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Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous Nacetylsphingosine (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 (DHC2), 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 doublechain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS and PDMPinduced 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 cellassociated ceramide is an early event in cells undergoing apoptosis.

Increase in Ceramide Level Alters the Lysosomal Targeting of Cathepsin D prior to Onset of Apoptosis in HT-29 Colon Cancer Cells