Phospholipids as cancer biomarkers: Mass spectrometry-based analysis.

Lipids, particularly phospholipids (PLs), are key components of cellular membrane. PLs play important and diverse roles in cells such as chemical-energy storage, cellular signaling, cell membranes, and cell-cell interactions in tissues. All these cellular processes are pertinent to cells that undergo transformation, cancer progression, and metastasis. Thus, there is a strong possibility that some classes of PLs are expected to present in cancer cells and tissues in cellular physiology. The mass spectrometric soft-ionization techniques, electrospray ionization (ESI), and matrix-assisted laser desorption/ionization (MALDI) are well-established in the proteomics field, have been used for lipidomic analysis in cancer research. This review focused on the applications of mass spectrometry (MS) mainly on ESI-MS and MALDI-MS in the structural characterization, molecular composition and key roles of various PLs present in cancer cells, tissues, blood, and urine, and on their importance for cancer-related problems as well as challenges for development of novel PL-based biomarkers. The profiling of PLs helps to rationalize their functions in biological systems, and will also provide diagnostic information to elucidate mechanisms behind the control of cancer, diabetes, and neurodegenerative diseases. The investigation of cellular PLs with MS methods suggests new insights on various cancer diseases and clinical applications in the drug discovery and development of biomarkers for various PL-related different cancer diseases. PL profiling in tissues, cells and body fluids also reflect the general condition of the whole organism and can indicate the existence of cancer and other diseases. PL profiling with MS opens new prospects to assess alterations of PLs in cancer, screening specific biomarkers and provide a basis for the development of novel therapeutic strategies. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:107-138, 2018.

Characterization of the changes in eicosanoid profiles of activated macrophages treated with 20(S)-ginsenoside Rg3.

In this study, we used ultra-performance liquid chromatography coupled with tandem mass spectrometry to assess the levels of eicosanoids from RAW264.7 macrophages treated with lipopolysaccharides (LPS) and 20(S)-ginsenoside Rg3 (Rg3). The production of nitric oxide (NO) and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in inflammatory macrophages treated with LPS. Rg3 treatment, however, decreased the levels of NO, TNF-α, and IL-6 in activated macrophages. Eicosanoids, known as major metabolites correlated with inflammation, have pro- or anti-inflammatory activities. For a detailed characterization of the eicosanoids altered by treatment with LPS and Rg3, the eicosanoids were profiled by multiple reaction monitoring. A total of 69 macrophage eicosanoids were analyzed and the profiling dataset was statistically analyzed. Principal component and hierarchical cluster analyses differentiated control cells from cells treated with LPS, Rg3, or LPS+Rg3 for 12 or 24h. Furthermore, 18 differentially regulated eicosanoids were found between macrophages treated with LPS for 24h and those treated with LPS+Rg3 for 24h (fold change>2, p value<0.05). These results indicate that Rg3 alters eicosanoid metabolism in activated macrophages treated with LPS. Furthermore, we also identified several eicosanoids correlated with the anti-inflammatory activity of Rg3.

UPLC-QqQ/MS-Based Lipidomics Approach To Characterize Lipid Alterations in Inflammatory Macrophages.

In this study, UPLC-QqQ/MS-based lipidomics was applied to profile various lipids from RAW264.7 macrophages treated with different concentrations of lipopolysaccharide (LPS). The degree of inflammation increased with the LPS concentration. To elucidate the altered lipid metabolism of inflammatory macrophages, we targeted to analyze 25 lipid classes from LPS-treated RAW264.7 cells. As a result, 523 lipid species were successfully profiled by using the optimal UPLC and MRM. Statistical data analyses such as PCA, PLS-DA, and HCA differentiated five RAW264.7 cells treated with different concentrations of LPS. VIP plot, heat map, and bar plot also provided lists of up- or down-regulated lipids according to the LPS concentration. From the results, 11 classes of lipids, TG, DG, ChE, PE, PS, PI, PA, LyPC, LyPE, Cer, and dCer, were increased, and three classes, cholesterol, PC, and LyPA, were decreased in an LPS concentration-dependent manner. Furthermore, the treatment of an anti-inflammatory compound recovered the levels of PC, PE, PI, PA, LyPE, LyPA, and Cer from the activated macrophages. Finally, these results demonstrate the correlation between inflammation and lipid metabolism in macrophages. The differentially regulated lipids also have the potential to be used as biomarkers for inflammation.

UPLC-MS/MS-Based Profiling of Eicosanoids in RAW264.7 Cells Treated with Lipopolysaccharide.

While both the pro- and anti-inflammatory effects of several eicosanoids have been widely studied, the degree of inflammation in cells that results from various eicosanoids has yet to be comprehensively studied. The objective of this study was to assess the effect of lipopolysaccharide (LPS) treatment on eicosanoid content in RAW264.7 cells. An Ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)-based profiling method was used to analyze the eicosanoid contents of RAW264.7 cells treated with different LPS concentrations. The profiling data were subjected to statistical analyses, such as principal component analysis (PCA) and hierarchical clustering analysis. LPS treatment increased nitric oxide production and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, in a concentration-dependent manner. In total, 79 eicosanoids were identified in the cells. RAW264.7 cells treated with different LPS concentrations were well differentiated in the PCA score plot. A heatmap was used to identify the eicosanoids that were up- or down-regulated according to the degree of inflammation and LPS concentration. Thirty-nine eicosanoids were upregulated and seven were down-regulated by LPS treatment in a concentration-dependent manner. Our novel UPLC-MS/MS technique can profile eicosanoids, and can evaluate the correlations between inflammation and eicosanoid metabolism.

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid-Mediated Autocrine Mechanism.

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat-stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non-CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere-forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC-associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1-specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA-stimulated CSC properties. Autotaxin, an LPA-producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA-producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA-dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin-LPA-LPA receptor 1-AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs.