Ceramides (Cers) have recently been identified as key signaling molecules that mediate biological functions such as cell growth, differentiation, senescence, apoptosis, and autophagy. However, the functions of Cer accumulation in necrotic cell death remain unknown. The aim of this study was to clarify the relationship between Cer accumulation with inhibition of the conversion pathway of Cer and concomitant necrotic cell death. In order to minimize the effect of apoptosis against necrotic cell death, A549 cells having the inhibiting effect of caspase 9 by survivin were used in this study. Consequently, Cer accumulation in A549 cells would likely be associated with a pathway other than the mitochondrial caspase-dependent pathway of apoptosis. Here, we showed that the dual addition of a glucosyl-Cer synthase inhibitor and a ceramidase inhibitor to A549 cell culture induced palmitoyl-Cer accumulation with Cer synthase 5 expression and necrotic cell death with lysosomal rupture together with leakage of cathepsin B/alkalization after 2-3 h, although it is unknown in this study whether the necrotic cell death was caused by the lysosomal rupture. This Cer accumulation was followed by a steep increase in sphinganine base levels via the activation of serine palmitoyltransferase activity brought about by the increase in palmitoyl-coenzyme A concentration as a substrate after 5-6 h. The increase in palmitoyl-coenzyme A concentration was achieved by activation of the fatty acid synthetic pathway from acetyl coenzyme A.
Keywords: ([13C16]C16:0-CoA, palmitoyl-13C16 coenzyme A; 4-HPR, N-(4-hydroxyphenyl)retinamide; A549 cells; APCI, atmospheric pressure chemical ionization; BSA, bovine serum albumin; C16:0-Cer, palmitoyl-ceramide; C16:0-CoA, palmitoyl-coenzyme A; C2:0-CoA, acetyl-coenzyme A; CHOP, CAAT/enhancer binding protein homologous protein; CathB, cathepsin B; Cer, ceramide; CerS, ceramide synthase; D-NMAPPD; D-NMAPPD, N-[(1R,2R)-2-hydroxy-1-(hydroxy-methyl)-2-(4-nitrophenyl)ethyl]tetradecanamide; DAPI, 4′,6-diamidino-2-phenylindole; DL-PDMP; DL-PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulfoxide; DTT, dithiothreitol; ER, endoplasmic reticulum; ESI, electrospray ionization; FATP1, fatty acid transport protein 1; FBS, fetal bovine serum; GlcCer, glucosylceramide; IS, internal standard; L-[2,3,3-D3]Ser, L-serine-2,3,3-D3; LC3, microtubule-associated protein 1 light chain 3B; LDH, lactate dehydrogenase; LMP, lysosomal membrane permeabilization; Lys, lysosomes; MAM, mitochondria-associated membrane; Myriocin, 2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxo-6-eicosenoic acid; Necrosis; Palmitoyl-ceramide; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM, selected-ion monitoring; SM, sphingomyelin; SPT, serine palmitoyltransferase; SPTLC, SPT-long chain base subunit; Ser, Serine; Sphinganine; [1,2,3,4-13C4]C16:0 acid, palmitic acid-1,2,3,4-13C4; [2-13C]C2:0 acid, sodium acetate-2-13C; [D7]d18:0, D-erythro-sphinganine-D7; [D7]d18:1, D-erythro-sphingosine-D7; acridine orange, 3,6-Bis(dimethylamino) acridine hydrochloride; d18:0, sphinganine; d18:1, sphingosine; d18:1-[D31]C16:0-Cer, N-palmitoyl [D31]-D-erythro-sphingosine.