Melanina y melanoma: Actualización de la estructura molecular y terapia fototérmica

Melanin pigment and melanoma are two fields of increasing interest and relevance in biomedical research. Melanins are ubiquitous biopigments with strong adaptive value and multiple functions. In mammals, melanin corresponds to eumelanin (brown-black) and pheomelanin (yellow-red), and is found mainly in the skin and derivatives, retinal pigmented epithelium, and central nervous systems (neuromelanin, in substantia nigra, locus coeruleus, etc.). Melanin also occurs in the malignant melanoma, which is one of the most aggressive and therapy-resistant tumors in veterinary and human medicine. Several chemical structures have been proposed for eumelanin, but there is still no agreement about its molecular organization. Two models, namely a flexible linear chain, and a rigid planar chain, are the structures that better agree with physico-chemical properties of eumelanin. The latter model, which appears as the most plausible structure, corresponds to a benzoquinone derivative of the porphycene ring, and explains the broad-band light absorption, antioxidant capacity, electric conductivity, and photothermal effect, as well as the multilayered and graphite-like organization shown by X-ray crystallography and electron microscopy. In addition to traditional oncologic treatments and recent immunological and gene therapy advances, photodynamic and photothermal approaches represent novel therapeutic modalities for melanoma. In the latter case, since eumelanin is practically the ideal photothermal sensitizer, the massive vibrational decay from photo-excited electronic states after NIR irradiation induces an immediate and highly efficient heating response that results in coagulative necrosis of the tumor. This allows repetitive treatments due to the remaining melanin contained in tumoral melanophages. Although the evolution and prognosis of the advanced melanoma is still a concern, new physical procedures can now be applied.Fil: Stockert, J.C. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal (INITRA). Buenos Aires, ArgentinaFil: Stockert, J.C. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Angel H. Roffo". Area Investigación. Buenos Aires, ArgentinaLa melanina y el melanoma son dos campos de interés y relevancia crecientes en investigación biomédica. Las melaninas son biopigmentos ubicuos con gran valor adaptativo y múltiples funciones. En mamíferos, la melanina corresponde a la eumelanina (marron-negro) y a la feomelanina (amarillo-rojo), y se encuentra en la piel y derivados, epitelio pigmentado de la retina, y sistema nervioso central (neuromelanina, en substantia nigra, locus coeruleus, etc). La melanina también está presente en el melanoma maligno, uno de los tumores más agresivos y resistentes a la terapia en medicina veterinaria y humana. Varias estructuras químicas han sido propuestas para la eumelanina, pero todavía no hay acuerdo sobre su organización molecular. Dos modelos, uno constituido por una cadena linear y flexible, y otro por una cadena plana y rígida son las estructuras que mejor encajan con las propiedades físico-químicas de la eumelanina. El último modelo, que parece ser la estructura más plausible, corresponde a un derivado benzoquinónico del porficeno y explica la absorción de luz de banda ancha, la capacidad antioxidante, la conductividad eléctrica y el efecto fototérmico, así como también la organización en multicapa similar al grafito mostrada por la cristalografía de rayos X y la microscopía electrónica. Sumados a los tratamientos oncológicos tradicionales y a los avances inmunológicos y de terapia génica más recientes, los abordajes fotodinámico y fototérmico representan novedosas modalidades terapéuticas para el melanoma. En este último caso, como la eumelanina es el sensibilizador fototérmico prácticamente ideal, el decaimiento vibracional masivo a partir de estados electrónicos foto-excitados por NIR induce un calentamiento inmediato y muy eficiente que produce la necrosis coagulativa del tumor. Esta respuesta permite tratamientos repetidos debido a la melanina remanente contenida en los melanófagos tumorales. Aunque la evolución y pronóstico del melanoma avanzado son todavía asuntos preocupantes, nuevos procedimientos físicos pueden ser ahora aplicados

Uptake and localization mechanisms of fluorescent and colored lipid probes. 1. Physicochemistry of probe uptake and localization, and the use of QSAR models for selectivity prediction

We outline the factors involved in precise targeting of lipids and membranes by probes, namely, lipid and probe chemistry, geometry/topography of probe delivery, and probe uptake kinetics. The special case of probe orientation within membranes also is considered. The varieties of commercially available fluorophores are described, and an overview of probe physicochemical properties (amphiphilicity, conjugated system size, electrical properties, head group size, lipophilicity and solubility) is provided together with notes on their parameterization. Probe-lipid physicochemical interactions, and their relations to parameterization, then are discussed including the nature and derivation of decision-rule QSAR models, partitioning and insertion of probes into bulk lipids and complications of this, partitioning and insertion of probes into membranes, and flip-flop of probes across membrane leaflets. A general QSAR algorithm for understanding lipid probe application then is set out. Problems and limitations are outlined. Biological issues include varied biomembrane composition, cell line effects and toxicity of fluorescent probes. Methodological issues include difficulties of estimating certain numerical structure parameters, the impure character of many fluorochromes and dyes, and the perturbation of biomembrane structure by fluorescent probes

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Uptake and localization mechanisms of fluorescent and colored lipid probes: part 3. protocols for predicting intracellular localization of lipid probes using QSAR models

We provide detailed protocols for applying the QSAR decision-rule models described in Part 2 of this paper. These procedures permit prediction of the intracellular localization of fluorescent probes or of any small molecular xenobiotic whether fluorescent or not. Also included is a set of notes that give practical advice on various possible problems and limitations of the methods, together with a flow chart that provides a graphical algorithmic summary of the QSAR models

Uptake and localisation of small-molecule fluorescent probes in living cells: a critical appraisal of QSAR models and a case study concerning probes for DNA and RNA

Small-molecule fluorochromes are used in biology and medicine to generate informative microscopic and macroscopic images, permitting identification of cell structures, measurement of physiological/physicochemical properties, assessment of biological functions and assay of chemical components. Modes of uptake and precise intracellular localisation of a probe are typically significant factors in its successful application. These processes and localisations can be predicted using quantitative structure activity relations (QSAR) models, which correlate aspects of the physicochemical properties of the probes (expressed numerically) with the uptake/localisation. Pay-offs of such modelling include better understanding and trouble-shooting of current and novel probes, and easier design of future probes (“guided synthesis”). Uptake models discussed consider adsorptive (to lipid or protein domains), phagocytic and pinocytotic endocytosis, as well as passive diffusion. Localisation models discussed include those for cytosol, endoplasmic reticulum, Golgi apparatus, lipid droplets, lysosomes, mitochondria, nucleus and plasma membrane. A case example illustrates how such QSAR modelling of probe interactions can clarify localisation and mode of binding of probes to intracellular nucleic acids of living cells, including not only eukaryotic chromatin DNA and ribosomal RNA, but also prokaryote chromosomes

Binding of cationic dyes to DNA: distinguishing intercalation and groove binding mechanisms using simple experimental and numerical models

Simple methods for predicting intercalation or groove binding of dyes and analogous compounds with double stranded DNA are described. The methods are based on a quantitative assessment of the aspect (width to length) ratio of the dyes. The procedures were validated using a set of 38 cationic dyes of varied chemical structures binding to well oriented DNA fibers and assessing binding orientation by linear dichroism and polarized fluorescence. We demonstrated that low aspect ratio dyes bound by intercalation, whereas more rod-like dyes were groove binders. Some problems that result and possible applications are discussed briefly

Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here