Abstract
Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of 1H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines.
Highlights
Figure 1. 1H Nuclear Magnetic Resonance (NMR) spectra of aqueous extracts obtained from mouse cell lines
It is evident upon simple inspection that all the metabolites demonstrated differences in concentration among the cell lines, and that differences in some metabolites concentrations were linked to major alterations in metabolism which occur in tumorigenic cells, examples of which are higher concentrations of lactic acid and lower concentrations of glycerol seen in the metastatic cell lines[20]
The majority of these studies, provide only 1D 1H or homonuclear 2D NMR assignments, which can result in erroneous assignments due to the high signal overlap in 1H NMR spectra obtained from aqueous extracts
Summary
Figure 1. 1H NMR spectra of aqueous extracts obtained from mouse cell lines. Melan-a (A), Tm1 (B), Tm5 (C) and B16-F10 (D). Established melanocytes cell lines are important for performing comparative studies[9], mitogenic agents such as tumour-promoting phorbol-ester must be added to the growth media in order to stimulate melanocyte growth[10,11]. Their mechanism of action involves the activation of a family of proteins named protein kinase C (PKC), which in turn play important roles in mediating cell growth, differentiation, and tumour promotion[12]. We investigated the effect of mitogenic agents on the metabolic profile of the melanocyte cell line
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