An improved human skin explant culture method for testing and assessing personal care products.

BACKGROUND: Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation. MATERIALS AND METHODS: Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application. RESULTS: This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing. CONCLUSION: Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.

The ß-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts.

ß-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of ß-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of ß-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal ß-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that ß-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important ß-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/ß-catenin activity in fibroblasts. Blocking Wnt/ß-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with ß-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the ß-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

UV cell stress induces oxidative cyclization of a protective reagent for DNA damage reduction in skin explants.

UV irradiation is a major driver of DNA damage and ultimately skin cancer. UV exposure leads to persistent radicals that generate ROS over prolonged periods of time. Toward the goal of developing long-lasting antioxidants that can penetrate skin, we have designed a ROS-initiated protective (RIP) reagent that, upon reaction with ROS (antioxidant activity), self-cyclizes and then releases the natural product apocynin. Apocynin is a known antioxidant and inhibitor of NOX oxidase enzymes. A key phenol on the compound 1 controls ROS-initiated cyclization and makes 1 responsive to ROS with a EC50 comparable to common antioxidants in an ABTS assay. In an in vitro DNA nicking assay, the RIP reagent prevented DNA strand breaks. In cell-based assays, the reagent was not cytotoxic, apocynin was released only in cells treated with UVR, reduced UVR-induced cell death, and lowered DNA lesion formation. Finally, topical treatment of human skin explants with the RIP reagent reduced UV-induced DNA damage as monitored by quantification of cyclobutane dimer formation and DNA repair signaling via TP53. The reagent was more effective than administration of a catalase antioxidant on skin explants. This chemistry platform will expand the types of ROS-activated motifs and enable inhibitor release for potential use as a long-acting sunscreen.

In vitro behavior and UV response of melanocytes derived from carriers of CDKN2A mutations and MC1R variants.

Coinheritance of germline mutation in cyclin-dependent kinase inhibitor 2A (CDKN2A) and loss-of-function (LOF) melanocortin 1 receptor (MC1R) variants is clinically associated with exaggerated risk for melanoma. To understand the combined impact of these mutations, we established and tested primary human melanocyte cultures from different CDKN2A mutation carriers, expressing either wild-type MC1R or MC1RLOF variant(s). These cultures expressed the CDKN2A product p16 (INK4A) and functional MC1R. Except for 32ins24 mutant melanocytes, the remaining cultures showed no detectable aberrations in proliferation or capacity for replicative senescence. Additionally, the latter cultures responded normally to ultraviolet radiation (UV) by cell cycle arrest, JNK, p38, and p53 activation, hydrogen peroxide generation, and repair of DNA photoproducts. We propose that malignant transformation of melanocytes expressing CDKN2A mutation and MC1RLOF allele(s) requires acquisition of somatic mutations facilitated by MC1R genotype or aberrant microenvironment due to CDKN2A mutation in keratinocytes and fibroblasts.

Utilization of Reactive Oxygen Species Targeted Therapy to Prolong the Efficacy of BRAF Inhibitors in Melanoma.

BRAF mutations occur in about 50% of melanoma patients. FDA approved BRAF and MEK inhibitors have improved the prognosis of patients with BRAF mutations. However, all responders develop resistance typically within one year of treatment. Recent observations demonstrate that BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells. A100, identified from a library screen, is a ROS-activated prodrug that self-cyclizes into a stable bicyclic ring and causes DNA double strand breaks. We proposed to examine if ROS activated therapy will inhibit tumor growth and evade resistance to BRAF inhibitors. In this study, the BRAF inhibitor dabrafenib was used to generate resistant cell lines (A375DR, SK-MEL-24DR and WM-115DR). Flow cytometry experiments showed that ROS levels are increased in these dabrafenib-resistant cells as compared to parental cells, assessed by both the H2DCFDA and MitoSOX assays. Furthermore, we observed that resistant cells had increased levels of the mitochondrial enzymes SOD2 and PRDX1, which function to reduce ROS levels in the mitochondria. We found that A100 sensitized the resistant melanoma cells to dabrafenib and induced DNA damage. Co-treatment of both A100 and dabrafenib significantly suppressed in vitro cell proliferation and three- dimensional (3D) matrigel growth. This study suggests that the combination of A100 with a BRAF inhibitor could be a potential strategy to treat melanoma patients with BRAF mutations.

