LC-MS based sphingolipidomic study on A549 human lung adenocarcinoma cell line and its taxol-resistant strain.

BACKGROUND: Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T). METHODS: A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis. RESULTS: Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 (p < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T. CONCLUSIONS: This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, "inhibition of the de novo synthesis pathway" and "activation of glycosphingolipid pathway" may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer.

Improved sphingolipidomic approach based on ultra-high performance liquid chromatography and multiple mass spectrometries with application to cellular neurotoxicity.

The emerging field of sphingolipidomics calls for accurate quantitative analyses of sphingolipidome. Existing analytical methods for sphingolipid (SPL) profiling often suffer from isotopic/isomeric interference, leading to the low-abundance, but biologically important SPLs being undetected. In the current study, we have developed an improved sphingolipidomic approach for reliable and sensitive quantification of up to 10 subclasses of cellular SPLs. By integratively utilizing high efficiency chromatographic separation, quadrupole time-of-flight (Q-TOF) and triple quadrupole (QQQ) mass spectrometry (MS), our approach facilitated unambiguous identification of several groups of potentially important but low-abundance SPLs that are usually masked by isotopic/isomeric species and hence largely overlooked in many published methods. The methodology, which featured a modified sample preparation and optimized MS parameters, permitted the measurement of 86 individual SPLs in PC12 cells in a single run, demonstrating great potential for high throughput analysis. The improved characterization, along with increased sensitivity for low-abundance SPL species, resulted in the highest number of SPLs being quantified in a single run in PC12 cells. The improved method was fully validated and applied to a lipidomic study of PC12 cell samples with or without amyloid ß peptide (Aß) treatment, which presents a most precise and genuine sphingolipidomic profile of the PC12 cell line. The adoption of the metabolomics protocol, as described in this study, could avoid misidentification and bias in the measurement of the analytically challenging low-abundance endogenous SPLs, hence achieving informative and reliable sphingolipidomics data relevant to discovery of potential SPL biomarkers for Aß-induced neurotoxicity and neurodegenerative disease.

Novel Fatty Acid in Cordyceps Suppresses Influenza A (H1N1) Virus-Induced Proinflammatory Response Through Regulating Innate Signaling Pathways.

Influenza virus (IV) infections usually cause acute lung injury characterized by exaggerated proinflammatory responses. The paucity of therapeutic strategies that target host immune response to attenuate lung injury poses a substantial challenge in management of IV infections. In this study, we chemically synthesized a novel fatty acid (2Z,4E)-deca-2,4-dienoic acid (DDEA) identified from Chinese Cordyceps by using UHPLC-Q-TOF-MS techniques. The DDEA did not inhibit H1N1 virus replication but attenuated proinflammatory responses by reducing mRNA and protein levels of TNF-α, IFN-α, IFN-ß, IL-6, CXCL-8/IL-8, CCL-2/MCP-1, CXCL-10/IP-10, CCL-3/MIP-1α, and CCL-4/MIP-1ß in A549 cells and U937-derived macrophages. The anti-inflammatory effect occurred through downregulations of TLR-3-, RIG-I-, and type I IFN-activated innate immune signaling pathways. Altogether, our results indicate that DDEA may potentially be used as an anti-inflammatory therapy for the treatment of IV infections.

Improved approach for comprehensive profiling of gangliosides and sulfatides in rat brain tissues by using UHPLC-Q-TOF-MS.

Gangliosides (GAs) and sulfatides (STs) are major acidic glycosphingolipids (GSLs) that are particularly abundant in the central nervous system and associated with substantial neurodegenerative diseases. In this study, we developed an improved approach for the comprehensive profiling of GAs and STs in rat brain tissues by adopting a pre-fractionation step before the LC-MS analysis. The pre-fractionation step allows the efficient enrichment of different types of acidic GSLs and the removal of high-abundance interferences, thereby greatly enhanced the detection sensitivity and accuracy of low-abundance acidic GSLs. By using this improved approach, a total of 340 acidic GSLs (from 281 compositions) were characterized in rat brain tissues, including 277 GAs (from 230 compositions) and 63 STs (from 51 compositions), among which 57 GAs and 14 STs were novel acidic GSLs that have not been reported previously. This study represented the most comprehensive profiling of acidic GSLs in rat brain tissues. The result of this study greatly enlarged our understanding of the structural diversity of natural acidic GSLs, and provided important chemical information for the exploration of biological function of acidic GSLs in the central nervous system.

