Glutamate Signaling And Molecular Mechanism Of Interaction With Cytoskeleton Protein In Melanoma Cell

Background:Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). It acts via a number of glutamate receptor classes to induce neuronal depolarization. Glutamate receptors can be subdivided into ionotropic receptors, and the metabotropic receptors. In addition to its central function, regulatory effects of glutamate have been described in peripheral tissues such as bone, pancreas and skin. In the epidermis, glutamate is believed to play a role in barrier homeostasis and wound healing via NMDA receptors on keratinocytes. Expression of a functional mGluR5 has been reported on melanocytes, where it is not only suggested to be involved in the control of melanocyte proliferation, but also harbours potential toxicity and may ontribute to cell damage. Glutamate toxicity has been studied extensively in neurones. Excessive stimulation of both ionotropic and metabotropic glutamate receptors leads to cell death. Blockage of NMDA receptors with MK801 caused a melanocyte morphology change, which was associated with disorganisation of actin and tubulin microfilaments. Whereas the role of the glutamate signalling system in neurones is well documented, the presence of the various glutamate receptors on melanoma cells and their effects on melanoma cells behaviour has not been studied in detail. It is also unclear whether melanoma cells defects, such as in the pigmentary disorder vitiligo where functional melanocytes are lost from the epidermis by unknown mechanisms, could be related to glutamate receptors. It is necessary to elucidate the glutamate receptors expression of human melanoma cells and the effects of glutamate receptor agonists and antagonists on melanoma cells morphology and growth.Microtubule-associated proteins (MAPs) are a family of proteins that bind to and stabilize microtubules (MTs). MAP2 is one isoform of MAPs. Expression of MAP2 in vitro in heterologous cells induces rapid formation of stable MT bundles and cellular processes. Vijayasaradhi et al found that MAP2, a neuronspecific protein, is expressed abundantly in early invasive primary melanoma lesions and primary melanoma cell lines but is absent in metastatic melanomas lesions and cell lines. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Melanoma exhibits plasticity of differentiation and is known to differentiate along multiple, including endothelial and neuronal, cellular pathways. However, the effects of MAP2 on melanoma cells on morphology and growth are not well understood.Objective: Study the Glutamate signaling in melanoma cell and molecular mechanism with cytoskeleton protein.Methods:To investigate the expression, subcellular localization ,effects on melanoma growth, invasion and cell morphology change of MAP2a, NMDAR2A and mGluR1,we performed double-immunofluorescence staining coupled with confocal microscopy analysis, three dimensional atomic force microscopy analysis, flow cytometry analysis, tumor cell growth assay and in vivo study, mainly used human metastatic melanoma cell line WM451LU.Results:The results demonstrate that MAP2a, NMDAR2A and mGluR1 have similar subcellular localization, and for the first time showed that combined adenovirus-mediated MAP2a and NMDAR antagonist MK-801 treated melanoma cells led to morphologic alteration, the tumor cell represented the secondary and tertiary branches resembled normal melanocytes. NMDAR antagonist MK-801 and mGluR1 antagonist CPCCOEt showed the significant tumor growth suppression of melanomas in vitro. Therefore, MAP2a, NMDAR2A and mGluR1 appear to be a promising target for melanoma therapy.