Abstract
Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of omega-3 fatty acids and ultimately affect their therapeutic potentials.
Highlights
While much effort has been devoted to genomic profiling leading to the identification of certain gene components of cancer, information on metabolomics and lipidomics in cancer cells or tissues is limited
When we treated two non-small cell lung cancer (NSCLC) A549 and H596 cells with eicosapentaenoic acid (EPA), EPA inhibited the proliferation of A549 cells (IC50,6.25 mM) more effectively than it did the H596 cells (IC50.50 mM) (Fig. 2A) even though these two cell lines showed similar COX-2 expression (Fig. 2B)
To delineate the mechanisms that would mediate the differential effects noted with EPA treatment, we evaluated the different mass spectral profiles of these cell lines using matrix-assisted laser desorption/ ionization (MALDI)-mass spectrometry (MS) followed by Principal component analysis (PCA) analysis (Fig. 3)
Summary
While much effort has been devoted to genomic profiling leading to the identification of certain gene components of cancer, information on metabolomics and lipidomics in cancer cells or tissues is limited. Advancements in commercially available instrumentation, including MALDI coupled to the linear ion trap Orbitrap instrument or quadrupole-time-of-flight (QTOF) mass spectrometers have allowed for the successful generation of mass spectra in the lower mass ranges (,1000 Daltons), allowing for MALDI-MS profiling of metabolites including lipids, not typically common with MALDI instrumentation due to matrix effects and source fragmentation [7,8,9] These advancements in MS and ionization instrumentation have moved MALDI-MS into numerous new research areas, including lipidomics [7,10,11,12], peptide [2], drug [13,14,15,16], and metabolite [17] analyses directly from biological samples, including tissues. These recently published studies highlight the expanded use of MALDI-MS for direct tissue analysis to characterize tissues on the basis of their metabolite, lipid, peptide, and protein profiles
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