Alterations of Sphingolipid Metabolism in Different Types of Polycystic Ovary Syndrome.

The roles of sphingolipids in polycystic ovary syndrome (PCOS) are still unknown. This study aimed to investigate the sphingolipid characteristics for different types of PCOS using liquid chromatography-mass spectrometry (LC-MS). A total of 107 women with PCOS and 37 healthy women as normal controls were studied. PCOS patients were further classified into non-obesity with insulin resistance (IR) (NOIR), obesity with IR (OIR), and non-obesity and non-IR (NIR) subgroups. A total of 87 serum sphingolipids, including 9 sphingosines, 3 sphinganines, 1 sphingosine-1-phosphate (S1P), 19 ceramides (Cers), 1 ceramide-1-phosphate, 44 sphingomyelins (SMs), 4 hexosylceramides, and 6 lactosylceramides (LacCers) were analyzed using an improved sphingolipidomic approach based on LC-MS. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients when compared with healthy women, and SM species with long saturated acyl chains showed potential as novel biomarkers of PCOS. In addition, the level of LacCer was only elevated in NIR, and there was almost no change in NOIR and OIR. This study is the first to report the comprehensive sphingolipidomic profiling of different subgroups of PCOS with or without IR or obesity and suggests that serum sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.

Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS.

In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia.

New Immunosuppressive Sphingoid Base and Ceramide Analogues in Wild Cordyceps.

A comprehensive identification of sphingoid bases and ceramides in wild Cordyceps was performed by integrating a sequential chromatographic enrichment procedure and an UHPLC-ultrahigh definition-Q-TOF-MS based sphingolipidomic approach. A total of 43 sphingoid bases and 303 ceramides were identified from wild Cordyceps, including 12 new sphingoid base analogues and 159 new ceramide analogues based on high-resolution MS and MS/MS data, isotope distribution, matching with the comprehensive personal sphingolipid database, confirmation by sphingolipid standards and chromatographic retention time rule. The immunosuppressive bioassay results demonstrated that Cordyceps sphingoid base fraction exhibits more potent immunosuppressive activity than ceramide fraction, elucidating the immunosuppressive ingredients of wild Cordyceps. This study represented the most comprehensive identification of sphingoid bases and ceramides from a natural source. The findings of this study provided an insight into therapeutic application of wild Cordyceps.

LC-MS Based Sphingolipidomic Study on A2780 Human Ovarian Cancer Cell Line and its Taxol-resistant Strain.

Drug resistance elicited by cancer cells continue to cause huge problems world-wide, for example, tens of thousands of patients are suffering from taxol-resistant human ovarian cancer. However, its biochemical mechanisms remain unclear. Sphingolipid metabolic dysregulation has been increasingly regarded as one of the drug-resistant mechanisms for various cancers, which in turn provides potential targets for overcoming the resistance. In the current study, a well-established LC-MS based sphingolipidomic approach was applied to investigate the sphingolipid metabolism of A2780 and taxol-resistant A2780 (A2780T) human ovarian cancer cell lines. 102 sphingolipids (SPLs) were identified based on accurate mass and characteristic fragment ions, among which 12 species have not been reported previously. 89 were further quantitatively analyzed by using multiple reaction monitoring technique. Multivariate analysis revealed that the levels of 52 sphingolipids significantly altered in A2780T cells comparing to those of A2780 cells. These alterations revealed an overall increase of sphingomyelin levels and significant decrease of ceramides, hexosylceramides and lactosylceramides, which concomitantly indicated a deviated SPL metabolism in A2780T. This is the most comprehensive sphingolipidomic analysis of A2780 and A2780T, which investigated significantly changed sphingolipid profile in taxol-resistant cancer cells. The aberrant sphingolipid metabolism in A2780T could be one of the mechanisms of taxol-resistance.