RESUMO
BACKGROUND: Both mesenchymal stromal cells (MSCs) and acellular dermal matrices (ADMs) represent fascinating therapeutic tools in the wound healing scenario. Strategies aimed at combining these two treatment modalities are currently under investigation. Moreover, scarcity of quantitative, nondestructive techniques for quality assessment of engineered tissues poses great limitations in regenerative medicine and collagen autofluorescence-based imaging techniques are acquiring great importance in this setting. OBJECTIVE: Our goals were to assess the in vitro interactions between ADSCs and ADMs and to analyze extracellular-matrix production. METHODS: Adipose-derived MSCs (ADSC) were plated on 8-mm punch biopsies of a commercially available ADM (Integra®). Conventional histology with hematoxylin-eosin staining, environmental scanning electron microscopy, and confocal-laser scanning microscopy were used to obtain imaging of ADSC-seeded ADMs. Collagen production by ADSCs was quantified by mean fluorescence intensity (MFI), expressed in terms of positive pixels/field, obtained through ImageJ software processing of three-dimensional projections from confocal scanning images. Control conditions included: fibroblast-seeded ADM, ADSC- and fibroblast-induced scaffolds, and Integra® alone. RESULTS: ADSCs were efficiently seeded on Integra® and were perfectly incorporated in the pores of the scaffold. Collagen production was revealed to be significantly higher when ADSCs were seeded on ADM rather than in all other control conditions. Collagen autofluorescence was efficiently used as a surrogate marker of ECM production. CONCLUSIONS: Combined therapies based on MSCs and collagenic ADMs are promising therapeutic options for chronic wounds. Not only ADSCs can be efficiently seeded on ADMs, but ADMs also seem to potentiate their regenerative properties, as highlightable from fluorescence confocal imaging.
Assuntos
Derme Acelular , Células-Tronco Mesenquimais , Colágeno , Imageamento Tridimensional , Microscopia ConfocalRESUMO
In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure.
Assuntos
Tecido Adiposo/citologia , Pele Artificial , Células-Tronco/citologia , Engenharia Tecidual/métodos , Tecido Adiposo/metabolismo , Colágeno Tipo IV/metabolismo , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrina alfaV/metabolismo , Queratinócitos/citologia , CicatrizaçãoRESUMO
Gold nanoparticles functionalized with 3-mercapto-1-propansulfonate (AuNPs-3MPS) have been prepared and loaded with Methotrexate (MTX), an immunosuppressive agent used in the systemic treatment of moderate-severe inflammatory diseases. The effects of the AuNPs-3MPS@MTX topically administered in vitro on skin model and in vivo on imiquimod-induced psoriasis-like mice model, have been studied. Clinical response, epidermal thickness, cell proliferation rate and inflammation were tested. AuNPs-3MPS@MTX treated mice showed a decreasing of scaling and erythema score, reduction of epidermal thickness, parakeratosis and hyperkeratosis, compared to AuNPs-3MPS treated mice. Immunohistochemistry analysis staining displayed that Ki67, K6 CD3 and CD8 stainings were reduced in AuNPs-3MPS@MTX treated mice. Blood evaluation showed no differences in blood count and in ALT and AST levels before and after AuNPs-3MPS or AuNPs-3MPS@MTX treatment. Topical AuNPs-3MPS@MTX treatment is able to induce a reduction of keratinocytes hyperproliferation, epidermal thickness and also inflammatory infiltrate in vivo on imiquimod-induced psoriasis like mice model.
Assuntos
Portadores de Fármacos/química , Ouro/química , Imunossupressores/administração & dosagem , Metotrexato/administração & dosagem , Psoríase/tratamento farmacológico , Compostos de Sulfidrila/química , Administração Tópica , Animais , Modelos Animais de Doenças , Imunossupressores/uso terapêutico , Nanopartículas Metálicas/química , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BLAssuntos
Células-Tronco Mesenquimais , Psoríase , Células Cultivadas , Humanos , Psoríase/diagnóstico , Psoríase/terapiaRESUMO
Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal-epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments.
