanocytes treated with 1, further suggesting that the N-capping of this tetrapeptide with pentadecanoyl or 4-phenylbutyryl results in increased potency. In Fig. 2A , the difference in the maximal achievable stimulation of tyrosinase activity in response to {alpha}-MSH vs. 2 and 3 is due to the variation in the responses of different primary melanocyte cultures, representing donors’ variability. As shown previously for {alpha}-MSH, 1, 2, or 3 resulted in dose-dependent stimulation of melanocyte proliferation (Fig. 2B ) The mitogenic effect of {alpha}-MSH and 1 was first evident at a concentration of 5 nM, while that of 2 and 3 was first detected at 10 pM.


Figure 1
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  		Figure 1. Primary structures of the native hormone {alpha}-MSH, the tetrapeptide analog 1, and the N-terminally modified analogs 2 and 3.


Figure 2
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  		Figure 2. Dose-dependent effects of {alpha}-MSH, 1, 2, and 3 on tyrosinase activity and proliferation of cultured human melanocytes. Melanocytes were plated into 60 mm culture dishes and treated for a total of 6 d with increasing doses of melanocortin peptides (0 [control]–100 nM), as described in Materials and Methods. Tyrosinase activity (in A) and cell number (in B) were determined in triplicate dishes/group. SE for each data point was less than 10%. Each peptide was tested at least twice on different melanocyte cultures, with similar results.

We compared the residual effects of 2 and 3 on tyrosinase activity to that of the best-known superpotent full-length {alpha}-MSH analog NDP-MSH, and the physiological hormone {alpha}-MSH (Fig. 3 ). Melanocytes were treated for 3 d with 1 nM of the respective analogs or {alpha}-MSH and then maintained in medium lacking any melanocortin peptide for 2 or 4 d. Treatment with 2, 3, or NDP-MSH for 3 d resulted in a greater stimulatory effect on tyrosinase activity than treatment with {alpha}-MSH. Two days after removal of melanocortin peptides, the effect of {alpha}-MSH on tyrosinase activity was greatly reduced, yet remained statistically higher than control. In comparison, the effect of NDP-MSH was reduced markedly, yet remained higher than that of {alpha}-MSH, while the effect of 2 was not diminished and that of 3 was only slightly decreased. Four days after removal of melanocortin peptides, the stimulatory effect of {alpha}-MSH was no more evident; that of 2 remained the same as on day 2; while the effects of NDP-MSH and 3 decreased further, yet remained statistically higher than control. The results of this experiment indicate that the residual effect of 3 was comparable with that of NDP-MSH, while 2 had the most prolonged effect.


Figure 3
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  		Figure 3. Comparison of the residual effects of 2, 3, NDP-{alpha}-MSH, and {alpha}-MSH on tyrosinase activity of cultured human melanocytes. Human melanocytes were plated into 60 mm dish and treated for 3 d with 1 nM {alpha}-MSH, NDP-{alpha}-MSH, 2 or 3, as described in Materials and Methods. Tyrosinase activity was determined after 3 d of treatment with, and 2 and 4 d after removal of, melanocortin peptides. The data were analyzed statistically using Student’s t test. *Statistically different from its respective control; {Delta} = statistically different from NDP-{alpha}-MSH at the respective day after peptide removal at P ≤ 0.001.

The ability of 2 and 3 to promote the survival of human melanocytes following UVR exposure was compared with that of {alpha}-MSH, as determined by Annexin V staining of apoptotic melanocytes (Fig. 4 ). The results of many experiments using different melanocyte cultures showed that maintenance of melanocytes in medium lacking BPE resulted in an average of 20% apoptosis. Exposure of melanocytes to105 mJ/cm2 UVB in the absence of treatment with any melanocortin resulted in 30% increase in apoptosis above control. Treatment with {alpha}-MSH reduced the apoptotic response to UVR to 20%. Treatment with 2 or 3 inhibited the apoptotic response to UVR more markedly than {alpha}-MSH and reduced the UVR-induced increase in apoptosis to 5 and 11%, respectively. In other experiments, 2 and 3 reduced the cytotoxic effect of UVR on melanocytes, evidenced by decrease in the number of dead melanocytes that incorporated trypan blue dye and increase in the number of viable melanocytes that excluded the dye (data not shown), as demonstrated previously for {alpha}-MSH treatment (8) . These results demonstrate that 2 and 3 are more potent than {alpha}-MSH in promoting the survival of human melanocytes following exposure to UVR.


