RESUMO
Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.
Assuntos
Melanoma/enzimologia , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Camundongos Pelados , Camundongos SCID , Terapia de Alvo Molecular , Invasividade Neoplásica , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Inibidores de Serina Proteinase/uso terapêutico , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carga TumoralRESUMO
Targeting neuroendocrine receptors can be considered as another interesting approach to treating fibrotic disorders. Previously, we could demonstrate that tropisetron, a classical serotonin receptor blocker, can modulate collagen synthesis and acts in vitro through the α7 nicotinic acetylcholine receptor (α7nAchR). Here, we used a pharmacologic approach with specific α7nAchR agonists to validate this hypothesis. PHA-543613, an α7nAchR-specific agonist, not only prevented but also reversed established skin fibrosis of mice injected with bleomycin. Interestingly, agonistic stimulation of α7nAchR also attenuated experimental skin fibrosis in the non-inflammation driven adenovirus coding for TGFß receptor Iact mouse model, indicating fibroblast-mediated and not only anti-inflammatory effects of such agents. The fibroblast-mediated effects were confirmed in vitro using human dermal fibroblasts, in which the α7nAchR-specific agonists strongly reduced the impact of TGFß1-mediated expression on collagen and myofibroblast marker expression. These actions were linked to modulation of the redox-sensitive transcription factor JunB and impairment of the mitochondrial respiratory system. Our results indicate that pharmacologic stimulation of the α7nAchR could be a promising target for treatment of patients with skin fibrotic diseases. Moreover, our results suggest a mechanistic axis of collagen synthesis regulation through the mitochondrial respiratory system.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinuclidinas/farmacologia , Escleroderma Sistêmico/tratamento farmacológico , Pele/patologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Adenoviridae/genética , Animais , Bleomicina/toxicidade , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Cultura Primária de Células , Quinuclidinas/uso terapêutico , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time-dependent manner (24, 48, 72 hours) in highly pigmented MNT-1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk-Mel-28 cells. Subsequently, MNT-1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N-phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10-3 mol/L, triggered the appearance of premelanosomes (stage I-II of melanosome) and MNT-1 cells synthesize de novo endogenous melatonin shown by LC-MS. In conclusion, these studies show a melanogenic-like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.
Assuntos
Melaninas/biossíntese , Melanoma/metabolismo , Melatonina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Transtornos da Pigmentação/tratamento farmacológico , Transtornos da Pigmentação/metabolismo , Transtornos da Pigmentação/patologiaRESUMO
Tropisetron is a serotonin receptor (5-HT-R)-modulating agent and approved as an antiemetic for patients undergoing chemotherapy. In the gut, it acts via specific serotonin receptors, 5-HT3 -R, to elicit its beneficial effects against nausea. We investigated whether tropisetron can affect inflammatory cell responses of human primary epidermal keratinocytes (NHK) which are key cells in the regulation of skin homoeostasis. Tropisetron significantly and dose-dependently suppressed tumor necrosis factor (TNF)-α-mediated mRNA expression and protein secretion of interleukin (IL)-6 and IL-8 in these cells. This effect of tropisetron was independent of p65/NF-κB as shown by various NF-κB signal transduction read-outs. Importantly, the anti-inflammatory tropisetron effect on NHK was neither mediated by 5-HT3 -R nor 5-HT4 -R since these receptors were absent in NHK. In contrast, NHK expressed α7 nicotinic acetylcholine receptors (α7nAchR) which previously were found to bind tropisetron. The α7nAchR antagonist α-bungarotoxin neutralized, whereas AR-R17779, a specific α7nAchR agonist, mimicked the suppressive effect of tropisetron on TNF-α-mediated IL-6 and IL-8 expression in NHK. Our findings suggest that tropisetron and probably other α7nAchR-activating agents could be useful for the future therapy of inflammatory skin diseases.
Assuntos
Queratinócitos/metabolismo , Tropizetrona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Anti-Inflamatórios/farmacologia , Homeostase , Humanos , Inflamação , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinócitos/efeitos dos fármacos , Fosforilação , Ligação Proteica , Psoríase/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Transdução de Sinais , Pele/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm² caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10-6 M with lower effects seen at 10-9 or 10-4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.
Assuntos
Melanoma/metabolismo , Melatonina/metabolismo , Melatonina/farmacologia , 5-Metoxitriptamina/farmacologia , Animais , Cálcio/metabolismo , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/efeitos da radiação , Melatonina/análogos & derivados , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação Oxidativa/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Raios UltravioletaRESUMO
Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-ß1 had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-ß1 -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-ß1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.
Assuntos
Fibroblastos/enzimologia , NADPH Oxidase 4/genética , Escleroderma Sistêmico/enzimologia , Pele/enzimologia , Pele/patologia , Actinas/genética , Adulto , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Simulação por Computador , Citocinas/genética , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fibronectinas , Fibrose , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Recém-Nascido , Isoenzimas/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Complexos Multienzimáticos/metabolismo , Miofibroblastos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1/genética , NADPH Oxidase 2/genética , NADPH Oxidase 4/metabolismo , Oniocompostos/farmacologia , Cultura Primária de Células , RNA Mensageiro/metabolismo , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta/farmacologia , Adulto JovemRESUMO
Prohormone convertases (PCs) are endoproteases that process many substrates in addition to hormone precursors. Although overexpression of PCs is linked to carcinogenesis in some solid tumors, the role of subtilisin-kexin isoenzyme-1 (SKI-1) in this context is unknown. We show that SKI-1 is constitutively expressed in human pigment cells with higher SKI activity in seven out of eight melanoma cell lines compared with normal melanocytes. SKI-1 immunoreactivity is also detectable in tumor cells of melanoma metastases. Moreover, tissue samples of the latter display higher SKI-1 mRNA levels and activity than normal skin. From various stimuli tested, 12-O-tetradecanoylphorbol-13-acetate and tunicamycin affected SKI-1 expression. Importantly, SKI-1 inhibition by the cell-permeable enzyme inhibitor decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) not only suppressed proliferation and metabolic activity of melanoma cells in vitro but also reduced tumor growth of melanoma cells injected intracutaneously into immunodeficient mice. Mechanistic studies revealed that dec-RRLL-CMK induces classical apoptosis of melanoma cells in vitro and affects expression of several SKI-1 target genes including activating transcription factor 6 (ATF6). However, ATF6 gene silencing does not result in apoptosis of melanoma cells, suggesting that dec-RRLL-CMK induces cell death in an ATF6-independent manner. Our findings encourage further studies on SKI-1 as a potential target for melanoma therapy.
Assuntos
Melanoma/metabolismo , Melanoma/secundário , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator 6 Ativador da Transcrição/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Humanos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Camundongos SCID , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Neoplasias Cutâneas/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-ß1 (TGF-ß1)/TGF-ß3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Pele/lesões , Cicatrização/fisiologia , Animais , Moléculas de Adesão Celular/genética , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Fibrose/prevenção & controle , Humanos , Injeções Intradérmicas , Macrófagos/patologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/patologiaRESUMO
OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.