Suppression of MAPK signaling in BRAF-activated PTEN-deficient melanoma by blocking ß-catenin signaling in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of ß-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking ß-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

Self-Cyclizing Antioxidants to Prevent DNA Damage Caused by Hydroxyl Radical.

Antioxidant therapy is a promising treatment strategy for protecting DNA from the damage caused by reactive oxygen species (ROS). Here, we report new self-cyclizing antioxidant reagents that are selective for the hydroxyl radical. Our mechanistic investigation revealed that the reagents react with three equivalents of oxidant in a cascade reaction to form a bicyclic final product. Among the reagents synthesized, 1 c showed favorable properties in vitro and in cellular studies. Using As2 O3 , which triggers ROS production, we showed that 1 c prevents formation of the guanine oxidation product 2,2,4-triamino-2H-oxazol-5-one-2'-deoxyribonucleoside and lowers cellular levels of reactive oxygen. The described self-cyclizing antioxidants are efficient, flexible, and tunable reagents with the potential to limit toxic oxidative stress.

YAP and WWTR1: New targets for skin cancer treatment.

The core components of the Hippo signaling pathway are a cascade of kinases that govern the phosphorylation of downstream transcriptional co-activators, namely, YES-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ). The Hippo signaling pathway is considered an important tumor-suppressor pathway, and its dysregulation has been noted in a variety of human cancers, in which YAP/WWTR1 enable cancerous cells to overcome contact inhibition, and to grow and spread uncontrollably. Interestingly, however, recent studies have told a somewhat different but perhaps more intriguing YAP/WWTR1 story, as these studies found that YAP/WWTR1 function as a central hub that integrates signals from multiple upstream signaling pathways, cell-cell interactions and mechanical forces and then bind to and activate different downstream transcriptional factors to direct cell social behavior and cell-cell interactions. In this review, we present the latest findings on the role of YAP/WWTR1 in skin physiology, pathology and tumorigenesis and discuss the statuses of newly developed therapeutic interventions that target YAP/WWTR1 in human cancers, as well as their prospects for use as skin cancer treatments.

Targeted deactivation of cancer-associated fibroblasts by ß-catenin ablation suppresses melanoma growth.

Cancer-associated fibroblasts (CAFs) are the crucial components of the dynamic tumor microenvironment, which not only supports the growth and metastasis of melanoma but also contributes to drug resistance in melanoma treatment. We recently discovered that loss of ß-catenin signaling deactivated stromal fibroblasts and reduced the production of paracrine factors and extracellular matrix proteins. Based on this finding, we aimed to determine whether melanoma growth could be suppressed by targeted deactivation of CAFs via ß-catenin ablation using a combination of in vitro and in vivo approaches. Using an in vitro three-dimensional (3D) tumor co-culture model, we showed that ß-catenin-deficient fibroblasts lost the ability to respond to melanoma cell stimulation and to support the growth of B16F10 melanoma cells. To determine the in vivo effects of CAF deactivation on melanoma growth, we designed a novel genetic approach to ablate ß-catenin expression in melanoma-associated fibroblasts only after melanoma tumor was formed. As expected, our observation showed that development of B16F10 melanoma was significantly delayed when ß-catenin expression was ablated in CAFs. We determined that inhibition of tumor growth was due to decreased melanoma cell proliferation and increased cell death. Further analysis revealed that CAF deactivation caused the downregulation of the MAPK/ERK signaling cascade and S and G2/M phase cell cycle arrest in B16F10 melanoma cells. Overall, our data emphasize the significance of targeting CAFs as a potential novel therapeutic approach to improve melanoma treatment by creating a tumor-suppressive microenvironment through tumor-stroma interactions.

Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes.

Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

Stepping up melanocytes to the challenge of UV exposure.

Exposure to solar ultraviolet radiation (UV) is the main etiological factor for skin cancer, including melanoma. Cutaneous pigmentation, particularly eumelanin, afforded by melanocytes is the main photoprotective mechanism, as it prevents UV-induced DNA damage in the epidermis. Therefore, maintaining genomic stability of melanocytes is crucial for prevention of melanoma, as well as keratinocyte-derived basal and squamous cell carcinoma. A critical independent factor for preventing melanoma is DNA repair capacity. The response of melanocytes to UV is mediated mainly by a network of paracrine factors that not only activate melanogenesis, but also DNA repair, anti-oxidant, and survival pathways that are pivotal for maintenance of genomic stability and prevention of malignant transformation or apoptosis. However, little is known about the stress response of melanocytes to UV and the regulation of DNA repair pathways in melanocytes. Unraveling these mechanisms might lead to strategies to prevent melanoma, as well as non-melanoma skin cancer.

alpha-MSH activates immediate defense responses to UV-induced oxidative stress in human melanocytes.

