RESUMEN
Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.
Asunto(s)
Melanoma , Fosfatidilinositol 3-Quinasas , Humanos , Animales , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , alfa-MSH/farmacología , Melanoma/metabolismo , Metabolismo Energético , Glucosa , Adenosina Trifosfato/metabolismoRESUMEN
Melanocortin-1 Receptor (MC1R), when stimulated by alpha-melanocyte-stimulating hormone (α-MSH), is a driver of eumelanogenesis. Brown/black eumelanin is an effective filter against ultraviolet radiation (UVR) and is a scavenger of free radicals. Several polymorphic variants of MC1R are frequent in red-head people. These polymorphisms reduce the ability of MC1R to promote eumelanogenesis after its activation and spontaneous pheomelanogenesis take place. Since pheomelanin can act as an endogenous photosensitizer, people carrying MC1R polymorphisms are more susceptible to skin cancer. Here, we summarize current knowledge on the biology of MC1R beyond its ability to drive eumelanogenesis. We analyze its capacity to cope with oxidative insult and consequent DNA damage. We describe its ability to transduce through different pathways. We start from the canonical pathway, the cAMP/protein kinase A (PKA) pathway mainly involved in promoting eumelanogenesis, and protection from oxidative damage, and we then move on to describe more recent knowledge concerning ERK pathways, phosphoinositide 3-kinase (PI3K) pathway/AKT, and α-MSH/Peroxisome proliferators activated receptor-γ (PPAR-γ) connection. We describe MC1R polymorphic variants associated with melanoma risk which represent an open window of clinical relevance.
Asunto(s)
Pleiotropía Genética , Receptor de Melanocortina Tipo 1/metabolismo , Pigmentación de la Piel , alfa-MSH/metabolismo , Animales , Daño del ADN , Reparación del ADN , Humanos , Receptor de Melanocortina Tipo 1/genética , Pigmentación de la Piel/genéticaRESUMEN
The melanocortin-1 receptor (MC1R) belongs to the family of the G protein-coupled receptor (GPCR). Activated GPCRs can promote the phosphoinositide 3-kinase (PI3K) pathway. Few studies deal with the role of the PI3K pathway activation in response to αMSH. On B16-F10 cell line, we investigated the αMSH-dependent modulation of pAKT/AKT, as a key element of the PI3K pathway after rapid and prolonged stimulation. We demonstrated that αMSH triggers a rapid modulation of AKT which culminates in an increase in its phosphorylation. We highlighted a comparable upregulation of pAKT after exposure to αMSH on primary cultures of normal human melanocytes (NHMs) expressing a wild-type MC1R. On B16-F10 cells, NHMs, and an ex vivo model of human skin biopsies, we explored the influence of PI3K/AKT signaling triggered by αMSH, focusing on the control of melanogenesis and pigment release. We showed that the αMSH-dependent PI3K/AKT pathway exerts a negative feedback on melanogenesis and promotes the extracellular release of pigment. We strengthened the role of the PI3K/AKT pathway triggered by αMSH in preserving redox equilibrium and genome integrity, highlighting its role in affecting cell survival.
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Retroalimentación Fisiológica , Melaninas/metabolismo , Melanocitos/citología , Melanoma Experimental/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , alfa-MSH/farmacología , Animales , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Pigmentación , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Bovine colostrum, the first milk secreted by the mammary glands of cows shortly after they have given birth, provides a natural source of bioactive substances helpful to promote tissue development and repair, and to maintain a healthy immune system. Owing to its properties, the use of colostrum in the treatment of human diseases is under investigation. We evaluated the biological activity of colostrum on human primary keratinocytes, focusing on its effects with regard to a proliferation/differentiation balance. Using cellular and molecular approaches, we showed that colostrum favors a cell cycle withdrawal by increasing the expression of p21/WAF1 and p27/KIP1. It also promotes the transition of keratinocytes from a proliferating to a differentiating state, as assessed by a decrease in keratin 5 and an increase in keratin 16. We demonstrated the ability of colostrum to induce the expression of early and late differentiation markers (keratin 1, involucrin, and filaggrin) and the synthesis of caspase 14 and bleomycin hydrolase, the two main enzymes involved in filaggrin maturation. Moreover, we showed that bovine colostrum is able to promote keratinocyte stratification and terminal differentiation not only in two-dimensional (2D), but also in a more physiological system of three-dimensional (3D) skin equivalents. Finally, we demonstrated that colostrum stimulates cell differentiation through the PI3K/PLC-γ1/PKCα pathways mainly associated to tyrosine kinase receptors. These results suggest the possibility to benefit from colostrum properties for the treatment of skin diseases characterized by altered differentiation and perturbed barrier function.
