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• W2014245712 abstract "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. 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. In current anticancer therapies, most cytostatic agents impair cell division by cross-linking DNA (e.g. cisplatin or alkylating agents), disrupting the cytoskeleton (e.g.vinblastine), or rectifying the cytoskeleton (e.g. Taxol). In a new approach to cancer chemotherapy, the cell membrane was described as a target for cytostatic agents. It is known that alkyllysophospholipids possess antineoplastic properties in vitro and in vivo (1Berdel W.E. Andreesen R. Munder P.G. Kuo J.F. Phospholipids and Cellular Regulation. II. CRC Press, Boca Raton, FL1985: 41-73Google Scholar), leading to the development of another class of antiproliferative phospholipid analogs, the alkylphosphocholines. The prototype of these phospholipid analogs, hexadecylphosphocholine (HePC) 1The abbreviations used are: HePC, hexadecylphosphocholine; HePS, hexadecylphosphoserine; ELISA, enzyme-linked immunosorbent assay; KGM, keratinocyte growth medium; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; PC, phosphatidylcholine; RPMI, Roswell Park Memorial Institute; SM, sphingomyelin; TUNEL, terminal UMP nick end labeling. 1The abbreviations used are: HePC, hexadecylphosphocholine; HePS, hexadecylphosphoserine; ELISA, enzyme-linked immunosorbent assay; KGM, keratinocyte growth medium; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; PC, phosphatidylcholine; RPMI, Roswell Park Memorial Institute; SM, sphingomyelin; TUNEL, terminal UMP nick end labeling. (for chemical structure see Fig. 1), has been shown to inhibit cell proliferation and tumor growth (2Unger C. Eibl H. Breiser H.W. van Heyden H.W. Engel J. Hilgard P. Sindermann H. Peukert M. Nagel G.S. Onkologie. 1988; 11: 295-296Crossref PubMed Scopus (50) Google Scholar, 3Geilen C.C. Haase R. Buchner K. Wieder T. Hucho F. Reutter W. Eur. J. Cancer. 1991; 27: 1650-1653Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 4Unger C. Fleer E.A.M. Kötting J. Neumüller W. Eibl H. Prog. Exp. Tumor Res. 1992; 34: 25-32Crossref PubMed Google Scholar). Due to its amphiphilic behavior and favorable penetration characteristics, HePC was expected to be a good candidate for topical treatment of skin metastases in breast cancer patients. Indeed, the first clinical studies showed promising results (5Unger C. Sindermann H. Peukert M. Hilgard P. Engel J. Eibl H. Prog. Exp. Tumor Res. 1992; 34: 153-159Crossref PubMed Google Scholar), and HePC is currently used for the treatment of cutaneous metastases of mammary carcinomas (6Clive S. Leonard R.C.F. Lancet. 1997; 349: 621-622Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In search for the cellular targets of HePC it was demonstrated that HePC inhibits the biosynthesis of phosphatidylcholine (PC) in different cell lines (7Haase R. Wieder T. Geilen C.C. Reutter W. FEBS Lett. 1991; 288: 129-132Crossref PubMed Scopus (63) Google Scholar, 8Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1992; 267: 6719-6724Abstract Full Text PDF PubMed Google Scholar, 9Wieder T. Geilen C.C. Reutter W. Biochem. J. 1993; 291: 561-567Crossref PubMed Scopus (29) Google Scholar, 10Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar). Subsequently, we and others systematically investigated the effects of different phospholipid analogs. Using alkylphosphocholines (11Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 12Posse de Chaves E. Vance D.E. Campenot R.B. Vance J.E. Biochem. J. 1995; 312: 411-417Crossref PubMed Scopus (37) Google Scholar), 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (13Wieder T. Haase A. Geilen C.C. Orfanos C.E. Lipids. 1995; 30: 389-393Crossref PubMed Scopus (37) Google Scholar, 14Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and N-acetylsphingosine-1-phosphocholine (15Wieder T. Perlitz C. Wieprecht M. Huang R.T.C. Geilen C.C. Orfanos C.E. Biochem. J. 1995; 311: 873-879Crossref PubMed Scopus (24) Google Scholar), it has been demonstrated that inhibition of PC biosynthesis was paralleled by inhibition of cell growth. Interestingly, all biologically active phospholipid analogs contained a phosphocholine head group. Investigating hexadecylphosphoethanolamine and hexadecylphosphoserine (HePS), neither cell proliferation nor PC biosynthesis was inhibited (11Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar). In further experiments, inhibition of the rate-limiting enzyme of PC biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), was shown to be the mechanism underlying the inhibition of PC biosynthesis for all different classes of phospholipid analogs tested so far. Incubation of cells with the different analogs reduced the active, membrane-bound form of cytidylyltransferase (8Geilen C.C. Wieder T. Reutter W. J. Biol. Chem. 1992; 267: 6719-6724Abstract Full Text PDF PubMed Google Scholar, 13Wieder T. Haase A. Geilen C.C. Orfanos C.E. Lipids. 