Oxidized phosphatidylcholines trigger ferroptosis in cardiomyocytes during ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner (P < 0.05). POVPC and PONPC also caused a significant decrease in Ca2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells (P < 0.05). PONPC depressed maximal respiration rate (P < 0.01; 54%) and spare respiratory capacity (P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease.NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages.

The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.

Toxicity of oxidized phosphatidylcholines in cultured human melanoma cells.

The oxidized phospholipids (oxPL) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are generated from 1-palmitoyl-2-arachidonoyl-phosphatidylcholine under conditions of oxidative stress. These oxPL are components of oxidized low density lipoprotein. They are cytotoxic in cells of the arterial wall thus playing an important role in the development and progression of atherosclerosis. The toxic lipid effects include inflammation and under sustained exposure apoptosis. The aim of this study was to find out whether such toxic effects, especially apoptosis, are also elicited by oxPL in melanocytic cells in order to assess their potential for therapeutic intervention. FACS analysis after staining with fluorescent markers was performed to identify the mode of lipid-induced cell death. Activation of sphingomyelinase which generates apoptotic ceramide was measured using an established fluorescence assay. Ceramide profiles were determined by mass spectrometry. We found that 50µM POVPC induce cell death in human melanoma cells isolated from different stages of tumor progression but affect primary human melanocytes to a much lesser extent. In contrast, 50µM PGPC was only apoptotic in two out of four cell lines used in this study. The toxicity of both compounds was associated with efficient lipid uptake into the tumor cells and activation of acid sphingomyelinase. In several but not all melanoma cell lines used in this study, activation of the sphingomyelin degrading enzyme correlated with an increase in the concentration of the apoptotic mediator ceramide. The individual patterns of the newly formed ceramide species were also cell line-specific. PGPC and POVPC may be considered potential drug candidates for topical skin cancer treatment. They are toxic in malignant cells. The respective oxidized phospholipids are naturally formed in the body and resistance to these compounds is not likely to occur.

Lipid nanoparticles protect from edelfosine toxicity in vivo.

Edelfosine, an alkyl-lysophospholipid antitumor drug with severe side-effects, has previously been encapsulated into lipid nanoparticles (LN) with the purpose of improving their toxicity profile. LN are made of lipids recognized as safe by the Food and Drug Administration (FDA) and, therefore, these systems are generally considered as nontoxic vehicles. However, toxicity studies regarding the use of LN as vehicles for drug administration are limited. In the present study, we investigated the in vivo toxicity of free edelfosine, and the protection conferred by LN. The free drug, non-loaded LN and edelfosine-loaded LN were orally administered to mice. Our results show that the oral administration of the free drug at 4 times higher than the therapeutic dose caused the death of the animals within 72h. Moreover, histopathology revealed gastrointestinal toxicity and an immunosuppressive effect. In contrast, LN showed a protective effect against edelfosine toxicity even at the higher dose and were completely safe. LN are, therefore, a safe vehicle for the administration of edelfosine by the oral route. The nanosystems developed could be further used for the administration of other drugs.

Dissociations in the effect of delay on object recognition: evidence for an associative model of recognition memory.

Rats were administered 3 versions of an object recognition task: In the spontaneous object recognition task (SOR) animals discriminated between a familiar object and a novel object; in the temporal order task they discriminated between 2 familiar objects, 1 of which had been presented more recently than the other; and, in the object-in-place task, they discriminated among 4 previously presented objects, 2 of which were presented in the same locations as in preexposure and 2 in different but familiar locations. In each task animals were tested at 2 delays (5 min and 2 hr) between the sample and test phases in the SOR and object-in-place task, and between the 2 sample phases in the temporal order task. Performance in the SOR was poorer with the longer delay, whereas in the temporal order task performance improved with delay. There was no effect of delay on object-in-place performance. In addition the performance of animals with neurotoxic lesions of the dorsal hippocampus was selectively impaired in the object-in-place task at the longer delay. These findings are interpreted within the framework of Wagner's (1981) model of memory.

Novel anti-inflammatory action of edelfosine lacking toxicity with protective effect in experimental colitis.