Asunto(s)
Diferenciación Celular , Calostro/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/citología , Piel/citología , Animales , Bovinos , Células Cultivadas , Femenino , Proteínas Filagrina , Humanos , Queratinocitos/metabolismo , Embarazo , Piel/metabolismoRESUMEN
Cerium oxide nanoparticles (CNPs) are potent radical scavengers protecting cells from oxidative insults, including ionizing radiation. Here we show that CNPs prevent X-ray-induced oxidative imbalance reducing DNA breaks on HaCat keratinocytes, nearly abating mutagenesis. At the same time, and in spite of the reduced damage, CNPs strengthen radiation-induced cell cycle arrest and apoptosis outcome, dropping colony formation; notably, CNPs do not possess any intrinsic toxicity toward non-irradiated HaCat, indicating that they act on damaged cells. Thus CNPs, while exerting their antioxidant action, also reinforce the stringency of damage-induced cell integrity checkpoints, promoting elimination of the "tolerant" cells, being in fact radio-sensitizers. These two contrasting pathways are mediated by different activities of CNPs: indeed Sm-doped CNPs, which lack the Ce3+/Ce4+ redox switch and the correlated antioxidant action, fail to decrease radiation-induced superoxide formation, as expected, but surprisingly maintain the radio-sensitizing ability and the dramatic decrease of mutagenesis. The latter is thus attributable to elimination of damaged cells rather than decreased oxidative damage. This highlights a novel redox-independent activity of CNPs, allowing selectively eliminating heavily damaged cells through non-toxic mechanisms, rather reactivating endogenous anticancer pathways in transformed cells.
RESUMEN
BACKGROUND: The α-Melanocyte Stimulating Hormone (αMSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. αMSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the αMSH/Peroxisome Proliferator Activated Receptor (PPARγ) pathway as a new pathway on the B16-F10 mouse melanoma cell line. αMSH induced the translocation of PPARγ into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the αMSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to αMSH by reducing proliferation and that PPARγ was involved in this effect. Due to its key role in the control of cell proliferation, PPARγ agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to αMSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPARγ pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to αMSH. METHODS: We employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the αMSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E). RESULTS: The αMSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the αMSH mediated effect on proliferation in NHMs but it did not mimic the αMSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the αMSH proliferative effect. Further experiments, treating melanoma cell lines with αMSH in the presence/absence of GW9662, as an inhibitor of PPARγ, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure. CONCLUSIONS: In both melanoma cell lines, αMSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPARγ as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.
Asunto(s)
Melanoma/metabolismo , PPAR gamma/metabolismo , Transducción de Señal , alfa-MSH/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expresión Génica , Genes Reporteros , Humanos , Melanoma/genética , Melanoma/patología , Melanoma Experimental , Ratones , PPAR gamma/genética , Activación TranscripcionalRESUMEN
Increasing attention is addressed to identify products able to enhance skin photoprotection and to prevent skin carcinogenesis. Several studies have demonstrated that the α-melanocyte stimulating hormone (αMSH), acting on a functional MC1R, provides a photoprotective effect by inducing pigmentation, antioxidants and DNA repair. We discovered a link between αMSH and the nuclear receptor Peroxisome Proliferator-Activated Receptor-γ (PPARγ), suggesting that some of the αMSH protective effects may be dependent on PPARγ transcriptional activity. Moreover, we demonstrated that the activation of PPARγ by the parrodiene 2,4,6-octatrienoic acid (Octa) induces melanogenesis and antioxidant defence in human melanocytes and counteracts senescence-like phenotype in human fibroblasts. In this study, we demonstrate that the activation of PPARγ by Octa exerts a protective effect against UVA- and UVB-induced damage on normal human keratinocytes (NHKs), the major target cells of UV radiation. Octa promotes the antioxidant defence, augments DNA repair and reduces the induction of proteins involved in UV-induced DNA damage response. Our results contribute to deepen the analysis of the αMSH/PPARγ connection and suggest perspectives for the development of new molecules and formulations able to prevent cutaneous UV damage by acting on the different skin cell populations through PPARγ activation.