1995; 30: 389-393Crossref PubMed Scopus (37) Google Scholar, 15Wieder T. Perlitz C. Wieprecht M. Huang R.T.C. Geilen C.C. Orfanos C.E. Biochem. J. 1995; 311: 873-879Crossref PubMed Scopus (24) Google Scholar), suggesting a link between the regulation of PC biosynthesis by cytidylyltransferase and the proliferative properties of cells. Experimental data supporting this hypothesis were provided by two recent studies which demonstrated that (i) inhibition of PC biosynthesis resulted in an arrest of the cell cycle (16Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 11612-11618Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and (ii) Chinese hamster ovary mutant 58 cells carrying a temperature-sensitive mutation in cytidylyltransferase were driven into apoptosis when shifted to the nonpermissive growth temperature (17Cui Z. Houweling M. Chen M.H. Record M. Chap H. Vance D.E. Tercé F. J. Biol. Chem. 1996; 271: 14668-14671Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In the present study, we investigated the influence of the anticancer drug HePC on apoptosis and sphingomyelin (SM) biosynthesis, a metabolic pathway that is coupled to PC biosynthesis (18Hampton R.Y. Morand O.H. Science. 1989; 246: 1050Crossref PubMed Scopus (48) Google Scholar, 19Hannun Y.A. Obeid L.M. Trends Biochem. Sci. 1995; 20: 72-77Abstract Full Text PDF Scopus (573) Google Scholar). It was shown that HePC inhibited incorporation of [3H]choline and [3H]serine into SM, thereby increasing cellular ceramide. Furthermore, using cell permeable ceramides and fumonisin B1, a potent, competitive inhibitor of ceramide synthase (20Merrill A.H. Liotta D.C. Riley R.T. Trends Cell Biol. 1996; 6: 218-223Abstract Full Text PDF PubMed Scopus (132) Google Scholar), experimental evidence was provided that HePC-induced apoptosis is mediated by an elevation of cellular ceramide. [methyl3H]Choline chloride (2.8–3.1 TBq/mmol), l-[3-3H]serine (1.11 TBq/mmol), and [γ-32P]ATP (185 TBq/mmol) were from Amersham (Braunschweig, Germany). Silica Gel 60 high performance thin layer chromatography plates and reagents were purchased from Merck (Darmstadt, Germany). Streptomyces sp. sphingomyelinase, phospholipids, and ceramide standards were from Sigma (München, Germany). The cytotoxicity detection kit (lactate dehydrogenase, LDH), the in situ cell death detection kit, fluorescein, and the cell death detection enzyme-linked Immunosorbent assay (ELISA)Plus were from Boehringer (Mannheim, Germany).sn-1,2-Diacylglycerol kinase from Escherichia coli was purchased from Lipidex, Inc. (Westfield, NJ). HePC was synthesized as described previously (21Geilen C.C. Samson A. Wieder T. Wild H. Reutter W. J. Label. Compd. Radiopharm. 1992; 31: 1071-1076Crossref Scopus (19) Google Scholar), and HePS was a gift from Asta Pharma (Frankfurt/Main, Germany). The purity of both substances was >95% as shown by high performance TLC. N-Acetylsphingosine (C2-ceramide) and fumonisin B1 were from Alexis (Grünberg, Germany). For quantification of radioactivity a radioscanner (LB 2821 HR; Berthold, Wildbad, Germany) was used. HaCaT cells (22Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3440) Google Scholar) were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated fetal calf serum, 0.35 g/liter glutamine, 100 000 IU/liter penicillin, and 0.1 g/liter streptomycin in plastic culture dishes (Nunc, Wiesbaden, Germany). Media and culture reagents were obtained from Life Technologies, Inc. (Karlsruhe, Germany). Penicillin and streptomycin were from Boehringer (Mannheim, Germany). Confluent cells were subcultured after detaching the cells with 0.1% trypsin, 0.02% ethylenediamine tetraacetic acid in phosphate-buffered saline (PBS). Unless otherwise stated, HaCaT cells were maintained in keratinocyte growth medium (KGM) for the duration of the experiments. KGM was prepared from keratinocyte basal medium by addition of 10 μg/liter epidermal growth factor, 5 mg/liter insulin, 0.5 μmol/liter hydrocortisone, 50 mg/liter bovine pituitary extract, 100 mg/liter penicillin/streptomycin, and 2.5 mg/liter Fungizone. Keratinocyte basal medium and supplements were purchased from Clonetics (San Diego, CA). 