Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin.

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

Elastase activated liposomal delivery to nucleated cells.

The specific activation of liposomes for delivery has been explored by enzyme mediated cleavage of a peptide substrate covalently conjugated to a fusogenic lipid. We have previously shown an elastase sensitive peptide conjugated to 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [corrected] (DOPE) could be activated by enzymatic cleavage, triggering liposome-liposome lipid mixing and fusion with erythrocyte ghosts (Pak et al., Biochim. Biophys. Acta, 1372 (1998) 13-27). Further optimization of this system has been aimed at obtaining substrate cleavage at or below physiological elastase levels and to demonstrate triggered delivery to living cells. Therefore a new peptide-lipid, MeO-suc-AAPV-DOPE (N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE), has been developed that exhibits greater sensitivity and selectivity for elastase cleavage and subsequent conversion to DOPE. This peptide-lipid was used with DODAP (dioleoyl dimethylammonium propane, a pH dependent cationic lipid) in a 1:1 mol ratio with the expectation that endocytosis would lead to a liposome with an overall positive charge if enzymatic cleavage had occurred. Elastase treated liposomes displayed pH dependent enhancement of binding, lipid mixing, and delivery of 10000 MW dextrans, relative to untreated liposomes, when incubated with HL60 human leukemic cells. Heat denatured elastase did not activate DODAP/MeO-suc-AAPV-DOPE liposomes, indicating enzymatic activity of elastase is necessary. Liposomes bound to ECV304 endothelial cells at physiological pH could be activated by elastase to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm. These results suggest enzyme substrate peptides linked to a fusogenic lipid may be used to elicit specific delivery from liposomes to cells.

Evidence for involvement of mitogen-activated protein kinase, rather than stress-activated protein kinase, in potentiation of 1-beta-D-arabinofuranosylcytosine-induced apoptosis by interruption of protein kinase C signaling.

The stress-activated protein kinase (SAPK) and mitogen-activated protein kinase (MAPK) cascades mediate cytotoxic and cytoprotective functions, respectively, in the regulation of leukemic cell survival. Involvement of these signaling systems in the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) and modulation of ara-C lethality by protein kinase C PKC inhibition/down-regulation was examined in HL-60 promyelocytic leukemia cells. Exposure to ara-C (10 microM) for 6 hr promoted extensive apoptotic DNA damage and cell death, as well as activation of PKC. This response was accompanied by downstream activation of the SAPK and MAPK cascades. PKC-dependent MAPK activity seemed to limit ara-C action in that the toxicity of ara-C was enhanced by pharmacological reductions of PKC, MAPK, or both. Thus, ara-C action was (1) partially attenuated by diradylglycerols, which stimulated PKC and MAPK, but (2) dramatically amplified by sphingoid bases, which inhibited PKC and MAPK. The cytotoxicity of ara-C also was substantially increased by pharmacological reductions of PKC, including down-regulation of PKC by chronic preexposure to the macrocyclic lactone bryostatin 1 or inhibition of PKC by acute coexposure to the dihydrosphingosine analog safingol. Significantly, both of these manipulations prevented activation of MAPK by ara-C. Moreover, acute disruption of the MAPK module by AMF, a selective inhibitor of MEK1, suppressed both basal and drug-stimulated MAPK activity and sharply increased the cytotoxicity of ara-C, suggesting the direct involvement of MAPK as a downstream antiapoptotic effector for PKC. None of these chemopotentiating agents enhanced ara-CTP formation. Ceramide-driven SAPK activity did not seem to mediate drug-induced apoptosis, given that (1) neutralization of endogenous tumor necrosis factor-alpha with monoclonal antibodies or soluble tumor necrosis factor receptor substantially reduced ceramide generation and SAPK activation by ara-C, whereas the induction of apoptosis was unaffected; (2) pharmacological inhibition of sphingomyelinase by 3-O-methoxysphingomyelin reduced ceramide generation and SAPK activation without limiting the drug's cytotoxicity; and (3) potentiation of ara-C action by bryostatin 1 or safingol was not associated with further stimulation of SAPK. These observations collectively suggest a primary role for decreased MAPK, rather than increased SAPK, in the potentiation of ara-C cytotoxicity by interference with PKC-dependent signaling.

Synthesis of ether-linked analogues of lysophosphatidate and their effect on the proliferation of human epithelial cancer cells in vitro.