Assuntos
Distrofina/metabolismo , Melanócitos/metabolismo , Mitocôndrias/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Pele/metabolismo , Biópsia , Northern Blotting , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Distrofina/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/ultraestrutura , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Oligomicinas/farmacologia , Rodaminas/metabolismo , Pele/efeitos dos fármacos , Pele/ultraestrutura , Fatores de Tempo , Utrofina/metabolismoRESUMO
BACKGROUND: MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. METHODS: Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. RESULTS: No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. CONCLUSIONS: Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.
RESUMO
Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Melanoma/patologia , Ácido Poliglutâmico/metabolismo , Timidilato Sintase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Cumarínicos/química , Cumarínicos/uso terapêutico , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/uso terapêutico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidilato Sintase/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Brooke-Spiegler syndrome represents an autosomal dominant disease characterized by the occurrence of multiple cylindromas, trichoepitheliomas and (sporadically) spiroadenomas. Patients with Brooke-Spiegler syndrome are also at risk of developing tumors of the major and minor salivary glands. Patients with Brooke-Spiegler syndrome have various mutations in the CYLD gene, a tumor-suppressor gene located on chromosome 16q. To date, 68 unique CYLD mutations have been identified. We describe two families with Brooke-Spiegler syndrome, one with familial cylindromatosis and one with multiple familial trichoepithelioma, which showed wide inter-family phenotypic variability. Analysis of germline mutations of the CYLD and PTCH genes was performed using peripheral blood. In addition, formalin-fixed paraffin-embedded tumor samples were analyzed for PTCH somatic mutations and cylindroma cell cultures were obtained directly from patients for further growth and analysis. Clinically, the major features of Brooke-Spiegler syndrome include the presence of heterogeneous skin tumors and wide inter- and intra-familial phenotypic variability. Histopathologically, both cylindromas and trichoepitheliomas were found in affected individuals. Mutations or loss of heterozygosity was not found in CYLD and PTCH genes. In CYLD and PTCH mutation-negative patients, other genes may be affected and further studies are needed to clarify whether these patients may be affected by de novo germline mutations.
Assuntos
Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias/genética , Receptores de Superfície Celular/genética , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/genética , Enzima Desubiquitinante CYLD , Família , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Síndromes Neoplásicas Hereditárias/patologia , Receptores Patched , Receptor Patched-1 , Neoplasias Cutâneas/patologiaRESUMO
Adipose derived mesenchymal stromal cells (ADSCs) represent a fascinating tool in the scenario of wound healing and regenerative medicine. Recent data already demonstrated that ADSCs could exert a stimulatory action on epithelial cells through secretion of soluble factors. The aim of the present study was to assess how ADSCs guide wound re-epithelization in vitro in the presence of keratinocytes. We used an organotypic model of wound healing and we seeded keratinocytes on a ADSC-induced dermal matrix. Conventional hematoxylin-eosin stain and immunohistochemistry staining for Ki67, p63 and pan-keratins were performed at different timepoints. Histological sections of organotypic cultures showed complete coverage of the ADSC-induced matrix by keratinocytes. Proliferation of basal stem cells was found to be the main mechanism responsible for epithelization of the dermis. In conclusion, ADSC do not only stimulate dermal regeneration through collagen deposition but also promote epithelization. HIGHLIGHTS: Mesenchymal stromal cells (MSCs) are widely used in regenerative medicine and wound healing. MSCs do not only stimulate dermal regeneration through collagen deposition but also promote epithelization. MSCs directly target the basal stem cell niche and promote its proliferation, migration and subsequent differentiation.
