(P16-016-23) Understanding the Impact of Altered Phosphatidylethanolamine (PE) Metabolism in Human Hepatoma Cells

Objectives: Altered phospholipid metabolism has been shown to induce non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). Phosphatidylethanolamine (PE) is the second most abundant glycerophospholipid in mammalian membranes and is synthesized primarily through the PE arm of the Kennedy Pathway. It is important for lipid storage/transport and cell proliferation. Ethanolamine phosphate phospholyase (ETNPPL) is a metabolic enzyme expressed in astrocytes and hepatocytes that catabolizes phosphoethanolamine (p-etn). In humans, low ETNPPL expression has been linked to reduced survivability in patients with hepatocellular carcinoma (HCC). Therefore, we investigated the impact of removing p-etn on PE synthesis and overall cellular health.

Methods: To investigate the biological effects of removing p-etn, human Huh7 hepatoma cells were transfected with a plasmid containing mouse ETNPPL cDNA. Immunofluorescence was conducted to visualize the localization of ETNPPL. Cells were treated with 3H-ethanolamine to assess incorporation into p-etn and PE. Proliferation rate of cells was measured using a WST-1 assay. mRNA expression was measured for many tumor suppressor genes, including p53.

Results: Immunofluorescence showed that ETNPPL colocalized with DAPI, indicating it is localized in the nucleus. Radiolabel experiments showed there was a decrease in ethanolamine incorporation into PE and an increased PC/PE ratio in cells expressing ETNPPL. Huh7 cells showed a significant decrease in 3H-p-etn when ETNPPL was expressed compared to control cells. ETNPPL expressing cells also had a decreased rate of cell proliferation with increased mRNA and protein expression of p53, a tumor suppressor gene. In addition to p53, an increase in expression of four other tumor suppressor genes (CDKN1A, BBC3, BAX, BRCA1) was observed. Finally, ETNPPL expressing cells exhibited an increase in levels of acetylated histones H3 and H4 when measured by Western blot.

Conclusions: ETNPPL observably removes p-etn and impacts PE metabolism in the cell. This alteration influences cellular changes that could link ETNPPL to NAFLD and HCC.

Funding Sources: Canadian Institutes of Health Research