To investigate whether lysophosphatidate analogues of alkyllysophospholipids were antiproliferative we synthesized three new ether-linked analogues of lysophosphatidic acid and investigated their antiproliferative activity on epithelial cancer cell lines derived from different tissues. The antiproliferative effects of the compounds on MCF-7 and T47D (breast), A549 and A427 (lung), A498 (kidney), SK-N-SH and SK-N-MC (neuroblastoma), and DU145 (prostate) cells were compared with the ability of 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine, the archetypic alkyllysophospholipid, to inhibit the proliferation of all the cell lines. 1-O-Hexadecyl-2-O-methyl-sn-glycero-3-phosphate and 4-thiohexadecyl-3(S)-O-methoxybutane-4-phosphate were unable to inhibit the proliferation of any of the cells to any degree, while slightly enhancing the proliferation of DU145 cells. In contrast 4-O-hexadecyl-3(S)-O-methoxybutanephosphonate was a potent antiproliferative agent that was on the whole more active than 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine. Since 1-Oleoyl-2-lyso-phosphatidate (LPA) was non-mitogenic in all the cell lines except the neuroblastoma line SK-N-SH, it is unlikely that the inhibition of cell proliferation by 4-O-hexadecyl-3(S)-O-methoxybutanephosphonate was a consequence of perturbation of cellular response to the mitogenic effects of LPA.

Induction of apoptosis and potentiation of ceramide-mediated cytotoxicity by sphingoid bases in human myeloid leukemia cells.

Prior studies demonstrated that ceramide promotes apoptotic cell death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77), and that this lethal process is potently suppressed by diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L., Gewirtz, D. A., Kolesnick, R. N., and Grant, S. (1994) J. Biol. Chem. 269, 31685-31692). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0. 001 10 microM) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations (

Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis.

We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.

Synthesis and evaluation of the antiproliferative effects of 1-O-hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-beta-D- glucopyranosyl)-sn-glycerol and 1-O-hexadecyl-2-O-methyl-3-0- (2'-amino-2'-deoxy-beta-D-glucopyranosyl)-sn-glycerol on epithelial cancer cell growth.

Two ether glucosyl diglyceride analogs were synthesized, and their antiproliferative activity against four epithelial cancer cell lines was evaluated. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-beta-D- glucopyranosyl)-sn-glycerol (4) was synthesized by reaction of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl chloride with 1-O-hexadecyl-2-O-methyl-sn-glycerol followed by deacetylation by methanolic hydrolysis. The N-acetyl group of 4 was removed by hydrolysis with ethanolic potassium hydroxide to form 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)- sn-glycerol (5). Compounds 4 and 5 inhibited the proliferation of MCF-7, A549, A427, and T84 cancer cell lines. The IC(50) values for 5 ranged from 6.5 to 12.2 microM, whereas 4 was more effective against A549 cells (IC(50) 9 microM) than against MCF-7 (IC(50) 17 microM) and A427 (IC(50) 25 microM) cells and was inactive against T84 cells. Under identical incubation conditions, compounds 4 and 5 were potent inhibitors of the proliferation of OVCAR-3 cells with IC(50) values of 12 and 4 microM, respectively, whereas ET-18-OCH(3), hexadecylphosphocholine, and erucylphosphocholine had IC(50) values of 24, >30, and >30 microM, respectively. The cell-inhibitory profile of these ether-linked glucosyl diglycerides strengthens the hypothesis that such glycolipids represent a distinct group of antitumor ether lipids, having antineoplastic activities that differ from the well-known alkylphosphocholines and alkyllysophospholipids.

Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner.

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol in the target membrane. Recently, we have demonstrated that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the present paper, we show that the activation of low-pH-dependent fusion of SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo-, and L-threo-ceramide did not support fusion of the virus. Similar results were obtained with the corresponding sphingomyelin stereoisomers. The stereospecificity of SFV fusion activation was confirmed by using an assay based on degradation of the viral capsid protein by trypsin encapsulated in the target liposomes. Fusion mediated by D-erythro-ceramide was not affected by the additional presence in the target liposomes of ceramide stereoisomers incapable of fusion activation. Binding of the virus to the liposomes, as assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes. The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the target membrane represents an essential step in the activation of the fusion capacity of SFV.

Sphingolipid-dependent fusion of Semliki Forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone.

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we demonstrate that sphingolipid-dependent fusion of SFV with cholesterol-containing liposomes exhibits remarkable molecular specificity, the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone being critical for the sphingolipid to mediate the process. This observation supports the notion that sphingolipids act as a cofactor in SFV fusion, interacting directly with the viral fusion protein to induce its ultimate fusion-active conformation.