Figure 4
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  		Figure 4. Comparison of the antiapoptotic effects of analogs 2 and 3, and {alpha}-MSH on human melanocytes. Human melanocytes were plated and treated with 0, 1nM {alpha}-MSH, 2 or 3 for 4 d, and were subsequently irradiated with 0, or 105 mJ/cm2 UVB, followed by treatment with the respective peptide for 24 h. Melanocytes were then harvested and duplicate samples/group were stained with APC-Annexin V and analyzed by flow cytometry, as described in Materials and Methods. Similar results were obtained in two independent experiments using two different melanocyte cultures.

To determine that the effects of 2 and 3 on human melanocytes are mediated by activating the MC1R, we tested whether or not their stimulation of tyrosinase activity could be blocked by ASIP-YY (Fig. 5 ). It was observed that the concomitant treatment of human melanocytes with either melanocortin analog or ASIP-YY resulted in total abrogation of the stimulatory effect of 3, and partial inhibition of the effect of 2 on tyrosinase activity. As expected, concomitant treatment with {alpha}-MSH and ASIP-YY completely abolished the {alpha}-MSH-induced increase in tyrosinase activity, as previously shown (23) .


Figure 5
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  		Figure 5. Abrogation of the effects of agonists 2 and 3 on tyrosinase activity by ASIP-YY. Human melanocytes were plated and treated with 0, 1 nM {alpha}-MSH, 2 or 3, with or without concomitant treatment with 50 nM ASIP-YY for 6 d. Tyrosinase activity was then determined as described in Materials and Methods. *Statistically different from untreated control; {Delta} = statistically different from {alpha}-MSH or the respective agonist alone at P ≤ 0.001, as determined by ANOVA followed by Newman-Kewls Multiple Comparison Test.

To further prove that 2 and 3 bind the MC1R, their effects on tyrosinase activity and UVB-induced apoptosis of human melanocytes expressing loss-of-function MC1R were tested (Figs. 6 and 7 ). Both analogs, as well as {alpha}-MSH, failed to stimulate tyrosinase activity or rescue these melanocytes from UVB-induced apoptosis, suggesting that similar to {alpha}-MSH, the action of 2 and 3 on human melanocytes requires functional MC1R. In these experiments, forskolin, a direct activator of adenylate cyclase, increased tyrosinase activity and remarkably abrogated the UVB-induced apoptosis. The results of forskolin treatment clearly indicated that inability of these melanocytes to respond to melanocortins is due to a defect in the MC1R and not in its downstream signaling pathway.


Figure 6
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  		Figure 6. Lack of stimulation of tyrosinase activity in melanocytes expressing loss-of-function MC1R by 2 or 3. Human melanocytes that express loss-of-function MC1R were plated and treated with 0, 1 nM {alpha}-MSH, 2, or 3 or with µM forskolin for 6 d and tyrosinase activity was determined as described in Materials and Methods. Similar results were obtained in three experiments using three different cultures with loss-of-function MC1R.


Figure 7
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  		Figure 7. The antiapoptotic effect of {alpha}-MSH analogs requires functional MC1R. Human melanocytes expressing loss-of-function MC1R were treated with 0, 1nM {alpha}-MSH, 2, or 3, or 1 µM forskolin, irradiated with 0, 105, or 120 mJ/cm2 UVB, and stained with APC-Annexin V 24 h thereafter, as described in Materials and Methods and in the legend for Fig. 4 .

