| Systems biology is a new and rapidly developing research area in which a systems-level understanding of a biological response of cells and organisms can be achieved through the quantitative analysis of all molecules in a cell or an organism and the study of the interactions among these molecules. Therefore, high-throughput technologies for biological molecules are required. For example, genomics and proteomics are high-throughput analysis methods for DNA/RNA and proteins, respectively. Recently, a new concept 'lipidomics' has been proposed. It is a high-throughput analysis technology for lipids using mass spectrometry (MS). With lipidomic technique, our laboratory had studied the effects of alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on sphingolipids metabolism. We have found that MNNG can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR). Such clustering mostly occurs at the lipid rafts, and sphingolipids play a major role in this process.In lipid research, various inhibitors of lipid metabolism are often utilized in order to understand the metabolic pathway of lipids. For instance, imipramine, an inhibitor of acid sphingomyelinase (ASM), can inhibit the hydrolyzation of sphingomyelin to ceramide, eventually reduces the concentration of ceramide in cells. The polyene antibiotics filipin and nystatin have been routinely used in lipid research; however, very few information is available for their effects on lipid metabolism, except that they can interfere with the structure of cellular lipid rafts. Therefore, we studied the effects of filipin and nystatin on cellular sphingolipids metabolism and how they influence the cellular responses to MNNG using the lipidomic approach. The effects of filipin and nystatin on sphingolipids metabolism in FL cellsCellular sphingolipids were extracted from FL cells after 30 min treatment with two different concentrations of filipin and nystatin. Matrix assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) was used to analyzechanges of cellular sphingolipids.Results: FL cells were treated with either 0.2 μg/mL or 10 μg/mL filipin. With low filipin concentration treatment, new peaks such as m/z 536, 580, 624, 660, 720 appeared, indicating that there were new ceramides synthesized. On the other hand, there were no significant changes in the m/z range of 750 - 900, which represents the sphingomyelin molecules. These data suggested that filipin had stronger effects on ceramide metabolism than on sphingomyelin metabolism. The high-concentration-treated group showed similar effects.For nystatin treatment, it was found that at a concentration of 50 μg/mL, the intensity of ions at m/z 812, 834, and 836 in the range of m/z 750 - 900 were increased, indicating that nystatin could activate the synthesis of sphingomyelin. In the range of m/z 500 - 750, ceramides at m/z 612, 660, 676, 720 appeared. 100 μg/mL nystatin also had similar effects. However, its had stronger effects on ceramide synthesis, as two new peaks m/z 552 and 658 appeared besides those induced by 50 μg/mL nystatin. The effects of filipin and nystatin on MNNG-induced changes of sphingolipidsPreviously, we have found that MNNG can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR), and we have also discovered sphingolipids play an important role in this process. Nystatin, which can disrupt lipid rafts, can inhibit the MNNG-induce receptor clustering . But by which way such inhibition occurs is not clear. Therefore, we analyzed whether filipin and nystatin could interfere with MNNG-induced changes of sphingolipids metabolism.Seven groups, i.e., Blank Control, DMSO-solvent control, MNNG (10μM), filipin (10μg/mL), nystatin (100μg/mL), MNNG (10μM) + filipin (10μg/mL), MNNG (1μM) + nystatin (100 μg/mL), were set in the experiment. After MS analysis, we found that MNNG, filipin, and nystatin can all induce changes in sphingolipids metabolism, with MNNG exhibiting the strongest effects. On the other hand, many sphingolipids induced by MNNG-treatment were inhibited by pre-incubation with either filipin or nystatin. For example, MNNG-induced m/z 567.5was inhibited by filipin; m/z 572.2 was inhibited by both filipin and nystatin; m/z 606.1 was inhibited by nystatin. The possible structures for some peaks were proposed based on the sphingolipids MS database built by our laboratory.The effects of MNNG, filipin, and nystatin on key enzymes in the sphingolipids metabolism in FL cellsSince MNNG, filipin and nystatin can all induce changes in cellular sphingolipids metabolism, it was of our interest to find out the cause for these changes. So we evaluated the expression of three key enzymes in sphingolipids biosynthesis pathway, e.g., serine palmitoyltransferase (SPT), acid sphingomyelinase (ASM), and sphingomyelin synthase (SMS) under various conditions as described above. It was found that there were no significant differences for the mRNA levels of SMS,SPT and ASM. Conclusion:1. Both filipin and nystatin can affect cellular sphingolipid metabolism. According to the different changes of ceramides and sphingomyelins after filipin and nystatin treatment, the possible targets in sphingolipids biosynthesis pathway for filipin and nystatin could be hypothesized, which might provide clues for further research.2. Filipin and nystatin can inhibit changes in FL cellular sphingolipids metabolism induced by MNNG The influences of MNNG, filipin and nystatin on the metabolism of lipids in FL cells were not through the changes of the expression of the three major enzymes. |
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