25 mmol/liter HePC and 30 mmol/liter C2-ceramide stock solutions were prepared in ethanol. A 25 mmol/liter HePS stock solution was prepared in dimethyl sulfoxide and sonicated for 2 min prior to use and a 10 mmol/liter fumonisin B1 stock solution was prepared in methanol. All stock solutions were diluted with KGM to give the final concentrations. Ethanol, dimethyl sulfoxide, and methanol (vehicles) were added to controls and were present at 0.1, 0.1, and 0.5%, respectively. The addition of the vehicles did not influence viability or proliferation of HaCaT cells (23Geilen C.C. Bektas M. Wieder T. Orfanos C.E. FEBS Lett. 1996; 378: 88-92Crossref PubMed Scopus (60) Google Scholar). Cytotoxicity of HePC was determined by the release of LDH from the cytosol of damaged cells into the supernatant. The LDH activity was measured in an enzymatic test. In the first step NAD+ is reduced to NADH/H+ by the LDH-catalyzed conversion of lactate to pyruvate. In the second step the catalyst (diaphorase) transfers H/H+ from NADH/H+ to 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride which is reduced to formazan and the reaction product is quantified photometrically at 492 nm. The LDH assay was performed with the components of the cytotoxicity detection kit (LDH) from Boehringer Mannheim according to the manufacturer's instructions. Confluent HaCaT cells which have been equilibrated in KGM for 24 h were incubated in KGM supplemented with different concentrations of HePC or HePS. After incubation, the cell culture supernatant was removed and clarified by centrifugation at 2000 rpm in an Eppendorf centrifuge for 5 min. 20 μl of the supernatant were diluted with 80 μl of KGM and transferred to microtiter plates. After addition of 100 μl of reaction mixture containing 2-[4-Iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride, diaphorase, NAD+, and sodium lactate the samples were incubated for 5 min at room temperature, protected from light. The reaction was stopped with 50 μl of 1 mol/liter HCl, and the absorbance of the samples was measured at 492 nm. HaCaT cells were seeded in RPMI medium containing 10% fetal calf serum at subconfluent (14,000 cells/cm2) densities. After an equilibration period of 24 h, medium was removed, the cells were washed with PBS, and serum-free KGM containing different concentrations of HePC or HePS was added. After 24 h, the number of cells was determined by crystal violet staining as described in detail previously (11Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar). Apoptosis was measured using the cell death detection ELISAPlus from Boehringer Mannheim according to the manufacturer's instructions. Confluent HaCaT cells that had been equilibrated in KGM for 24 h were incubated in KGM supplemented with different concentrations of HePC or HePS. After 20 h of incubation, the media were removed and the cells were trypsinized using 250 μl of 0.1% trypsin, 0.02% ethylenediamine tetraacetic acid in PBS. The reaction was stopped by the addition of 500 μl of RPMI medium containing 10% fetal calf serum, and the cell suspensions were combined with the respective media. The cells were pelleted in an Eppendorf centrifuge for 5 min at 2000 rpm and washed with 500 μl of RPMI medium containing 10% fetal calf serum. The cell pellet was incubated in 1 ml of lysis buffer (component of the assay kit) for 30 min at 4 °C, and the lysate was centrifuged for 10 min at 13,000 rpm in an Eppendorf centrifuge. Then, the cytosolic nucleosomes in the supernatant were detected using the cell death detection ELISAPlus from Boehringer Mannheim. For this, 20 μl of the probe were transferred to a streptavidin-coated microtiter plate, and 80 μl of immunoreagent containing 1 part biotinylated anti-histone antibody, 1 part peroxidase-conjugated anti-DNA-antibody, and 18 parts incubation buffer were added. After 2 h of incubation at room temperature, the microtiter plate was washed three times with incubation buffer, and the amount of peroxidase retained in the immunocomplex was determined photometrically at 405 nm with 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) as a substrate. The specificity of the method was checked by the use of known inducers of apoptosis, such as cell permeable ceramides, and the results of the ELISA were confirmed by typical morphological changes of apoptotic