Effects of ACSVL3 Knockout on Lipid and Glucose Metabolism in Malignant Glioma Cells

glioma;brain cancer;lipid metabolism;ACSVL3;acyl-CoA synthetases;Biochemistry

Kolar, Elizabeth AnneRaben, Daniel M. ;

Johns Hopkins University

关键词: glioma;    brain cancer;    lipid metabolism;    ACSVL3;    acyl-CoA synthetases;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39716/KOLAR-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Gliomas are the largest category of primary central nervous system tumors.Glioblastoma multiforme (GBM) is a World Health Organization Grade IV glioma that comprises 70% of all gliomas.Prognosis is very poor once diagnosed, and current treatments cannot prolong survival after relapse.Very long-chain acyl-CoA synthetase 3 (ACSVL3) is overexpressed in malignant glioma, and depleting ACSVL3 in GBM cells (e.g. U87MG) diminishes their tumorigenic properties and affects signaling through receptor tyrosine kinases.An ACSVL3-deficient knockout (KO) U87MG cell line was generated to study how ACSVL3 contributes to the malignant properties of glioma cells.Acyl-CoA synthetase enzyme activity was measured with long- and very long-chain fatty acids: palmitic acid (C16:0), stearic acid (C18:0), behenic acid (C22:0), and lignoceric acid (C24:0).There were significant decreases in the activation of stearic and behenic acids, while the activation of palmitic acid and lignoceric acid did not change.Ceramide synthesis assays and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis revealed a decrease in C18:0 and C22:0 ceramides, reinforcing the enzyme activity assay results.LC/MS-MS analysis also revealed a decrease in sphingosine 1-phosphate, an important signaling molecule that affects growth and proliferation. Proteomic analysis showed lower protein levels for enzymes involved in ceramide synthesis in the ACSVL3 KO line.Fluorescent microscopy and thin layer chromatography analyses show that ACSVL3 deficiency affects lipid rafts and ganglioside synthesis.Proteomic analysis also predicted changes in glycolysis and the tricarboxylic acid cycle (TCA) when ACSVL3 is depleted in U87MG cells.Glycolytic enzymes were higher while TCA enzymes were lower in ACSVL3 KO cells.Immunofluoresence using an antibody that detects Tom20, a mitochondrial outer membrane marker, revealed differences in mitochondrial morphology in the ACSVL3 KO cells when compared to the U87MG cells.From these studies, we conclude that ACSVL3 is important for the synthesis of structural and signaling sphingolipids that contribute to the growth and proliferation of the GBM cells, and that this enzyme likely contributes to mitochondrial-involved carbohydrate metabolism.