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NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery.

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.

SUBMITTER: Santana-Filho AP

PROVIDER: S-EPMC5309734 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery.

Santana-Filho Arquimedes Paixão de AP Jacomasso Thiago T Riter Daniel Suss DS Barison Andersson A Iacomini Marcello M Winnischofer Sheila Maria Brochado SM Sassaki Guilherme Lanzi GL

Scientific reports 20170215

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 metabolo ...[more]

PMID: 28198377

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Project description:The two most common melanoma histopathologic subtypes, superficial spreading (SSM) and nodular melanoma (NM), are believed to represent sequential phases of linear progression from radial to vertical growth. Studies suggest, however, that SSM and NM are biologically distinct. We utilized an integrative genomic approach to examine the possibility that SSM and NM are the result of independent pathways characterized by unique molecular alterations. Cell lines including SSM, NM, metastatic melanoma, and melanocyte controls were evaluated for copy number changes and differential mRNA expression using single nucleotide polymorphism array (SNP 6.0, Affymetrix) and gene array (U133A 2.0, Affymetrix). Data sets were integrated to identify copy number alterations that correlated with gene expression, and array results were validated using immunohistochemistry on human tissue microarrays (TMAs) and an external data set. The functional effect of genomic deletion was assessed by lentiviral overexpression. Integrative genomics revealed 8 genes in which NM/SSM-specific copy number alterations were correlated with NM/SSM differential gene expression (P<0.05, Spearman’s rank). Pathways analysis of differentially expressed genes (N=114) showed enrichment for metabolic-related processes. SSM-specific genomic deletions (DIS3, MTAP, G3BP2, SEC23IP, USO1) were verified in an expanded panel of cell lines, and forced overexpression of MTAP in SSM resulted in reduced cell growth. Metabolism-related gene ALDH7A1 was verified as overexpressed in NM using human TMAs.The identification of recurrent genomic deletions in SSM not present in NM challenges the linear model of melanoma progression and supports the unique molecular classification of SSM and NM. Gene expression profiling using Affymetrix U133A 2.0 arrays was performed on 18 melanoma cell lines including 2 primary superficial spreading melanoma, 2 primary nodular melanoma, 2 metastatic nodular melanoma, and 12 metastatic cell lines. Four melanocyte control lines were also evaluated including 2 immortalized melanocyte cell lines (Hermes 1 and 2B) and 2 normal melanocyte lines cultured from neonatal foreskin (HEM-N and HEM-LP).

Project description:BackgroundThe ?-Melanocyte Stimulating Hormone (?MSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. ?MSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the ?MSH/Peroxisome Proliferator Activated Receptor (PPAR?) pathway as a new pathway on the B16-F10 mouse melanoma cell line. ?MSH induced the translocation of PPAR? into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the ?MSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to ?MSH by reducing proliferation and that PPAR? was involved in this effect. Due to its key role in the control of cell proliferation, PPAR? agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to ?MSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPAR? pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to ?MSH.MethodsWe employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the ?MSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E).ResultsThe ?MSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the ?MSH mediated effect on proliferation in NHMs but it did not mimic the ?MSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the ?MSH proliferative effect. Further experiments, treating melanoma cell lines with ?MSH in the presence/absence of GW9662, as an inhibitor of PPAR?, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure.ConclusionsIn both melanoma cell lines, ?MSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPAR? as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.

Dataset Information

NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery.

Publications

NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery.

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