cells seen at the ultrastructural level, e.g. membrane blebbing and condensation of the chromatin. Apoptosis leads to double strand DNA fragments, which can be detected by labeling of the free 3′-OH-ends with modified nucleotides in an enzymatic reaction. In the in situ cell death detection kit, fluorescein (Boehringer Mannheim, Germany) the terminal deoxynucleotidyltransferase is used to catalyze the labeling of the free ends of the DNA-strand breaks with fluorescein-containing nucleotides. The reaction was carried out according to the manufacturer's instructions. Confluent HaCaT cells that have been equilibrated in KGM for 24 h were incubated in KGM supplemented with 25 μmol/liter HePC or 25 μmol/liter HePS. After 20 h, cells were washed with PBS and trypsinized. 100 μl of cell suspension were applied to a slide and centrifuged for 5 min at 500 rpm. The cytospins were dried for 30 min at room temperature, and the slides were put in a 4% paraformaldehyde solution for 30 min. For permeabilization, 200 μl of 0.1% Triton X-100 in 0,1% sodium citrate were added to the cells and incubated at 4 °C for 2 min. Subsequently, cells were washed with PBS and 50 μl of TUNEL solution were added to the cells. Then, the cells were covered with a cover glas and the slides were put in a humid chamber and incubated at 37 °C for 1 h. After washing with PBS, Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) was added, and the cells were again covered with a cover glass. Finally, the cytospins were analyzed under a fluorescence microscope. Incorporation of [methyl-3H]choline into PC and SM was measured as described previously (15Wieder T. Perlitz C. Wieprecht M. Huang R.T.C. Geilen C.C. Orfanos C.E. Biochem. J. 1995; 311: 873-879Crossref PubMed Scopus (24) Google Scholar). Confluent HaCaT cells that had been equilibrated in KGM for 24 h were incubated in KGM containing 2 μCi/ml [methyl-3H]choline supplemented with different concentrations of HePC or HePS. After incubation, cells were washed twice with ice-cold PBS, harvested, and freeze-dried. Lipids in the samples were extracted by a modified method of Bligh and Dyer (24Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar) as described previously (15Wieder T. Perlitz C. Wieprecht M. Huang R.T.C. Geilen C.C. Orfanos C.E. Biochem. J. 1995; 311: 873-879Crossref PubMed Scopus (24) Google Scholar). Then, 10-μl aliquots of the chloroform phases were taken for scintillation counting, and 15-μl aliquots of the chloroform phases were separated by high performance TLC using the solvent system chloroform/methanol/triethylamine/water (30:35:34:8, by volume). Radioactivity was quantified by radioscanning and phospholipids were identified by calibrating the scanner with PC and SM standards. For lipid quantification, all lipids were stained with a CuSO4 solution (156 g/liter in 8.5% H3PO4) by the method of Touchstone et al. (25Touchstone J.C. Chen J.C. Beaver K. Lipids. 1980; 15: 61-62Crossref Scopus (439) Google Scholar) and quantified by video densitometry. Staining was linear in the range 0.5–6 μg of lipid per band. The specific radioactivities of PC and SM were calculated from the radioactivity and the amount of lipid found in the corresponding bands. The incorporation ofl-[3H]serine into SM and ceramide was determined as described elsewhere (23Geilen C.C. Bektas M. Wieder T. Orfanos C.E. FEBS Lett. 1996; 378: 88-92Crossref PubMed Scopus (60) Google Scholar) with some modifications. Confluent HaCaT cells that had been equilibrated 24 h in serine-free minimal essential medium (Life Technologies, Inc., Karlsruhe, Germany) containing 5% fetal calf serum were radiolabeled by the addition of 2 μCi/ml l-[3H]serine in the presence of different concentrations of HePC, HePS, or fumonisin B1. After incubation, medium was removed and the cells were washed twice with ice-cold PBS, harvested, and freeze dried. Lipids in the samples were extracted by a modified method of Bligh and Dyer (24Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar). Then, 15-μl aliquots of the chloroform phases were separated by high performance TLC using the solvent system chloroform/methanol (9:1, v/v), and radioactivity was quantified by radioscanning. Ceramide was identified by calibrating the scanner with known standards. Additionally, an internal, radiolabeled ceramide standard was prepared by incubation of serine-labeled cellular extracts with 0.1 unit/ml sphingomyelinase. The ceramide-bound radioactivity was measured as disintegrations/min