title: Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells.
abstract: Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.
authors.affiliation: Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: De Stefanis
authors.forename: Patrizia
authors.initials: P
authors.lastname: Reffo
authors.forename: Gabriella
authors.initials: G
authors.lastname: Bonelli
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Baccino
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Sala
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ghidoni
authors.forename: Patrice
authors.initials: P
authors.lastname: Codogno
authors.forename: Ciro
authors.initials: C
authors.lastname: Isidoro
mesh_terms: D019732:Amide Synthases / Q000037:antagonists & inhibitors; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002402:Cathepsin D / Q000494:pharmacology*; D002518:Ceramides / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D008247:Lysosomes / Q000187:drug effects*; D008928:Mitochondria / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009707:Nucleosomes / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D061945:Caspase Inhibitors; D002518:Ceramides; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D009025:Morpholines; C064769:N-acetylsphingosine; D009707:Nucleosomes; C033110:RV 538; D020169:Caspases; D002402:Cathepsin D; D019732:Amide Synthases; D013110:Sphingosine