Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells

Abstract

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.

Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells

“…1d). These findings agreed with the previous reports of tumor cells containing appreciable populations of dihydrospecies of Cers and sphingomyelins [7][8][9] and their composition were elevated under hypoxia [3,10]. (t18:0-C22:0h), and 1030.9 (t18:0-C24:0h).…”

“…Sphingolipids were purified from 5×10 7 cells as previously described [3,4]. Briefly, lipids were extracted with a chloroformmethanol mixture.…”

“…Mass spectrometry was performed as previously described [3,4]. Lipids were prepared as 10-350 pmol/μl solutions in chloroform:methanol (2:1).…”

“…All mass spectrometric data were acquired and analyzed using MALDI-MS software (Shimadzu/Kratos, UK). The compositions of molecular species from each class of lipid were calculated from the peak areas obtained from spectra as previously described [3,4].…”

Glycobiology Modifications in Intratumoral Hypoxia: The Breathless Side of Glycans Interaction

“…These changes are also affected by hypoxia, as demonstrated by Tanaka et al in human colorectal cancer cells (Caco-2) under hypoxic conditions (O 2 1%) for three days [129]. The authors showed that, in normoxia, major constituent ceramide species of neutral glycosphingolipids (NGSLs) were composed of the usual long-chain base of sphingosine (d18: 1) and hydroxy fatty acid (HFA); while, under hypoxic conditions, all the Cers, and nearly 80% of the sphingomyelins, were dihydrosphingolipids, composed of d18: 0-NFAs, but a significant proportion of d18: 1-HFAs still remained in the NGSLs [129]. …”