ceramide/disintegrations/min total lipid, and ceramide-bound radioactivity of control samples was set as 100%. After radioscanning, the plates were developed using the solvent system chloroform/methanol/triethylamine/water (30:35:34:8, by volume) to separate SM from glycosphingolipids in the samples. The SM peak in the resulting radioscan was identified by the use of standard SM. Additionally, it was shown that this peak was digested by sphingomyelinase treatment. The SM-bound radioactivity was measured as disintegrations/min SM/disintegrations/min total lipid, and SM-bound radioactivity of control samples was set as 100%. Determination of cellular ceramide was carried out as described in detail previously (26Wieder T. Geilen C.C. Kolter T. Sadeglar F. Sandhoff K. Brossmer R. Ihrig P. Perry D. Orfanos C.E. Hannun Y.A. FEBS Lett. 1997; 411: 260-264Crossref PubMed Scopus (35) Google Scholar) using sn-1,2-diacylglycerol kinase. Briefly, after incubation of the cells with HePC, lipids were extracted according to Bligh and Dyer (24Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar), and cellular ceramide in the samples was measured as described elsewhere (26Wieder T. Geilen C.C. Kolter T. Sadeglar F. Sandhoff K. Brossmer R. Ihrig P. Perry D. Orfanos C.E. Hannun Y.A. FEBS Lett. 1997; 411: 260-264Crossref PubMed Scopus (35) Google Scholar). Statistical comparisons were made in these studies with Student's t test. Due to its amphiphilic structure the cytotoxic effect of HePC might be a simple consequence of its lytic properties. On the other hand, it has been proposed that cells treated with HePC die by apoptosis (27Engelmann J. Henke J. Willker W. Kutscher B. Nossner G. Engel J. Leibfritz D. Anticancer Res. 1996; 16: 1429-1439PubMed Google Scholar). To characterize the cytotoxic effects of HePC in our experimental system we measured the release of LDH from the cytosol of HaCaT cells after 4 h of incubation with different concentrations of HePC. As shown in Fig. 2 A, HePC did not induce LDH release at concentrations ≤25 μmol/liter. However, at 50 μmol/liter a significant increase of LDH in the cell culture supernatant of 390% as compared with controls was observed, indicating that the cells were lysed. To exclude lytic effects, HePC concentrations ≤25 μml/liter were used in the following experiments. Furthermore, the synthetic phospholipid HePS, which has been shown to be biologically inactive and nontoxic at concentrations ≤50 μmol/liter (11Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar), was used in our studies as a negative control. As expected, HePS did not influence LDH release at concentrations below 50 μmol/liter (Fig. 2 A), whereas nonspecific permeabilization of HaCaT cells was observed when the cells were treated with 250 μmol/liter HePS (LDH release from HePS-treated cells was 664 ± 8% (n = 3) as compared with control cells). In further experiments, we tested the ability of HePC to induce apoptosis by determination of cytosolic nucleosomes as a result of apoptotic DNA damage (28Jarvis W.D. Kolesnick R.N. Fornari F.A. Traylor R.S. Gewirtz D.A. Grant S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 73-77Crossref PubMed Scopus (483) Google Scholar). We could show that treatment of HaCaT cells with nonlytic concentrations of 7.5 and 25 μmol/liter HePC significantly increased cytosolic nucleosomes to about 240 and 450% of control, respectively, whereas the control phospholipid HePS did not induce DNA damage (Fig. 2 B). Since the procedure of the cell death detection ELISA depends on intact plasma membrane function of the cells, the increase of cytosolic nucleosomes clearly distinguished the apoptotic effect of HePC at concentrations ≤25 μmol/liter from its lytic effect at 50 μmol/liter. The apoptotic capacity of HePC was confirmed on single cell level by TUNEL staining. As shown in Fig. 3, control and HePS-treated cells showed only a weak and diffuse staining, with 2 ± 1% (n= 3) of the cell population being fluorescent (apoptotic), whereas treatment of HaCaT cells with 25 μmol/liter HePC significantly increased the number of fluorescent (apoptotic) cells to about 26 ± 11% (n = 3) of the cell population. The antiproliferative effect of HePC has already been well documented in different tumor models (29Zeisig R. Fichtner I. Arndt D. Jungmann S. Anticancer Drugs. 