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV-signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose-dependent generation of hydrogen peroxide in UV-irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with alpha-melanocortin (alpha-MSH) reduced the UV-induced generation of 7,8-dihydro-8-oxyguanine (8-oxodG), a major form of oxidative DNA damage. Pretreatment with alpha-MSH also increased the protein levels of catalase and ferritin. The effect of alpha-MSH on 8-oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss-of-function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP-YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by alpha-MSH. Our data unravel some mechanisms by which alpha-MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss-of-function MC1R with melanoma.

alpha-MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV-induced DNA damage in human melanocytes.

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of alpha-melanocortin (alpha-MSH) that were more potent and stable than the physiological alpha-MSH, and mimicked its photoprotective effects against UV-induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified alpha-MSH core His(6)-d-Phe(7)-Arg(8), which contained different N-capping groups, C-terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C-terminal modifications. The most effective C-terminal tripeptide mimicked alpha-MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non-functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.

The melanocortin 1 receptor and the UV response of human melanocytes--a shift in paradigm.

Cutaneous pigmentation is the major photoprotective mechanism against the carcinogenic and aging effects of UV. Epidermal melanocytes synthesize the pigment melanin, in the form of eumelanin or pheomelanin. Synthesis of the photoprotective eumelanin by human melanocytes is regulated mainly by the melanocortins alpha-melanocortin (alpha-MSH) and adrenocorticotropic hormone (ACTH), which bind the melanocortin 1 receptor (MC1R) and activate the cAMP pathway that is required for UV-induced tanning. Melanocortins stimulate proliferation and melanogenesis and inhibit UV-induced apoptosis of human melanocytes. Importantly, melanocortins reduce the generation of hydrogen peroxide and enhance repair of DNA photoproducts, independently of pigmentation. MC1R is a major contributor to the diversity of human pigmentation and a melanoma susceptibility gene. Certain allelic variants of this gene, namely R151C, R160W and D294H, are strongly associated with red hair phenotype and increased melanoma susceptibility. Natural expression of two of these variants sensitizes melanocytes to the cytotoxic effect of UV, and increases the burden of DNA damage and oxidative stress. We are designing potent melanocortin analogs that mimic the effects of alpha-MSH as a strategy to prevent skin cancer, particularly in individuals who express MC1R genotypes that reduce but do not abolish MC1R function, or mutations in other melanoma susceptibility genes, such as p16.

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes.

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.

Cutaneous photoprotection and melanoma susceptibility: reaching beyond melanin content to the frontiers of DNA repair.

Melanoma is the most devastating form of skin cancer. The steady increase in the incidence of melanoma, its resistance to chemotherapy, together with its high potential to metastasize, have emphasized the importance of its prevention. It is becoming clear that solar ultraviolet radiation is a main culprit in the etiology of melanoma, the same as in basal and squamous cell carcinomas. It is commonly accepted that skin pigmentation and melanin content are principal determinants of the susceptibility to melanoma and other sun-induced skin cancers. Although this is generally true, however, prediction of melanoma risk based solely on pigmentary phenotype is not always precise and fails to identify high-risk individuals with dark skin color. Other important risk factors need to be considered and better defined, particularly DNA repair capacity. Emerging studies have revealed the role of melanoma susceptibility genes in regulating DNA repair, and indicated that melanoma patients have a lower DNA repair capacity than the general population. As the response of human melanocytes to ultraviolet radiation is modulated by an array of paracrine factors, we have focused our investigation on the role of melanocortins and the melanocortin 1 receptor, as well as endothelin-1, in this response. We have discovered novel roles for melanocortins and endothelin-1 as survival factors that rescue human melanocytes from ultraviolet radiation-induced apoptosis, and importantly enhance repair of DNA photoproducts and reduce the release of hydrogen peroxide that can cause oxidative stress. Our findings, together with epidemiological data showing that loss-of-function mutations in the melanocortin-1 receptor gene increase the risk of melanoma, substantiate the role of DNA repair in melanoma genesis, and suggest that responsiveness to melanocortins and endothelin-1 is important for melanoma prevention.

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

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.