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 growth and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC, e.g. protein 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 correlates with inhibition of phosphatidylcholine biosynthesis, which is tightly coupled to sphingomyelin biosynthesis, we tested the hypothesis that treatment of cells with the anticancer drug leads to increased cellular ceramide and subsequently to apoptotic cell death. In the present study, we showed that 25 μmol/liter HePC induced apoptosis. In further experiments, we demonstrated that HePC inhibited the incorporation of radiolabeled choline into phosphatidylcholine and at a later time point into sphingomyelin. This was confirmed by metabolic labeling of the lipid backbone using radiolabeled 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 with 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 B1, an inhibitor of ceramide synthesis. Furthermore, we found that membrane-permeable ceramides additively increased the apoptotic effect of HePC.

[1]  H. Sindermann,et al.  Hexadecylphosphocholine (D 18506) in the topical treatment of skin metastases: a phase-I trial. , 1988, Onkologie.

[2]  M. Shoji,et al.  The effect of edelfosine on CTP:cholinephosphate cytidylyltransferase activity in leukemic cell lines. , 1996, Leukemia research.

[3]  C. Unger,et al.  Hexadecylphosphocholine, a new ether lipid analogue. Studies on the antineoplastic activity in vitro and in vivo. , 1989, Acta oncologica.

[4]  W. Berdel,et al.  Inhibition of protein kinase C, (sodium plus potassium)-activated adenosine triphosphatase, and sodium pump by synthetic phospholipid analogues. , 1990, Cancer research.

[5]  C. Rock,et al.  Lysophosphatidylcholine and 1-O-Octadecyl-2-O-Methyl-rac- Glycero-3-Phosphocholine Inhibit the CDP-Choline Pathway of Phosphatidylcholine Synthesis at the CTP:Phosphocholine Cytidylyltransferase Step (*) , 1995, The Journal of Biological Chemistry.

[6]  W. Reutter,et al.  The phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis in Madin—Darby canine kidney cells , 1991, FEBS letters.

[7]  C. Unger,et al.  Antitumoral activity of alkylphosphocholines and analogues in human leukemia cell lines. , 1992, Progress in experimental tumor research.

[8]  R. Campenot,et al.  Alkylphosphocholines inhibit choline uptake and phosphatidylcholine biosynthesis in rat sympathetic neurons and impair axonal extension. , 1995, The Biochemical journal.

[9]  A. Giuliano,et al.  The antitumor phospholipid analog, hexadecylphosphocholine, activates cellular phospholipase D. , 1996, Cancer letters.

[10]  F. Re,et al.  Induction of apoptosis in human leukemic cells by the ether lipid 1‐octadecyl‐2‐methyl‐RAC‐glycero‐3‐ phosphocholine. A possible basis for its selective action , 1993, International journal of cancer.

[11]  Y. Hannun,et al.  Bcl‐2 antagonizes apoptotic cell death induced by two new ceramide analogues , 1997, FEBS letters.

[12]  T. Wieder,et al.  The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes , 1996, FEBS letters.

[13]  W. Reutter,et al.  Phospholipid analogues: side chain- and polar head group-dependent effects on phosphatidylcholine biosynthesis. , 1994, Journal of lipid research.

[14]  L. Verdonck,et al.  Alkyllysophospholipid ET‐18‐OCH3 acts as an activator of protein kinase C in HL‐60 cells , 1991, FEBS letters.

[15]  W. D. Bennett,et al.  Effect of alkyl-lysophospholipids on phosphatidylcholine biosynthesis in leukemic cell lines. , 1985, Experimental hematology.

[16]  W. Reutter,et al.  Synthesis of hexadecylphospho[methyl-14C]-choline , 1992 .

[17]  H. Grunicke,et al.  Hexadecylphosphocholine inhibits inositol phosphate formation and protein kinase C activity. , 1991, Cancer research.

[18]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[19]  K. Scotto,et al.  Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals , 1995, Cell.

[20]  S. Grant,et al.  Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Detmar,et al.  Phospholipid analogue hexadecylphosphocholine inhibits proliferation and phosphatidylcholine biosynthesis of human epidermal keratinocytes in vitro. , 1994, The Journal of investigative dermatology.

[22]  Y. Nishizuka,et al.  The molecular heterogeneity of protein kinase C and its implications for cellular regulation , 1988, Nature.

[23]  W. Reutter,et al.  Uptake, subcellular distribution and metabolism of the phospholipid analogue hexadecylphosphocholine in MDCK cells. , 1994, Biochimica et biophysica acta.

[24]  C. Unger,et al.  Inhibition of calcium-dependent protein kinase C by hexadecylphosphocholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine do not correlate with inhibition of proliferation of HL60 and K562 cell lines. , 1994, European journal of cancer.

[25]  C. Rock,et al.  Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine , 1995, The Journal of Biological Chemistry.

[26]  H. Sindermann,et al.  Hexadecylphosphocholine in the topical treatment of skin metastases in breast cancer patients. , 1990, Cancer treatment reviews.

[27]  A. Merrill,et al.  Fumonisins: fungal toxins that shed light on sphingolipid function. , 1996, Trends in cell biology.

[28]  R. Hampton,et al.  Sphingomyelin synthase and PKC activation. , 1989, Science.

[29]  F. Hucho,et al.  The phospholipid analogue, hexadecylphosphocholine, inhibits protein kinase C in vitro and antagonises phorbol ester-stimulated cell proliferation. , 1991, European journal of cancer.

[30]  S. Clive,et al.  Miltefosine in recurrent cutaneous breast cancer , 1997, The Lancet.

[31]  D. Vance,et al.  A Genetic Defect in Phosphatidylcholine Biosynthesis Triggers Apoptosis in Chinese Hamster Ovary Cells* , 1996, The Journal of Biological Chemistry.

[32]  Y. Hannun,et al.  Ceramide: an intracellular signal for apoptosis. , 1995, Trends in biochemical sciences.

[33]  D. Leibfritz,et al.  Early stage monitoring of miltefosine induced apoptosis in KB cells by multinuclear NMR spectroscopy. , 1996, Anticancer research.

[34]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[35]  R. Bell,et al.  Elevated ceramide levels in GH4C1 cells treated with retinoic acid. , 1992, Biochimica et biophysica acta.

[36]  W. Reutter,et al.  Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells. , 1992, The Journal of biological chemistry.

[37]  J. Hornung,et al.  Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line , 1988, The Journal of cell biology.

[38]  I. Fichtner,et al.  Antitumor effects of alkylphosphocholines in different murine tumor models: use of liposomal preparations. , 1991, Anti-cancer drugs.

[39]  D. Brindley,et al.  Phosphatidate phosphohydrolase and signal transduction. , 1996, Chemistry and physics of lipids.

[40]  Y. Hirabayashi,et al.  Induction of apoptotic DNA fragmentation and cell death by natural ceramide , 1995, FEBS letters.

[41]  W. Reutter,et al.  Antagonism of phorbol-ester-stimulated phosphatidylcholine biosynthesis by the phospholipid analogue hexadecylphosphocholine. , 1993, The Biochemical journal.