Disruption of sphingolipid and glycosphingolipid profiles in human bronchial epithelial cells and extracellular vesicles during gradual benzo[a]pyrene-induced epithelial-to-mesenchymal transition.

In this study, effects of environmental carcinogen benzo[a]pyrene (BaP) on deregulation of sphingolipid (SL) and glycosphingolipid (GSL) metabolism were studied during BaP-induced transformation of normal human bronchial epithelial HBEC-12KT cells. After 2-weeks of exposure, BaP altered their morphology, while it downregulated sphingosine-1-phosphate (S1P) and upregulated sphingosine, gangliosides, GM3 and Lc3 GSLs. A longer, 8-week exposure to BaP, further increased cell migratory capacity, induced epithelial-to-mesenchymal transition (EMT) markers and EMT-related transcriptional regulators (SNAI1, ZEB1 and ZEB2), and it increased intracellular sphingosine, ceramide-1-phosphate, as well as a series of GSLs (glucosylceramide, lactosylceramide, GM1a, GD3, Lc3 and Gb3). A distinct profile of SL/GSL levels was observed in fully transformed cells established via exposure to BaP for 12 weeks. Increased sphingosine, S1P, ceramide-1-phosphate, GD3, Lc3 and Gb3 levels were paralleled by a decrease of other SL/GSLs, including GM3 pathway. These alterations were also partly reflected within extracellular vesicles and microvesicles, particularly in those released from fully transformed cells. Significantly altered enzymes of SL/GSL metabolism included a downregulation of S1P lyase and increased S1P, downregulation of GM-synthetic enzymes, and upregulation of enzymes of GD3 and Gb3 synthesis. Using siRNA-mediated knockdown of individual EMT transcriptional regulators, we then found them to play only a partial role in regulation of S1P, GM, GD3, Lc3 or Gb3, via deregulation of expression of the respective enzymes, suggesting that their enzymatic activities can be regulated also by other mechanisms. The mechanisms underlying deregulation of SL/GSL levels elicited by carcinogenic environmental pollutants and functions of individual SL/GSL species deserve further attention.