The effects of 1, 2, and 3 on the release of hydrogen peroxide by UVB-irradiated melanocytes were investigated by measuring the hydrogen peroxide-induced luminescence of luminol (Fig. 8 ). Irradiation of melanocytes with UVB resulted in an instant increase in hydrogen peroxide release, which reached a maximum concentration by 15 min, declined thereafter, yet remained higher than control concentration 60 min after irradiation. Treatment with 1 nM {alpha}-MSH, 1, 2, or 3 markedly reduced the maximal concentration of hydrogen peroxide released 15 min after irradiation. At all the time points investigated, 1, 2, and 3 were effective in abrogating the increased release of hydrogen peroxide in response to UVB exposure.


Figure 8
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  		Figure 8. Reduction of UVB-induced release of hydrogen peroxide from human melanocytes by {alpha}-MSH and melanocortin analogs. Human melanocytes were plated onto 100 mm dishes, irradiated with 105 mJ/cm2 UVB and immediately treated with 0, 1 nM {alpha}-MSH, 1, 2, or 3. Hydrogen peroxide release was measured at 0, 15, 45, and 60 min after irradiation, as described in Materials and Methods. Each data point is representative of triplicate determinations ± SE. The data were analyzed using Student’s t test. {Delta} = Statistically different from the respective UVB-irradiated group at P ≤ 0.001. This experiment was repeated three times with similar findings.

We recently reported that {alpha}-MSH enhances the repair of UVR-induced CPD (8) . As 2 had "irreversible" effects on melanocytes, as shown in Fig. 3 , we selected 3 for investigating further whether or not it would mimic {alpha}-MSH in enhancing NER (Fig. 9 ). Using Southwestern blot analysis, we observed that 3 markedly enhanced the rate of removal of CPD 12 h after irradiation of melanocytes with UVR. Interestingly, 3 also reduced the induction of CPD immediately after UVR exposure. In other experiments, we found that both 1 and 2 had similar but less dramatic effects than 3 on CPD repair (data not shown).


Figure 9
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  		Figure 9. Reduction of CPD in UVR-irradiated human melanocytes treated with 3.

Melanocytes were plated onto 100 mm dishes, treated with 0 or 1 nM 3 for 4 d, followed by irradiation with 21 mJ/cm2 UVR. DNA was extracted immediately (T0) to measure induction of CPD, or 12 h after UVR exposure (T12) to determine CPD repair, using Southwestern blot analysis. Triplicate lanes were loaded for each group, and the mean ± SE of three different densitometry measurements are presented in the graph. The effect of 3 at T0 and T12 on the UVR irradiated group was statistically different from the effect of UVR alone at P < 0.05, as determined by ANOVA, followed by Newman-Kewls Multiple Comparison Test. This experiment was repeated three times using two different melanocyte cultures with similar findings.

DISCUSSION

The melanocortins {alpha}-MSH and ACTH are important paracrine/autocrine regulators of cutaneous pigmentation (24 , 25) . The two hormones are also important participants in the response of melanocytes to UVR, and via activation of the cAMP pathway, rescue human melanocytes from UVR-induced growth arrest and stimulate melanogenesis (15) . Moreover, we recently reported that melanocortins reduce the UVR-induced damage by decreasing oxidative stress, evidenced by diminished release of hydrogen peroxide, and enhancing the repair of DNA photoproducts, consequently counteracting the apoptotic effect of UVR on human melanocytes (8) . These effects of melanocortins suggest that they potentially protect melanocytes from UVR-induced carcinogenesis and malignant transformation to melanoma, the most fatal form of skin cancer. The importance of melanocortins and the MC1R in melanoma prevention is underscored by the compelling evidence that loss-of-function mutations in the MC1R are associated with increased risk for melanoma (26) .

