INDUCTION OF CERAMIDE-MEDIATED APOPTOSIS BY THE ANTICANCER PHOSPHOLIPID ANALOG, HEXADECYLPHOSPHOCHOLINE

The prototype of a new class of antiproliferative phospholipid analogs , hexadecylphosphocholine (HePC), has been shown to inhibit tumor grow th and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC, e.g. pr otein kinase C and CTP:phosphocholine cytidylyltransferase, have been proposed, the mechanisms of HePC-induced anticancer activity are still unclear, Considering that the antiproliferative effect of HePC correl ates with inhibition of phosphatidylcholine biosynthesis, which is tig htly coupled to sphingomyelin biosynthesis, we tested the hypothesis t hat treatment of cells with the anticancer drug leads to increased cel lular ceramide and subsequently to apoptotic cell death. In the presen t study, we showed that 25 mu mol/liter HePC induced apoptosis, In fur ther experiments, we demonstrated that HePC inhibited the incorporatio n of radio-labeled choline into phosphatidylcholine and at a later tim e point into sphingomyelin. This was confirmed by metabolic labeling o f the lipid backbone using radio-labeled serine, and it was shown that HePC decreased the incorporation of serine into sphingomyelin by 35% and simultaneously increased the incorporation of serine into ceramide by 70%. Determination of the amount of ceramide revealed an increase of 53% in HePC-treated cells compared with controls. In accordance wit h the hypothesis that elevated ceramide levels may be the missing link between the metabolic effects of HePC and its proapoptotic properties , HePC-induced apoptosis was blocked by fumonisin B-1, an inhibitor of ceramide synthesis, Furthermore, we found that membrane-permeable cer amides additively increased the apoptotic effect of HePC.