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Melanoma prevention strategy based on using tetrapeptide -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, Leonid N. Koikov, Joseph C. McNulty, Pilgrim J. Jackson, Glenn L. Millhauser, Sandy Schwemberger^||, George Babcock^||, Carrie Haskell-Luevano and James J. Knittel

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

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

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

SPECIFIC AIMS

The goal of this study is to demonstrate the potential efficacy of -melanocortin (-MSH) tetrapeptide analogs for prevention of sun-induced skin cancer, particularly melanoma. The specific aims are: 1) To compare the potencies of three tetrapeptide analogs of -MSH, namely 1 Ac-His-D-Phe-Arg-Trp-NH₂ and 2, and 3, respectively, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH₂, to that of -MSH in stimulating the activity of tyrosinase, the rate-limiting enzyme in the melanogenic pathway, in cultured normal human melanocytes; 2) To compare the residual effects of 2 and 3 to those of -MSH and the well-known potent melanocortin analog Ac-[Nle⁴, D-Phe⁷]--MSH (NDP-MSH) on tyrosinase activity; 3) To investigate the effects of 2 and 3 on the survival of UVR-irradiatated human melanocytes; 4) To demonstrate that 2 and 3 act as MC1R agonists; and 5) To investigate whether 1, 2, and 3 reduce the burden of UVR-induced DNA damage by affecting the generation of hydrogen peroxide and the repair of DNA photoproducts in human melanocytes.

PRINCIPAL FINDINGS

1. Comparison of potencies
Comparison of the potencies of 1, 2, and 3 to that of -MSH in stimulating the activity of tyrosinase in cultured normal human melanocytes revealed that 1 was less potent than -MSH with EC₅₀ of 10 nM, compared with EC₅₀ value of 0.9 nM for -MSH. Analogs 2 and 3 were equally potent, yet more potent than -MSH, with EC₅₀ values of 0.16 and 0.18 nM, respectively.

2. Comparison of residual effects
Comparison of the residual effects of 2 and 3 to those of -MSH and the well-known potent melanocortins analog Ac-[Nle⁴, D-Phe⁷]--MSH (NDP-MSH) on tyrosinase activity revealed that 2 and 3 had markedly greater residual effects than -MSH on tyrosinase activity. The residual effect of 2 was the greatest, and surpassed that of the potent -MSH analog NDP-MSH.

3. Investigation of survival effects
Investigation of the survival effects of 2 and 3 on UVR-induced human melanocytes showed that 2 and 3 had greater antiapoptotic effects than -MSH on UVR-irradiated human melanocytes, as determined by Annexin V staining.

4. Activation of the MC1R mediated the effects of 2 and 3
The effects of 2 and 3 were mediated by activation of the MC1R, as their stimulation of tyrosinase activity was abrogated by ASIP YY, an analog of agouti signaling protein. Additionally, the stimulatory effects of these analogs on tyrosinase activity and survival were absent in human melanocytes that expressed loss-of-function MC1R.

5. Effects of 1, 2, and 3
We found that similar to -MSH, 1, 2, and 3 reduced the release of hydrogen peroxide from UVR-irradiated human melanocytes. As reported previously for -MSH, we found that the potent analog 3, with reversible effects on tyrosinase activity, enhanced the repair of cyclobutane pyrimidine dimers in UVR-irradiated human melanocytes. These effects are expected to reduce the genotoxic and cytotoxic effects of UVR, thus preserving the genomic stability and viability of melanocytes.

CONCLUSIONS AND SIGNIFICANCE

Melanoma is the deadliest from of skin cancer and one of the most challenging human cancers. The incidence of melanoma has reached epidemic levels, and continues to rise at an annual rate of 4%. 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. The resistance of advanced melanoma tumors to chemotherapy and their high metastatic potential have sparked interest in devising effective prevention strategies to curtail the increase in the incidence of the disease. This study is significant since it introduces a novel strategy based on the use of fragment analogs of -melanocortin (-MSH) that can potentially protect against sun-induced skin cancer, particularly melanoma, the most fatal.