Assuntos
Tecido Adiposo , Células-Tronco Mesenquimais , Proliferação de Células , Colágeno , Células-Tronco , CicatrizaçãoRESUMO
The association between the metabolic profile and inflammatory cytokines in psoriasis is poorly understood. We analyzed the metabolic and cytokine/chemokine profiles in serum and skin from patients with new-onset psoriasis and healthy subjects (n = 7/group) by HR-MAS NMR and Bio-Plex immunoassay. Immuno-metabolic correlation matrix was analyzed in skin and serum to identify a potential immune-metabolic signature. Metabolomics analysis showed a significant increase in ascorbate and a decrease in scyllo-inositol, and a trend towards an increase in eight other metabolites in psoriatic skin. In serum, there was a significant increase of dimethylglycine and isoleucine. In parallel, psoriatic skin exhibited an increase of early inflammatory cytokines (IL-6, IL-8, TNF-α, IL-1ß) and correlation analysis highlighted some major clusters of immune-metabolic correlations. A cluster comprising scyllo-inositol and lysine showed correlations with T-cell cytokines; a cluster comprising serine and taurine showed a negative correlation with early inflammatory cytokines (IL-6, G-CSF, CCL3). A strong positive correlation was enlightened between glutathione and inflammatory cytokines/angiogenesis promoters of psoriasis. The integration of metabolic and immune data indicated a molecular signature constituted by IL-6, IL1-ra, DMG, CCL4, Ile, Gly and IL-8, which could discriminate patients and healthy subjects and could represent a candidate tool in the diagnosis of new-onset psoriasis.
Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Metabolômica , Estudos de Casos e Controles , Citocinas/sangue , Humanos , Mediadores da Inflamação/sangue , Isoleucina/sangue , Espectroscopia de Ressonância Magnética/métodos , Sarcosina/análogos & derivados , Sarcosina/sangue , Pele/metabolismoRESUMO
Cell-based strategies are today widely studied as possible therapies for wound healing. In this setting, fibroblasts play a key role since they are the main dermal cellular component and are responsible for extracellular matrix secretion. Several works report on the possibility of using fibroblast-derived extracellular matrix scaffolds for wound healing in skin injuries. While fibroblast-based substitutes have already been intensively studied by other groups, we focused our attention on the possibility of creating an adipose-derived stem cell (ADSC)-induced dermal scaffold for wound healing. ADSCs are a particular subset of mesenchymal stem cells present in the stromal vascular fraction of the adipose tissue. The aim of our work was to compare the ability of ADSCs and fibroblast to produce in vitro a scaffolding material, both in terms of collagen and fibronectin production. ADSCs turned out to be capable of efficiently producing a collagen and fibronectin-containing dermal matrix upon stimulation with ascorbic acid. We observed fibronectin and collagen production by ADSCs to be even more abundant when compared to fibroblasts'. Our results support the use of ADSC-induced sheets instead of fibroblast-based dermal substitutes as wound-healing strategies in full-thickness skin injuries.
Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Pele/lesões , Células-Tronco/metabolismo , Alicerces Teciduais , Tecido Adiposo/citologia , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Cultura Primária de Células , CicatrizaçãoRESUMO
BACKGROUND: Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis. METHODS: Thirty patients with histologically proven PCa, 33 with benign prostate hyperplasia (BPH) and 21 healthy controls were enrolled. cfDNA was extracted from seminal fluid samples. cfDNA quantification and analysis were performed using Qubit ssDNA Kit and Agilent 2100 Bioanalyzer. Statistical analysis included: Levene's test, Shapiro-Wilk, Kolmogorov-Smirnov and Kruskal Wallis tests. RESULTS: Median cfDNA was significantly higher in PCa patients 428.45â¯ng/mL (173.93-1159.62) compared to BPH patients 77.4â¯ng/mL (18.23-501) and healthy controls 25.4â¯ng/mL (15.37-76.62). scfDNA fragments longer than 1000 base-pairs were more common in patients with PCa compared to those with BPH and controls. CONCLUSIONS: scfDNA concentration and fragment size differed significantly in the three groups of PCa, BPH and healthy controls. Both parameters are potential clinical biomarkers for PCa and can be used in both early diagnosis and follow-up. Using automated systems for high-throughput cfDNA quantification could improve the reproducibility of the method and facilitate the implementation of liquid biopsies in the clinical setting.
Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Neoplasias da Próstata/diagnóstico , Sêmen/química , Idoso , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/sangue , Neoplasias da Próstata/terapiaRESUMO
A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.