Asunto(s)
Ácidos Grasos Insaturados/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Queratinocitos/efectos de la radiación , PPAR gamma/agonistas , Protectores contra Radiación/farmacología , Rayos Ultravioleta/efectos adversos , Antioxidantes/metabolismo , Células Cultivadas , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Receptor de Melanocortina Tipo 1/genética , Receptor de Melanocortina Tipo 1/metabolismo , alfa-MSH/metabolismoRESUMEN
Mushrooms represent a formidable source of bioactive compounds. Some of these may be considered as biological response modifiers; these include compounds with a specific biological function: antibiotics (e.g. plectasin), immune system stimulator (e,g, lentinan), antitumor agents (e.g. krestin, PSK) and hypolipidemic agents (e.g. lovastatin) inter alia. In this study, we focused on the Chinese medicinal mushroom "yun zhi", Trametes versicolor, traditionally used for (cit.) "replenish essence and qi (vital energy)". Previous studies indicated the potential activity of extracts from culture filtrate of asexual mycelia of T. versicolor in controlling the growth and secondary metabolism (e.g. mycotoxins) of plant pathogenic fungi. The quest of active principles produced by T. versicolor, allowed us characterising an exo-polysaccharide released in its culture filtrate and naming it Tramesan. Herein we evaluate the biological activity of Tramesan in different organisms: plants, mammals and plant pathogenic fungi. We suggest that the bioactivity of Tramesan relies mostly on its ability to act as pro antioxidant molecule regardless the biological system on which it was applied.
Asunto(s)
Polisacáridos/química , Polisacáridos/farmacología , Trametes/química , Conformación de Carbohidratos , Cromatografía en Gel , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Cutaneous phototype is considered mainly related to cutaneous pigmentation and to the eumelanin/pheomelanin ratio, which is mostly genetically determined by the melanocortin 1 receptor (MC1R) polymorphisms. However, data in literature indicate that, in addition to stimulation of eumelanin synthesis, the MC1R signalling activates antioxidant, DNA repair and survival pathways. New emerging aspects regarding photoprotection and skin phototypes are going beyond those features connected to the melanin content in the skin. Important new findings link the MC1R to nuclear receptors activation, shedding light on new extra-melanogenic effects dependent on the α-melanocyte-stimulating hormone (α-MSH) activity and new ways through which such functions are modulated. These evidences indicate that several factors including melanin play a part in defining the basis for individual sun sensitivity, suggesting that the cutaneous phototype represents a 'biochemical fingerprint'.
Asunto(s)
Piel/patología , Piel/efectos de la radiación , Rayos Ultravioleta , Humanos , Melaninas/biosíntesis , Estrés Oxidativo/efectos de la radiación , Receptor de Melanocortina Tipo 1/metabolismo , Transducción de Señal/efectos de la radiación , Pigmentación de la Piel/efectos de la radiaciónRESUMEN
We have discovered a new α-melanocyte stimulating hormone (α-MSH)/peroxisome proliferator activated receptor-γ (PPAR-γ) connection in B16-F10 cells. Both PPAR-γ up-regulation and its induction as an active transcription factor were observed in response to α-MSH. The α-MSH/PPAR-γ connection influenced both pigmentation and proliferation. The forskolin-stimulated cAMP/PKA pathway was not able to induce either PPAR-γ translocation into the nucleus or PPAR-γ transcriptional activity. As the melanocortin-1 receptor, the specific receptor for the α-MSH, is a G-protein coupled receptor, we wondered whether the phosphatidylinositol [PI(4,5)P(2) /PLC(ß) ] signal pathway was involved in mediating the α-MSH-dependent PPAR-γ activation. Employing inhibitors of PI(4,5)P(2) /PLC(ß) pathway, the results of our experiments suggested that this pathway was promoted by α-MSH and that α-MSH played a role in mediating PPAR-γ activation. We have demonstrated, for the first time, that α-MSH induces the PI(4,5)P(2) /PLC(ß) pathway, through analysis of the basic steps of the pathway. The α-MSH effect on PPAR-γ was independent of animal species and was not correlated with the physio-pathological status.