For many years, there has been a wide interest in developing melanocortin analogs that can be used for "sunless tanning", to avoid the deleterious effects of UVR. Although analogs of {alpha}-MSH with potent pigmentary effects have been developed (27 , 28) , yet they were not efficacious as tanning agents due to their inability to be delivered topically due to their size, and their nonselectivity for the MC1R. In this study, we have tested on cultured human melanocytes the effects of 2 N-capped tetrapeptide analogs of {alpha}-MSH (2 and 3) and compared them to those of the tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 (1) and the native hormone {alpha}-MSH. The amino acid sequence His-Phe-Arg-Trp is conserved in the structure of all melanocortins ({alpha}-, ß-, and {gamma}-MSH and ACTH) and is considered the active site and minimal sequence for the melanogenic effect (10 , 29 , 30) . Substitution of the L-Phe (7) by D-Phe in the structure of {alpha}-MSH resulted in a more potent and stable peptide (31) . We suggest that this substitution accounts for the potency of the tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2. Our results show that N-capping of 1 with pentadecanoyl or 4-phenylbutyryl results in superpotent analogs of {alpha}-MSH, 2, and 3, respectively, with EC50 values that are ~5-fold lower than that of {alpha}-MSH, as determined using the tyrosinase activity assay. Further comparison of 3 and 1 showed that maximal tyrosinase activity was achievable after treatment of melanocytes with 5 nM 3, compared with 50 nM 1. All three analogs stimulated melanocyte proliferation, however, using 3 at a concentration of 1 pM resulted in a two-fold increase in tyrosinase activity above control and only a trivial (13%) increase in melanocyte number.

A melanocortin analog intended for topical use is expected to mimic the effects of {alpha}-MSH on human melanocytes, have potent and reversible effects, and to be selective for the MC1R. These requirements should be fulfilled in order to avoid other nonpigmentary effects of melanocortins that result from activating the four other melanocortin receptors (MC2-MC5 R; 32 ), as observed in subjects injected with NDP-MSH (27) . It was reported that increasing the number of N-terminal capping group methylene units results in increased selectivity for the MC1R vs. MC3R or MC4R (17) . This suggests that 2 might be more selective for MC1R than MC3R or MC4R. Also, in pharmacological studies on heterologous cells transfected with the various MCR genes, 3 proved to be considerably more selective for the human MC1R than for either MC3R or MC4R (21) . Introduction of fatty acid residues into peptides is expected to contribute to their lipophilicity, and increase their stability and resistance to biodegradation by proteolytic enzymes (33 , 34) . Acylated peptides might have increased receptor selectivity due to modification of physiological transport properties (35 , 36) . These known effects of fatty acid residues on ligand-receptor interaction might explain the higher potency of 2 and 3, compared to 1 or the physiological ligand {alpha}-MSH.

Analogs 2 and 3 had remarkable residual effects, which was comparable with, or even surpassed, that of the best-known potent melanocortin analog NDP-{alpha}-MSH. The stimulatory effect of 2 on tyrosinase activity persisted and did not decrease even 4 d after removal of the peptide (Fig. 3) . However, the effect of 3, similar to that of NDP-MSH, diminished gradually after cessation of treatment, suggesting that this effect is reversible. Additionally, analogs 2 and 3 had greater antiapoptotic effects than {alpha}-MSH, and mimicked its effects on reducing the release of hydrogen peroxide and enhancing the repair of DNA photoproducts in UVR-irradiated human melanocytes (Figs. 4 , 8 , and 9) . We propose that analogs 2 and 3 restore genomic stability of human melanocytes after irradiation with UVR, and as a consequence, reduce UVR-induced apoptosis. Collectively, these results suggest that the fragment analogs of {alpha}-MSH, 2 and 3, can potentially protect human melanocytes from the damaging and mutagenic effects of UVR by reducing the burden of UVR-induced damage and restoring genomic stability. These fragment analogs of {alpha}-MSH can potentially protect human melanocytes from the damaging and mutagenic effects of UVR by reducing the burden of UVR-induced damage and restoring genomic stability, in addition to increasing melanogenesis that confers photoprotection.