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH(3)) is an antitumor alkyl-lysophospholipid analog that binds lipid rafts, altering their protein composition (J Exp Med 200:353-365). Because L-selectin locates in lipid rafts and plays a crucial role in the recruitment of leukocytes into inflamed tissues, we hypothesized that edelfosine might affect inflammation by modulating L-selectin and inflammatory cell migration. Here, we have found that edelfosine inhibited neutrophil-endothelium interaction through L-selectin shedding. Oral treatment of edelfosine diminished inflammation in two murine animal models. Edelfosine showed a higher antiinflammatory effect than the nonsteroidal anti-inflammatory drug (NSAID) indomethacin in the bentonite mouse-paw edema model. Using a rat model of experimental colitis, edelfosine oral administration ameliorated the clinical and histopathologic severity of the inflammatory colitis with a dramatic decrease in mucosal damage and neutrophil infiltration. Colon sections from edelfosine-treated rats showed a remarkable reduction in ulcer formation, edema, and inflammatory cell infiltration. Edelfosine enhanced lipopolysaccharide-induced expression of anti-inflammatory interleukin-10 in mouse macrophages. Edelfosine oral treatment in rats, at doses 8-fold higher than those displaying anti-inflammatory action, lacked toxicity. Edelfosine treatment showed no any significant cardiotoxicity, hepatotoxicity or renal toxicity. Unlike NSAIDs, edelfosine did not inhibit prostaglandin E(2) synthesis in gastrointestinal mucosal biopsies, and no histologic alteration in gastrointestinal tract was detected after drug treatment. Thus, edelfosine shows a potent in vitro and in vivo anti-inflammatory activity while sparing gastric mucosa. Our data identify edelfosine as a novel anti-inflammatory drug by abating neutrophil infiltration through L-selectin shedding and may provide a new therapeutic approach for inflammatory bowel disease free from toxicity.

Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor.

Neurologic impairment in HIV-1-associated dementia (HAD) and other neuroinflammatory diseases correlates with injury to dendrites and synapses, but how such injury occurs is not known. We hypothesized that neuroinflammation makes dendrites susceptible to excitotoxic injury following synaptic activity. We report that platelet-activating factor, an inflammatory phospholipid that mediates synaptic plasticity and neurotoxicity and is dramatically elevated in the brain during HAD, promotes dendrite injury following elevated synaptic activity and can replicate HIV-1-associated dendritic pathology. In hippocampal slices exposed to a stable platelet-activating factor analogue, tetanic stimulation that normally induces long-term synaptic potentiation instead promoted development of calcium- and caspase-dependent dendritic beading. Chemical preconditioning with diazoxide, a mitochondrial ATP-sensitive potassium channel agonist, prevented dendritic beading and restored long-term potentiation. In contrast to models invoking excessive glutamate release, these results suggest that physiologic synaptic activity may trigger excitotoxic dendritic injury during chronic neuroinflammation. Furthermore, preconditioning may represent a novel therapeutic strategy for preventing excitotoxic injury while preserving physiologic plasticity.

Cytotoxicity of an anti-cancer lysophospholipid through selective modification of lipid raft composition.

Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts.

Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug diffusion.

Inhibitors of choline uptake and metabolism cause developmental abnormalities in neurulating mouse embryos.

BACKGROUND: Choline is an essential nutrient in methylation, acetylcholine and phospholipid biosynthesis, and in cell signaling. The demand by an embryo or fetus for choline may place a pregnant woman and, subsequently, the developing conceptus at risk for choline deficiency. METHODS: To determine whether a disruption in choline uptake and metabolism results in developmental abnormalities, early somite staged mouse embryos were exposed in vitro to either an inhibitor of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), or an inhibitor of phosphatidylcholine synthesis, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3)). Cell death following inhibitor exposure was investigated with LysoTracker Red and histology. RESULTS: Embryos exposed to 250-750 microM DMAE for 26 hr developed craniofacial hypoplasia and open neural tube defects in the forebrain, midbrain, and hindbrain regions. Embryos exposed to 125-275 microM ET-18-OCH(3) exhibited similar defects or expansion of the brain vesicles. ET-18-OCH(3)-affected embryos also had a distended neural tube at the posterior neuropore. Embryonic growth was reduced in embryos treated with either DMAE (375, 500, and 750 microM) or ET-18-OCH(3) (200 and 275 microM). Whole mount staining with LysoTracker Red and histological sections showed increased areas of cell death in embryos treated with 275 microM ET-18-OCH(3) for 6 hr, but there was no evidence of cell death in DMAE-exposed embryos. CONCLUSIONS: Inhibition of choline uptake and metabolism during neurulation results in growth retardation and developmental defects that affect the neural tube and face.

ET-18-OCH(3)-induced cytotoxicity and DNA damage in rat astrocytes.