Asunto(s)
Melanoma Experimental/metabolismo , PPAR gamma/metabolismo , Neoplasias Cutáneas/metabolismo , alfa-MSH/farmacología , Animales , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diglicéridos/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Hidrólisis/efectos de los fármacos , Fosfatos de Inositol/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , PPAR gamma/genética , Fosfolipasa C beta/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Interest in colorless intermediates of melanocyte metabolism has traditionally been related to their role as melanin precursors, though several lines of evidence scattered in the literature suggested that these compounds may exert an antioxidant and protective function per se unrelated to pigment synthesis. Herein, we disclose the remarkable protective and differentiating effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible dopachrome tautomerase (DCT)-dependent eumelanin intermediate, on primary cultures of human keratinocytes. At micromolar concentrations, DHICA induced: (a) time- and dose-dependent reduction of cell proliferation without concomitant toxicity; (b) enhanced expression of early (spinous keratins K1 and K10 and envelope protein involucrin) and late (loricrin and filaggrin) differentiation markers; (c) increased activities and expression of antioxidant enzymes; and (d) decreased cell damage and apoptosis following UVA exposure. The hitherto unrecognized role of DHICA as an antiproliferative, protective, and antiapoptotic endogenous cell messenger points to a reappraisal of the biological functions of melanocytes and DCT in skin homeostasis and photoprotection beyond the mere provision of melanin pigments, and provides, to our knowledge, a previously unreported possible explanation to the higher resistance of the dark-skinned eumelanic phenotypes to sunburn and skin cancer.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epidérmicas , Epidermis/efectos de los fármacos , Indoles/farmacología , Melaninas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Comunicación Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Epidermis/metabolismo , Proteínas Filagrina , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinas/metabolismo , Melanocitos/citología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Precursores de Proteínas/metabolismo , Factores de TiempoRESUMEN
The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by alpha-melanocyte-stimulating hormone (alpha-MSH) and ultraviolet irradiation. Previous studies have shown that alpha-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and alpha-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.
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Sistema de Señalización de MAP Quinasas/fisiología , Monofenol Monooxigenasa/metabolismo , Pigmentación/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Imidazoles/metabolismo , Melaninas/biosíntesis , Melaninas/genética , Melanoma Experimental/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Piridinas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Ubiquitinadas/metabolismo , Rayos Ultravioleta , alfa-MSH/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
We demonstrated a direct correlation between melanogenic and catalase activities on in vitro and ex vivo models. Here, we investigated whether the stimulation of Melanocortin-1 Receptor (MC1R) could influence catalase expression, activity and cellular localization. For this purpose, we treated B16-F0 melanoma cells with alpha-Melanocyte Stimulating Hormone (alpha-MSH) and we showed a rapid induction of catalase through a cAMP/PKA-dependent, microphthalmia-associated transcription factor (MITF) independent mechanism, acting at post-transcriptional level. Moreover, alpha-MSH promoted a partial re-distribution of catalase to the cell periphery and dendrites. This work strengthens the correlation between melanogenesis and anti-oxidants, demonstrating the induction of catalase in response to a melanogenic stimulation, such as alpha-MSH-dependent MC1R activation. Moreover, this study highlights catalase regulatory mechanisms poorly known, and attributes to alpha-MSH a protective role in defending melanocytes, and possibly keratinocytes, not only on the basis of its pigmentary action, but also for its capacity to stimulate a quick anti-oxidant defence.
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Catalasa/metabolismo , Membrana Celular/efectos de los fármacos , Dendritas/efectos de los fármacos , Melanoma/metabolismo , Receptor de Melanocortina Tipo 1/metabolismo , alfa-MSH/farmacología , Animales , Catalasa/biosíntesis , Membrana Celular/enzimología , AMP Cíclico/metabolismo , Proteína Quinasa Tipo II Dependiente de AMP Cíclico/metabolismo , Dendritas/enzimología , Melanoma/patología , Ratones , Transporte de Proteínas/efectos de los fármacos , Receptor de Melanocortina Tipo 1/agonistas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation. METHODOLOGY/PRINCIPAL FINDINGS: By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7. CONCLUSIONS/SIGNIFICANCE: This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.
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Gutatión-S-Transferasa pi/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Transformación Celular Neoplásica , Regulación Viral de la Expresión Génica , Humanos , Queratinocitos/virología , Espectrometría de Masas/métodos , Conformación Molecular , Mutación , Proteínas E7 de Papillomavirus , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Represoras/metabolismoRESUMEN
Malignant melanoma incidence is increasing rapidly in Western countries. Its prevention requires a deep knowledge of the biological basis of the neoplasm leading to the identification of new biological risk markers. In in-vitro and ex-vivo models we demonstrated that catalase was modified not only in its activity but also in its charge properties after ultraviolet A irradiation through pheomelanin. Here we focus on the electrophoretic behaviour of catalase in the human skin in vivo, in association with cutaneous phototype. Zymographic analysis of the enzyme on skin biopsies from Caucasian population (phototype I-IV), collected from the trunk in autumn-winter, to exclude possible influences of an acute photoexposure, evidenced a protein doublet, representing the coexistence of two active isoforms of catalase with different charge properties. In the skin from low-phototype subjects, the percent contribution of the more acidic component of the doublet was prevalent, inversely correlated with total melanin concentration in hair, and associated with a high number of melanocytic nevi. In summary, this study shows for the first time the existence of an acidic catalase in association with clinically defined risk characteristics in low phototype skin in vivo, contributing to the knowledge of a new biochemical marker of cutaneous photosusceptibility.