Generation of ROS leads to imbalance of the redox state of cells and causes damage to lipids, proteins, and DNA (37) . We have observed that irradiation of human melanocytes with UVR results in dose-dependent generation of 8-hydroxydeoxyguanosine, a major form of oxidative DNA damage, and in increased expression of hemeoxygenase-1 (data not shown). Oxidative DNA damage is known to lead to mutations and carcinogenesis, and we expect this form of DNA damage to contribute to melanomagenesis.

The association of DNA photoproducts with increased skin cancer and melanoma is exemplified in xeroderma pigmentosum patients, who have reduced NER capacity (5) . Although "UV-signature" mutations, due to unrepaired DNA photoproducts, are rarely observed in melanoma, there is increasing evidence for the role of photoproducts in melanoma. First, melanoma patients seem to have reduced NER capacity, compared with normal control subjects (6) . Second, mutations in the melanoma-associated genes p16 and Arf result in reduced NER capacity (7) . Exposure of cultured human melanocytes to UVR resulted in the formation of pyrimidine, 6,4-pyrimidone photoproducts, which are mainly repaired by transcription coupled repair, in addition to CPD that are mainly repaired by global genomic repair (38) . However, the concentration of the former photoproducts was less, and their repair rate was faster than the latter. The persistence of CPD, relative to 6,4-photoproducts, led us to investigate their rate of repair in the presence or absence of {alpha}-MSH analogs.

Our results suggest that 2 and 3 are potent MC1R agonists that elicit their effects on human melanocytes by specifically binding and activating the MC1R. Importantly, their effects, particularly that of 3, on tyrosinase activity could be blocked by ASIP-YY, (Fig. 5) . Previously, we reported that ASIP completely abrogated the stimulatory effects of {alpha}-MSH on tyrosinase activity and proliferation of human melanocytes, and competed with {alpha}-MSH for binding to the MC1R (23) . Here, we showed that concomitant treatment with ASIP-YY totally abrogated the effect of 3, but only partially inhibited the effect of 2 on tyrosinase activity (Fig. 5) . These results might be attributed to the differences in the lipophilic properties of 2 and 3, which might affect their displacement from the MC1R by ASIP-YY. Additionally, neither 2 nor 3 had any effect on tyrosinase activity or survival of human melanocytes that express loss-of-function MC1R (Figs. 6 and 7) . Collectively, these findings indicate that 2 and 3 are MC1R agonists and require functional MC1R to elicit their effects on human melanocytes.

Expression of functional MC1R is important for prevention of photocarcinogenesis, particularly melanoma (26 , 39) . The steady increase in the incidence of melanoma, particularly in young adults, and the challenge of treating advanced melanoma, have led to increased focus on melanoma prevention (2) . We propose that potent fragment analogs of {alpha}-MSH, such as 3, with reversible effects on human melanocytes, can be further developed as topical agents to protect the skin from photocarcinogenesis, not only by inducing a tanning response, but importantly by limiting the extent of UVR-induced DNA damage, thus reducing the chance for mutagenesis and malignant transformation to melanoma. This potential preventive strategy should benefit most individuals with a high risk for melanoma, particularly those heterozygous for melanoma-associated MC1R alleles, or expressing mutations in other melanoma susceptibility genes, such as p16 or BRAF (26 , 40) .

ACKNOWLEDGMENTS

This work was partially supported by the National Institutes of Health grants (R01 ES-09110, Z.A.M.), Dean’s Discovery Fund, University of Cincinnati (Z.A.M.), Johnson and Johnson Skin Research Center Training Grant (Z.A.M. and A.L.K.), NIH (DK 064265, G.L.M.), NIH (DK 57080, C.H.L.), American Heart Association Predoctoral Fellowship (A.T.) and Skin Cancer Foundation (J.J.K.). We thank Dr. Toshio Moro, Ph.D. (Radioisotope Research Center, Nara Medical University) for his generous donation of the TDM2 antibody against cyclobutane pyrimidine dimers.

Received for publication December 30, 2005. Accepted for publication February 16, 2006.

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