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.