Assuntos
Analgésicos Opioides/farmacologia , Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Piperidinas/farmacologia , Receptores sigma/agonistas , Compostos de Espiro/farmacologia , Analgésicos Opioides/síntese química , Analgésicos Opioides/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Masculino , Melanoma/patologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3-bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR35) and 6-bromo-3,3-bis(3-chloro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [3H]-thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [3H]-thymidine test was the best in predicting toxicity against melanoma cells.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Naftalenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Naftalenos/química , Timidina/metabolismoRESUMO
Gold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Water-soluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Metotrexato/farmacologia , Psoríase/tratamento farmacológico , Compostos de Sulfidrila/química , Administração Cutânea , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fármacos Dermatológicos/administração & dosagem , Fármacos Dermatológicos/química , Fármacos Dermatológicos/farmacologia , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/ultraestrutura , Metotrexato/química , Metotrexato/farmacocinética , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Pele/metabolismo , Absorção Cutânea , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
It is known that liquorice root is rich in compounds which exert several pharmacological actions. In the present study, we evaluated the effect of glycyrrhizin (the main constituent of liquorice root) and of its metabolite aglycone, 18beta-glycyrrhetinic acid, on UVB-irradiated human melanoma cells: SKMEL-2 from metastatic tissue and SKMEL-28 from primary malignant melanoma. Tests performed (Trypan blue exclusion test, MTT and Western blot) showed that glycyrrhizin is not toxic for both types of cells. In SKMEL-28 cells, Bcl-2 expression was low after UVB irradiation, but it was increased when treated with glycyrrhizin. On the contrary, in the SKMEL-2 cell culture, Bcl-2 expression was not modified by the substances under study. The results show that glycyrrhizin treatment might offer protection from the damage induced in humans by UVB radiation, while it seems to be ineffective on metastatic cells. Further studies must be performed to understand the mechanism of the protective effect.
Assuntos
Citoproteção/fisiologia , Ácido Glicirrízico/farmacologia , Inibidores do Crescimento/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Raios Ultravioleta/efeitos adversos , Apoptose/efeitos da radiação , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Glicirretínico/farmacologia , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Since 1986, there have been reports in the literature that glycyrrhizin (GL), 18 alpha-glycyrrhetinic acid (18 alpha-GA) and 18 beta-glycyrrhetinic acid (18 beta-GA) can inhibit the growth of some murine tumours. Recently, testing the activity of GL and 18 alpha-GA on human tumour cells, we observed that both substances are good anti-proliferative agents, especially on those cells whose replication rate is slow. In the present study, the MTT test was performed on MCF-7, Hep-2 and VERO cells treated with increasing concentrations of GL and of its derivatives. Results showed that the substances do not have a toxic effect on VERO cells and that their anti-proliferative effect is evident only on the cell line (MCF-7) that evolves slowly. Finally, the TUNEL test revealed the presence of apoptosis in MCF-7 cells treated with dosages of GL and 18 alpha-GA, thereby confirming that these natural phytoestrogens could be considered as interesting new agents for cancer therapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ácido Glicirretínico/farmacologia , Ácido Glicirrízico/farmacologia , Neoplasias Laríngeas/tratamento farmacológico , Animais , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Chlorocebus aethiops , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Laríngeas/patologia , Células VeroRESUMO
Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches.
Assuntos
Melanoma/patologia , Células-Tronco Neoplásicas/fisiologia , Neoplasias Cutâneas/patologia , Adipogenia , Variação Antigênica , Antígenos de Diferenciação/metabolismo , Carcinogênese , Técnicas de Cultura de Células , Linhagem da Célula , Separação Celular , Células Cultivadas , Transição Epitelial-Mesenquimal , Citometria de Fluxo , Humanos , Melanoma/terapia , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Nestina/metabolismo , Osteogênese , Neoplasias Cutâneas/terapia , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND: The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts. RESULTS: Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors. CONCLUSION: Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets.