RESUMEN
To identify proteins involved in melanoma metastasis mechanisms, comparative proteomic studies were undertaken on B16F10 and B16Bl6 melanoma cell lines and their subsequent syngenic primary tumours as pulmonary metastases were present only in the mice bearing a B16Bl6 tumour. 2DE analyses followed by MALDI-TOF identification showed variations of 6 proteins in vitro and 13 proteins in vivo. Differential expressed proteins in tumours were related to energy production and storage. Two differentially expressed proteins which had not been previously associated to melanoma progression, annexin A1 (ANXA1) and creatine kinase B (CKB), were found both in cells and in tumours. To characterize ANXA1 involvement in melanoma B16 dissemination, we reduced ANXA1 protein level by siRNA and observed a significant decrease of B16Bl6 cell invasion through Matrigel coated chambers. We further demonstrated that the presence of several formyl peptide receptors (FPR1, FPRrs1 and 2) revealed by qRT-PCR, played a role in B16 invasion: incubation of B16Bl6 cells with the FPR agonist (fMLP) or antagonist (tBOC) enhanced or decreased Matrigel coated chamber invasion respectively, with a correlation of ANXA1 levels in both treatments. As ANXA1 could bind to FPRs, this should amplify invasion and enhance melanoma dissemination.
Asunto(s)
Anexina A1/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteómica , Animales , Secuencia de Bases , Línea Celular Tumoral , Creatina Quinasa/metabolismo , Electroforesis en Gel Bidimensional , Ratones , Metástasis de la Neoplasia , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
In recent years, there has been dramatic worldwide increase in incidence of malignant melanoma. Although localised disease is often curable by surgical excision, metastatic melanoma is inherently resistant to most treatments. In this context, targeted radionuclide therapy could be an efficient alternative. After pharmacomodulation study, we selected a quinoxaline derivative molecule (ICF01012) for its high, specific and long-lasting uptake in melanoma with rapid clearance from nontarget organs providing suitable dosimetry parameters for targeted radiotherapy. Aim of this study was to investigate, in vivo, efficacy of [(131)I]ICF01012 on nonmetastatic B16F0, metastatic B16Bl6 or human M4Beu melanoma tumours. First, colocalisation of ICF01012 with melanin by SIMS imaging was observed. Second, we showed that treatment drastically inhibited growth of B16F0, B16Bl6 and M4beu tumours whereas [(131)I]NaI or unlabelled ICF01012 treatment was without significant effect. Histological analysis and measure of PCNA proliferation marker expression showed that residual B16 tumour cells exhibit a significant loss of aggressiveness after treatment. This effect is associated with a lengthening of the treated-mice survival time. Moreover, with B16Bl6 model, 55% of the untreated mice had lung metastases whereas no metastasis was counted on treated group. Our data demonstrated a strong anti-tumoural effect of [(131)I]ICF01012 for radionuclide therapy on murine and human in vivo pigmented melanoma models, whatever their dissemination profiles and their melanin content be. Further studies will attempt to optimise therapy protocol by increasing the balance between the anti-tumoural effect and the safety on non-target organs.
Asunto(s)
Antineoplásicos/farmacología , Radioisótopos de Yodo , Melanoma Experimental/diagnóstico por imagen , Melanoma Experimental/terapia , Quinoxalinas/farmacología , Animales , Antineoplásicos/uso terapéutico , Western Blotting , Línea Celular Tumoral , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/secundario , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Antígeno Nuclear de Célula en Proliferación/sangre , Quinoxalinas/uso terapéutico , Cintigrafía , Radiofármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa de Ion SecundarioRESUMEN
BACKGROUND AND OBJECTIVES: Skin without significant dyschromia is an aesthetic goal of people worldwide. Current options for lightening skin could have significant drawbacks. The antisense strategy may be a viable alternative. The reactions in melanogenesis are catalyzed mainly by tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. Activation of tyrosinase is associated with phosphorylation by protein kinase C-betaI (PKC-betaI) and formation of a complex between phosphorylated tyrosinase and TRP-1. The aim of this study was to use 2 antisense oligonucleotides to modulate the synthesis of the tyrosinase/TRP-1 complex, PKC-beta, or both by interacting with the targeted mRNA, thus whitening skin by interfering with melanogenesis at the translational level. METHODS/STUDY DESIGN: In the in vitro study, the effect of the antisense oligonucleotides was evaluated by measuring the rate at which dihydroxyphenylalanine (DOPA) oxidase transforms L-DOPA to DOPAchrome in the pathway for melanin biosynthesis. A reduction in the reaction rate compared to the controls corresponded to a decrease in the enzyme activity and, consequently, to a reduction of the formation of melanin pigments. To evaluate the in vivo lightening effect of the antisense oligonucleotides, 30 Asian women volunteers with pigmented spots on both hands applied the test product twice daily for 8 weeks. The test product was applied to 2 marked-off areas of the hand: a pigmented spot (to evaluate the effect of the test product on the color of the spot) and a nonpigmented spot area (to evaluate the effect of the test product on normal skin pigmentation). The lightening effect was evaluated by comparing chromametric and mexametric parameters before treatment, after 4 weeks, and after 8 weeks. RESULTS: In vitro DOPA-oxidase activity was inhibited by 13% in melanocytes treated with the antisense sequence for PKC-BI alone, by 16% with the antisense sequence for TRP-1 alone, and by 36% with the association of 2 sequences. The inhibiting effect with both sequences required the specific sequences with nonreversed polarities. In vivo clinical results showed statistically significant whitening in both pigmented spots and nonpigmented spots when the test product was applied twice daily for 8 weeks by up to 30 Asian women. CONCLUSIONS: The association of TRP-1 and PKC-betaI antisense molecules significantly increased the inhibition of tyrosinase activity on human melanocytes. Antisense oligonucleotides are a new generation of active cosmetic ingredients that offer unprecedented specificity, biological stability, and safety in lightening skin. This is the first report of positive results in a cosmetic based on the use of these new active agents.
Asunto(s)
Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Oxidorreductasas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Envejecimiento de la Piel/efectos de los fármacos , Pigmentación de la Piel/efectos de los fármacos , Adulto , Femenino , Humanos , Persona de Mediana Edad , Monofenol Monooxigenasa/genética , Oxidorreductasas/genética , Proteína Quinasa C/genética , Proteína Quinasa C betaRESUMEN
BACKGROUND/OBJECTIVES: [corrected] The signs of aging may originate from natural processes or from exposure to the sun, wind, or other environmental factors. To evaluate the anti-aging effects of potential agents researchers must first identify and be able to quantify epidermal markers that change with aging. This paper summarizes the results of studies conducted to evaluate the transcriptional effects of an Aframomum angustifolium seed extract and Malva Sylvestris extract, and the antiaging efficacy of a skin care product containing the Aframomum angustifolium seed extract. METHODS: The transcriptional effect of an Aframomum angustifolium seed extract on normal human keratinocytes (NHKs) and normal human fibroblasts (NHF) was evaluated in vitro with the use of a low-density DNA array technology. The Malva Sylvestris extract was studied with a commercial DNA macroarray and by a real-time quantitative reverse transcriptase-polymerase chain reaction. The in vitro anti-aging activities of the Malva sylvestris extract were compared with those of all-trans retinoic acid (RA), a well-established topical therapy for photodamage and wrinkles. The genes studied were known to be modified by RA. The anti-aging efficacy of a facial skin care product containing Aframomum angustifolium seed extract was evaluated in a single-center study using image processing analysis and in a 2-center study by evaluation of the photographs by the investigator, independent evaluators, and subjects. RESULTS: In general, the Aframomum angustifolium seed extract strongly modified the gene expression profiles of NHKs and weakly modified the gene expression profiles of NHFs. After incubation with Aframomum angustifolium seed extract, the expressions of 3 antioxidant genes (metallothionein 1, metallothionein 2, and thioredoxin) were increased in NHKs, while expressions of 1 antioxidant gene (glutathione peroxidase) was increased in NHFs. Concerning the Malva sylvestris extract, a cDNA macro-array technology experiment with the reconstructed human epidermis model showed that some genes modulated by treatment with the Malva sylvestris extract are also regulated by RA treatment indicating a similar activity at the mRNA level. In the single-center study, a facial skin care product containing the Aframomum angustifolium seed extract significantly improved the homogeneity of the skin. The areas of the detected objects (skin imperfections) decreased significantly on each studied area of the face and the variance decreased significantly over the entire face. In the 2-center study, 28% percent of the subjects reported a greater than 50% overall global improvement in their skin by the end of the study compared to 11% of the subjects after 4 weeks of treatment. Seventy-six percent of subjects said they would purchase the cream. CONCLUSIONS: The authors developed a low-density DNA chip method that permitted the study of the transcriptional effect of Malva Sylvestris extract and of Aframomum angustrifolium seed extract. The gene expression profiles obtained demonstrate the anti-aging properties of these compounds. An in vivo single-center study, performed and analyzed with an assay based on image processing analysis, demonstrated the antiwrinkle activity of a formulation containing the Aframomum angustifolium seed extract. The data obtained in the 2-center study suggests that the cosmeceutical containing Aframomum angustifolium seed extract produces a global rejuvenation effect in terms of redness, pigmentation, and fine lines similar to that noted utilizing an intense pulse light source.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Malva , Extractos Vegetales/farmacología , Envejecimiento de la Piel/efectos de los fármacos , Zingiberaceae , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Semillas , Piel/efectos de los fármacos , Piel/metabolismoAsunto(s)
Células Dendríticas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transcripción Genética , Análisis de Varianza , Células Dendríticas/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Lipopolisacáridos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Transducción de Señal , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
This study reports a series of 14 new iodinated and fluorinated compounds offering both early imaging ((123)I, (124)I, (18)F) and systemic treatment ((131)I) of melanoma potentialities. The biodistribution of each (125)I-labeled tracer was evaluated in a model of melanoma B16F0-bearing mice, using in vivo serial γ scintigraphic imaging. Among this series, [(125)I]56 emerged as the most promising compound in terms of specific tumoral uptake and in vivo kinetic profile. To validate our multimodality concept, the radiosynthesis of [(18)F]56 was then optimized and this radiotracer has been successfully investigated for in vivo PET imaging of melanoma in B16F0- and B16F10-bearing mouse model. The therapeutic efficacy of [(131)I]56 was then evaluated in mice bearing subcutaneous B16F0 melanoma, and a significant slow down in tumoral growth was demonstrated. These data support further development of 56 for PET imaging ((18)F, (124)I) and targeted radionuclide therapy ((131)I) of melanoma using a single chemical structure.
Asunto(s)
Radioisótopos de Flúor/uso terapéutico , Radioisótopos de Yodo/uso terapéutico , Melanoma Experimental/radioterapia , Tomografía de Emisión de Positrones , Tomografía Computarizada de Emisión de Fotón Único , Animales , Radioisótopos de Flúor/farmacocinética , Radioisótopos de Yodo/farmacocinética , Melanoma Experimental/diagnóstico por imagen , Ratones , Distribución TisularAsunto(s)
Dermatitis Fototóxica/patología , Epidermis/patología , Envejecimiento de la Piel/inmunología , Envejecimiento de la Piel/patología , Luz Solar/efectos adversos , Anciano , Animales , Dermatitis Fototóxica/fisiopatología , Tejido Elástico/patología , Tejido Elástico/ultraestructura , Epidermis/metabolismo , Humanos , Queratinocitos/metabolismo , Pronóstico , Medición de RiesgoRESUMEN
INTRODUCTION: The use of radiolabeled molecules allows the study of in vivo biodistribution, target organs, and kinetic profile after systemic administration by 1) radioactive organ counting and 2) quantitative autoradiographic analysis of whole-body slices (WBA). However, these techniques are time- and animal consuming for several times studied. So, in vivo scintigraphic imaging should appear of interest for a first screening of compounds, as it is able to rapidly demonstrate tumoral uptake and kinetics by serial examinations in the same mice. MATERIALS AND METHODS: In this study, the tumoral distribution and kinetics of six molecules considered as potential melanoma tracers radiolabeled with (125)I were analyzed by gamma-scintigraphy comparatively to the results obtained by WBA. Tumoral uptake has been quantified and expressed by: 1) tumor-to-background ratios and 2) standardized tumoral uptake (STU) in percent injected dose per gram, with tumor weight being extrapolated from the measurement of the two diameters. RESULTS: Results from STU analysis showed good agreement (correlation coefficient = 0.92) with those of WBA, and the same classification of compounds (on the basis of their melanoma affinity) was obtained, with two compounds (of six) being rejected. CONCLUSIONS: [(125)I] scintigraphic imaging appeared as a relevant, easy-going method for a first pharmacologic selection in mice.
Asunto(s)
Radioisótopos de Yodo/farmacología , Melanoma/diagnóstico por imagen , Cintigrafía/métodos , Neoplasias Cutáneas/diagnóstico por imagen , Animales , Ensayos de Selección de Medicamentos Antitumorales , Vectores Genéticos , Cinética , Masculino , Melanoma/tratamiento farmacológico , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Fantasmas de Imagen , Neoplasias Cutáneas/tratamiento farmacológico , Imagen de Cuerpo EnteroRESUMEN
BACKGROUND: Studying photoexposed and photoprotected skin biopsies from young and aged women, it has been found that a specific zone, composed of the basal layers of the epidermis, the dermal epidermal junction, and the superficial dermis, is major target of aging and reactive oxygen species. We showed that this zone is characterized by significant variations at a transcriptional and/or protein levels. AIMS: Using low-density DNA chip technology, we evaluated the effect of a natural mixture of Aframomum angustifolium seed extract containing labdane diterpenoids on these aging markers. METHODS: Expression profiles of normal human fibroblasts (NHF) were studied using a customized cDNA macroarray system containing genes covering dermal structure, inflammatory responses, and oxidative stress defense mechanisms. For normal human keratinocyte (NHK) investigations, we chose OLISA technique, a sensitive and quantitative method developed by BioMérieux specifically designed to investigate cell death, proliferation, epidermal structure, differentiation, and oxidative stress defense response. RESULTS: We observed that this extract strongly modified gene expression profiles of treated NHK, but weakly for NHF. This extract regulated antioxidant defenses, dermal-epidermal junction components, and epidermal renewal-related genes. CONCLUSION: Using low-density DNA chip technology, we identified new potential actions of A. angustifolium seed extract on skin aging.