The ether lipid 1-octadecyl-2-methyl-rac-glicero-3-phosphocholine (ET-18-OCH(3)) is known to be selectively cytotoxic toward several types of tumor cells, in which it seems to activate a process of apoptotic cell death. Moreover, the drug has been demonstrated to be active in normal cells too, particularly in rat astrocytes. In these cells at low dosage (from 1 to 6 microg/ml of medium) ET-18-OCH(3) stimulates maturation and protective responses, whereas at increasing dosages (from 8 to 20 microg/ml) it shows cytotoxic effects. The present study demonstrates that when ET-18-OCH(3) is added to astrocytes, it activates, in a time- and concentration-dependent manner, an oxidative process by increasing both the generation of reactive oxygen species (ROS), including nitric oxide, and lipid peroxidation. When there is a high ET-18-OCH(3) concentration or the time of treatment is prolonged, the increased oxidative condition seems to trigger DNA fragmentation (monitored by COMET assay) as well as loss in cell viability. These cytotoxic effects indicate that ROS may be considered, in our experimental model, as executioners of a program of cell death. In addition, ET-18-OCH(3) being a promising molecule in antitumor therapy, our data, while reinforcing the importance of monitoring the therapeutic drug dosage employed, also suggest that it may be useful to associate some antioxidants with antitumor treatments.

Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid-induced apoptosis.

Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.

Intravitreal toxicology in rabbits of two preparations of 1-O-octadecyl-sn-glycerol-3-phosphonoformate, a sustained-delivery anti-CMV drug.

PURPOSE: To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA), as a long-acting, nontoxic intravitreous injectable drug delivery system for cytomegalovirus (CMV) retinitis. METHODS: ODG-PFA was synthesized by coupling the phosphonate residue of PFA to the 3 hydroxyl of 1-O-octadecyl-sn-glycerol and formulated as micelles and liposomes at concentrations so that, after injection into the rabbit vitreous, the resultant intravitreal concentrations were 0.2 mM, 0.63 mM, and 2 mM in micellar formulation and 0.02 mM, 0.063 mM, 0.2 mM, and 0.63 mM for liposomal formulation. The compounds were injected, and toxicology evaluations were performed. RESULTS: Intravitreal injections of micellar ODG-PFA resulted in aggregation of the material in vitreous and variable local retinal damage. Intravitreal injections of the liposomal ODG-PFA revealed even dispersion of the compounds and a clear vitreous, using final concentration in the vitreous of 0.2 mM. No intraocular toxicity was found with the 0.632 mM final concentration. The 50% inhibitory concentration (IC50) for CMV of ODG-PFA was 0.43+/-0.27 microM, and the therapeutic index of ODG-PFA after intravitreal injection was estimated to be 1470:1. CONCLUSIONS: Lipid-derivatized foscarnet liposome formulations may be a useful long-acting delivery system for the therapy of CMV retinitis.

Functional interactions between synthetic alkyl phospholipids and the ABC transporters P-glycoprotein, Ste-6, MRP, and Pgh 1.

The ABC superfamily of transporters includes the mammalian P-glycoprotein family (Class I and Class II P-gps), the multidrug resistance-associated protein (MRP), the Pgh-1 product of Plasmodium falciparum gene pfmdr1, all of which are associated with cellular pleiotropic drug resistance phenomena. STE6, the yeast transporter for the farnesylated peptide pheromone a, is also a member of this family. Structural similarities in this family translate into functional homology as expression of mouse Mdr3S (P-gp), P. falciparum Pgh-1, and human MRP partially restore mating in a sterile yeast mutant lacking a functional STE6 gene. The demonstration that Class II P-gps function as phosphatidylcholine (PC) translocators raise the possibility that other ABC transporters may also interact with physiological lipids. We report the identification of the synthetic lipid and PC analog ET-18-OCH3 (edelfosine) as a substrate for not only Class II P-gp but also for Class I P-gps and surprisingly for the other ABC transporters MRP, Pgh-1, and STE6. Expression of these proteins in the yeast Saccharomyces cerevisiae JPY201 was found to confer cellular resistance to cytotoxic concentrations of this lipid by a factor of 4-20-fold in a growth inhibition assay. The noted activity of ABC transporters toward this synthetic lipid was specific as a mutant variant of Mdr3 (Mdr3F) with reduced activity could not convey cellular resistance to ET-18-OCH3. ET-18-OCH3 was also found capable of blocking a-peptide pheromone transport and STE6 complementation by these ABC proteins. The inhibitory effect of ET-18-OCH3 on cell growth and a-factor transport could be abrogated by incubation with the lipid acceptor protein BSA or by enzymatic cleavage by microsomal alkylglycerol mono-oxygenase (MAMO). MAMO and BSA reversal of the ether lipid effect was only seen in the presence of a functional transporter. These results suggest that the group of cytotoxic synthetic PC analogs studied reveal possible structural and functional aspects common to the ABC transporters tested. Furthermore, the studies with BSA and MAMO suggest that the mechanism of transport of ET-18-OCH3 by these ABC transporters may be related to the flippase mechanism of PC transport by Mdr2.

