Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

• Published in: Biochemistry and biophysics reports Vol. 11; no. C; pp. 174 - 181
• Main Authors: Yamane, Mototeru, Moriya, Shota, Kokuba, Hiroko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier 01.09.2017
• Subjects: MS, mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; D-NMAPPD, N-[(1R,2R)-2-hydroxy-1-(hydroxy-methyl)-2-(4-nitrophenyl)ethyl]tetradecanamide; LC3B, autophagosomal membrane bound-microtubule-associated protein 1 light chain 3B; CA-74Me, [(2S,3S)-3-Propylcarbamoyloxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester; DTT, dithiothreitol; ECL, enhanced chemiluminescence reagent; DMEM, Dulbecco's modified Eagle's medium; SIM, selected ion monitoring; Bax, BCL2-associated X protein; DL-PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; [D7]d18:1, D-erythro-sphingosine-D7; d18:1, sphingosine; LMP, lysosomal membrane permeability; NAC, N-acetyl-L-cysteine; FBS, fetal bovine serum; CerS, ceramide synthase; PBS, phosphate buffered saline; d18:0, sphinganine; C16:0-Cer, palmitoyl-Cer; GlcCer, glucosyl ceramide; DAPI, 4′,6-diamidino-2-phenylindole; d18:1-[D31]C16:0-Cer, N-palmitoyl [D31]-D-erythro-sphingosine; acridine orange, 3,6-Bis(dimethylamino) acridine hydrochloride; Cer, ceramide; ISs, internal standards; LAMP-2, lysosome-associated membrane protein 2; APCI, atmospheric pressure chemical ionization; DMSO, dimethyl sulfoxide; [D7]d18:0, D-erythro-sphinganine-D7
• ISSN: 2405-5808; 2405-5808
• DOI: 10.1016/j.bbrep.2017.02.010

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10-20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.