We were among the first to demonstrate that the melanocortins -MSH and ACTH stimulate melanogenesis and proliferation of cultured normal human melanocytes. We also demonstrated that these melanocortins play a critical role in regulating the melanogenic response of human melanocytes to UVR. Others have shown that melanocortins stimulate the synthesis of eumelanin, the brown-black pigment known to confer photoprotection to the skin. Recently, we identified an important novel role for -MSH and ACTH as survival factors that rescue human melanocytes from the DNA damaging and cytotoxic effects of UVR by enhancing the repair of DNA photoproducts and reducing the generation of hydrogen peroxide and apoptosis. These findings are expected to maintain genomic stability and survival, and to prevent malignant transformation of melanocytes to melanoma. Based on these findings, we designed three tetrapeptide analogs of -MSH, namely 1 Ac-His-D-Phe-Arg-Trp-NH₂, and 2, and 3, respectively, n-capped peptides n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH₂, and tested their effects on cultured human melanocytes. Analog 1 represents a modification of the minimal effective sequence that is required for the pigmentary effect of melanocortins and is common for the melanocortins -, ßbeta;-, and -MSH and ACTH. N-capping of this peptide is expected to increase lipophilicity, stability, and resistance to degradation by proteolytic enzymes. All three peptides proved effective in stimulating the activity of tyrosinase, the rate-limiting enzyme in the melanin synthetic pathway, with 1 having at least five-fold higher EC₅₀ value, and 2 and 3 having a ten-fold lower EC₅₀ value than the physiological hormone -MSH. We demonstrated that 2 and 3 have greater residual effects and were more potent than -MSH in preventing apoptosis of UVR-irradiated human melanocytes. We attributed the survival effects of 2 and 3 to their ability to reduce the generation of hydrogen peroxide and enhance the repair of DNA photoproducts. We demonstrated that 2 and 3 are MC1R agonists, as their stimulatory effects on tyrosinase activity could be abrogated by an analog of ASIP, the physiological antagonist of the MC1R. Additionally, the effects of these two melanocortin analogs on tyrosinase activity and survival of normal human melanocytes were absent in melanocytes expressing loss-of-function MC1R. While 2 and 3 were equally potent, only 3 had reversible effects. A previous study showed greater selectivity of 3 for the MC1R than the remaining MC receptors. Given its potency and lipophilicity, selectivity for the MC1R, and reversibility of its effect on tyrosinase activity, we propose that 3 can be further developed into a topical agent that can be used for melanoma prevention. In addition to its melanogenic activity, this analog has the added benefit of reducing the burden of DNA damage resulting from exposure of melanocytes to UVR that might lead to melanoma formation. As the melanocyte plays an important photoprotective role in the skin, the use of such a melanocortin analog that increases melanogenesis might also prevent nonmelanoma skin cancer. We predict that this prevention strategy can be of particular benefit to individuals with known high risk for skin cancer in general, and should ultimately reduce the incidence of melanoma.

View larger version (10K): [in this window] [in a new window]   Figure 1. Dose-dependent effects of -MSH, 1, 2, and 3 on tyrosinase activity 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). Tyrosinase activity was 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.

View larger version (18K): [in this window] [in a new window]   Figure 2. 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.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic representation of the effects of -MSH analogs that act as MC1R agonists on human melanocytes. Recently, we reported that the melanocortins -MSH and ACTH modulate the responses of human melanocytes to UVR by enhancing the repair of DNA photoproducts, reducing the generation of hydrogen peroxide, and promoting survival, in addition to their known stimulatory effects on melanogenesis. We have designed, based on these findings, tetrapeptide analogs of -MSH and compared their effects to those of the native hormone -MSH. We found that these analogs are MC1R agonists, and that 2 and 3 are more potent with prolonged effect on melanogenesis, and greater antiapoptotic effect than -MSH. These two agonists also mimic the effects of -MSH on nucleotide excision repair and generation of hydrogen peroxide. We expect these agonists to restore the genomic stability of UVR-exposed human melanocytes and increase photoprotection of the skin, and thus prevent their malignant transformation to melanoma.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5655fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5655fjev1 20/9/1561    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abdel-Malek, Z. A. Articles by Knittel, J. J. Search for Related Content PubMed PubMed Citation Articles by Abdel-Malek, Z. A. Articles by Knittel, J. J.