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Catalasa/metabolismo , Cabello/enzimología , Melanoma/enzimología , Melanoma/metabolismo , Piel/enzimología , Adolescente , Adulto , Anciano , Biomarcadores de Tumor , Femenino , Cabello/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Melaninas , Persona de Mediana Edad , Nevo Pigmentado , Isoformas de Proteínas , Piel/metabolismo , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/metabolismo , Rayos Ultravioleta , Adulto JovenRESUMEN
The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H(2)O(2) treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H(2)O(2)-induced mitochondrial alterations.
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Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , 3-Yodobencilguanidina/farmacología , Benzamidas/farmacología , Fragmentación del ADN/efectos de los fármacos , Desoxiglucosa/farmacología , Guanidinas/farmacología , Humanos , Microscopía Fluorescente , Factores de Tiempo , Células U937RESUMEN
Glycogen synthase kinase 3beta (GSK3beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and the insulin response. GSK3beta also plays a key role in the Wnt/beta-catenin pathway. The master regulator of the pigmentation microphthalmia-associated transcription factor (MITF) is a target for the Wnt pathway, however, to date, the regulatory role of GSK3beta in the control of melanogenesis has not been elucidated. In this study, we evaluated the effect of inhibiting GSK3beta activity on the regulation of melanocyte differentiation. Exposure of the murine melanoma cell line B16 and normal human melanocytes to GSK3beta specific inhibitors (SB216763, SB415286, BIO, and LiCl) resulted in a dose-dependent accumulation of beta-catenin. This is associated with the induction of melanocyte differentiation-associated markers such as melanin synthesis, tyrosinase activity, and expression of tyrosinase and the microphthalmia-associated transcription factor. Attenuation of GSK3beta activity has an inhibitory effect on cell growth, and this was accompanied by morphological changes. Moreover, treatment of B16 cells with a siRNA targeted against beta-catenin completely abolished the promelanogenic effect of GSK3beta inhibition, however, the overexpression of a constitutively active mutant form of beta-catenin (pCS2beta-cat-mut) only slightly increased the degree of pigmentation. These results demonstrated that GSK3beta is implicated in the regulation of melanogenesis and that pharmacological inhibition of its activity could increase melanin synthesis through mechanisms probably not restricted to Wnt/beta-catenin pathway activation.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Melaninas/biosíntesis , Melanocitos/enzimología , Melanoma Experimental/enzimología , Animales , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Glucógeno Sintasa Quinasa 3 beta , Humanos , Melanocitos/citología , Melanocitos/efectos de los fármacos , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/metabolismo , Pigmentación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.
Asunto(s)
Catalasa/metabolismo , Melaninas/metabolismo , Melanocitos/metabolismo , Monofenol Monooxigenasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Pigmentación de la Piel , Western Blotting , Catalasa/genética , Células Cultivadas , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/enzimología , Monofenol Monooxigenasa/genética , Oxidantes/farmacología , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pigmentación de la Piel/fisiologíaRESUMEN
The amount of information on tumor biology has expanded enormously, essentially due to the completion of the human genome sequencing and to the application of new technologies that represent an exciting breakthrough in molecular analysis. Often these data spring from experimental procedures, such as a serial analysis of gene expression (SAGE) and DNA microarrays, which cannot be defined as hypothesis-driven: it may appear to be a "brute force" approach through which no information can be directly generated concerning the specific functions of selected genes in a definite context. However, interesting results are fruitfully generated, and thus it is important to consider the enormous potential these new technologies possess and to learn how to apply this novel form of knowledge in the emerging field of molecular medicine. This review, after a limited outline regarding several classic aspects of human cutaneous melanoma biology, genetics, and clinical approaches, will focus on the proficient use of up-to-date technologies in the study of the neoplastic disease and on their capability to provide effective support to conventional approaches in melanoma diagnosis, prognosis, and treatment.
Asunto(s)
Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Antígenos de Neoplasias/metabolismo , Apoptosis , Biomarcadores de Tumor/metabolismo , Ciclo Celular/fisiología , Humanos , Melaninas/biosíntesis , Melanoma/genética , Melanoma/prevención & control , Invasividad Neoplásica/prevención & control , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estrés Oxidativo/fisiología , Fagocitosis , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/prevención & controlRESUMEN
The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products.