1991; 2: 411-417Crossref PubMed Scopus (29) Google Scholar) and cell lines (11Geilen C.C. Haase A. Wieder T. Arndt D. Zeisig R. Reutter W. J. Lipid Res. 1994; 35: 625-632Abstract Full Text PDF PubMed Google Scholar, 30Unger C. Damenz W. Fleer E.A. Kim D.J. Breiser A. Hilgard P. Engel J. Nagel G. Eibl H. Acta Oncol. 1989; 28: 213-217Crossref PubMed Scopus (105) Google Scholar), including primary human keratinocytes (10Detmar M. Geilen C.C. Wieder T. Orfanos C.E. Reutter W. J. Invest. Dermatol. 1994; 102: 490-494Abstract Full Text PDF PubMed Google Scholar). However, no data were available on the immortalized human keratinocyte cell line HaCaT. As shown in Fig. 4 A, HePC inhibited the proliferation of HaCaT cells with a half-inhibitory concentration (IC50) of approximately 3 μmol/liter, whereas the control phospholipid HePS had no significant effect. At the nonlytic HePC concentration of 25 μmol/liter cell proliferation was almost completely blocked (6% of control), confirming our assumption that the antiproliferative effect is not mediated by cell lysis. The antiproliferative effect of HePC was paralleled by its ability to inhibit the incorporation of [methyl-3H]choline into PC, thereby decreasing the specific radioactivity of PC in the presence of 7.5 and 25 μmol/liter HePC after 4 h of incubation by 58 and 71%, respectively (Fig. 4 B). Inhibition of PC biosynthesis was also observed after 20 h of incubation (Fig. 4 C), indicating that this metabolic effect of HePC persists over a longer period of time. Consistent with the experiments described above, HePS did not significantly reduce the specific radioactivity of PC as compared with the dimethyl sulfoxide control. SM is synthesized by the transfer of a phosphocholine head group from PC to ceramide, a reaction catalyzed by the enzyme phosphatidylcholine:ceramide phosphocholinetransferase. Although the exact mechanism of the enzyme reaction remains unclear, more than 95% of overall SM synthesis is attributed to this pathway (31Spence M.W. Vance D.E. Phosphatidylcholine Metabolism. CRC Press, Boca Raton, FL1989: 185-203Google Scholar). Due to this precursor-product relationship, biosynthesis of SM might be influenced by inhibition of PC biosynthesis. First of all, we measured the time-dependent incorporation of [methyl-3H]choline into SM and found that the percentage of label in SM increased with incubation time (after 4 h of incubation only 1.5% of total radioactivity were SM-bound, whereas after 20 h of incubation SM-bound radioactivity reached 5.7% of total radioactivity), indicating that the choline head group originates from PC. This time course was also observed when the cells were treated with HePC; whereas HePC had already inhibited PC biosynthesis after 4 h, incorporation of [methyl-3H]choline into SM was not significantly influenced after 4 h (data not shown). However, incubation of HaCaT cells with 25 μmol/liter HePC for 20 h decreased the specific radioactivity of SM by 52% (Fig. 5 A), whereas the control lipid HePS was inactive. From the specific radioactivities of PC and the radioactive counts in SM the actual synthesis of SM was calculated, and we found that 1.38 nmol of SM/106 cells were formed in control cells after 20 h of incubation and 0.81 nmol of SM/106 cells in HePC-treated cells (59% of control). In a second approach, we metabolically labeled the lipid backbone of sphingo" @default.
• W2014245712 created "2016-06-24" @default.
• W2014245712 creator A5002457623 @default.
• W2014245712 creator A5034016144 @default.
• W2014245712 creator A5039656345 @default.
• W2014245712 date "1998-05-01" @default.
• W2014245712 modified "2023-09-28" @default.
• W2014245712 title "Induction of Ceramide-mediated Apoptosis by the Anticancer Phospholipid Analog, Hexadecylphosphocholine" @default.
• W2014245712 cites W1561474071 @default.
• W2014245712 cites W1561554950 @default.
• W2014245712 cites W1965990791 @default.
• W2014245712 cites W1966752040 @default.
• W2014245712 cites W1970044546 @default.
• W2014245712 cites W1972090215 @default.
• W2014245712 cites W1980805940 @default.
• W2014245712 cites W1983069935 @default.
• W2014245712 cites W1984961720 @default.
• W2014245712 cites W1986030520 @default.
• W2014245712 cites W1991865867 @default.
• W2014245712 cites W1992122620 @default.
• W2014245712 cites W1993282564 @default.
• W2014245712 cites W2003933642 @default.
• W2014245712 cites W2017069757 @default.
• W2014245712 cites W2018866317 @default.
• W2014245712 cites W2026922767 @default.
• W2014245712 cites W2027151723 @default.
• W2014245712 cites W2029799890 @default.
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