Enhanced therapeutic effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

The ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) has anticancer activity, but systemic toxicity has restricted its therapeutic use. In this report "free" ET-18-OCH3 and a stable, well-characterized, liposome-based formulation of ET-18-OCH3 (ELL-12) were compared for in vivo toxicity in normal mice and for therapeutic efficacy in three mouse tumor model systems. The entrapment of ET-18-OCH3 in liposomes decreased the acute toxicity of ET-18-OCH3 after i.v. administration. The maximum tolerated dose for a single i.v. dose of free ET-18-OCH3 was found to be approximately 25 mg/kg, whereas the maximum tolerated dose for ELL-12 was approximately 200 mg/kg. ELL-12 was much less hemolytic in vivo than ET-18-OCH3. The therapeutic efficacy of free ET-18-OCH3 and ELL-12 was investigated against i.p. P388 leukemia, Lewis lung cancer lung metastases, and B16/F10 melanoma (lung tumor nodules) in mice. Although ET-18-OCH3 had some anticancer activity, it was found that ELL-12 was more effective than ET-18-OCH3 in all three tumor models at lower and nontoxic dose schedules. These results suggest that association of ET-18-OCH3 in stable, well-characterized liposomes transforms it into an effective antitumor agent.

Cytokinetic and morphologic differences in ovarian cancer cells treated with ET-18-OCH3 and the DNA-interacting agent, etoposide.

New antineoplastic agents with different cytotoxic mechanisms are of interest for their ability to overcome resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro and in vivo, and the cell membrane is believed to be the target of their antitumor activity. In this study we have investigated the different cytokinetic and morphologic responses of human ovarian carcinoma cells (BG-1) to one of the ether lipids (ET-18-OCH3) and to etoposide. Etoposide induced a significantly greater G2/M block. However, the proportion of the cycling cell fraction decreased significantly in cells treated by ET-18-OCH3 and induction of the hypodiploid traction was strongly correlated with reduction of the cycling cell fraction. On the other hand, the hyperdiploid fraction was found to correlate with reduction of the cycling cell fraction in etoposide treated cells. Despite the significant appearance of the hypodiploid fraction, apoptosis was not observed by DNA-gel assay. Microscopic study showed that the hyperdiploid fraction represented cells with multiple nuclei. These observations support the unique lethal effect of ET-18-OCH3 on ovarian carcinoma cells, distinguishing it from the action of a typical DNA-interacting agent. The membrane-targeted ether lipids deserve consideration for the future chemotherapy of ovarian carcinoma, perhaps in combination with the appropriate DNA-interacting agent. New antineoplastic agents with different cytotoxic mechanisms are of interest not only for their unique inhibitory properties but also for their potential of overcoming resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro (1, 2, 3) and in vivo (4, 5), and the cell membrane is believed to be the target of their antitumor activity. Etoposide, a DNA-interacting agent, is also active against human ovarian cancer cells in vitro (6) or in clinical trials either as a single agent (7) or in combination with cisplatin (8). We have reported that a cytotoxic dose of one of the ether lipids, ET-18-OCH3, induces a G2/M block in BG-1 human ovarian cancer cells, and also a hypodiploid fraction as shown on DNA analysis by flow cytometry (FCM) (9). The G2/M block was also observed in BG-1 cells following etoposide treatment (6). In the present study, we have investigated the differences in the cytokinetic and morphologic responses of BG-1 cells to ET-18-OCH3 and to etoposide.

Synthesis and growth inhibitory properties of glycosides of 1-O-hexadecyl-2-O-methyl-sn-glycerol, analogs of the antitumor ether lipid ET-18-OCH3 (edelfosine).

Glycosylated antitumor ether lipids (GAELs), analogs of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (1, ET-18-OCH3, edelfosine), were synthesized in good overall yields by glycosylation of 1-O-alkyl-2-O-methyl-sn-glycerol and tested for in vitro antineoplastic activity against a variety of murine and human tumor cell lines. Stereospecific glycosylation was achieved by the use of 2-O-acetyl-3,4,6-tri-O-benzylglucopyranosyl and -mannopyranosyl trichloroacetimidates as donors, with trimethylsilyl trifluoromethanesulfonate as catalyst in the presence of molecular sieves at -78 degrees C. The GAELs differ from 1 in having the sn-3-phosphocholine residue replaced by one of the following monosaccharide residues: beta- and alpha-2-deoxy-D-arabino-hexopyranosyl, alpha-D-mannopyranosyl, 2-O-methyl-beta-D-glucopyranosyl, and 2-O-methyl-alpha-D-mannopyranosyl. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-deoxy-beta-D-arabino-hexopyranosyl)- sn-glycerol (2) was more effective than 1 in inhibiting the growth of MCF-7 (human breast cancer) and its adriamycin-resistant form MCF-7/adriamycin, and murine Lewis lung cancer cells. 2-Deoxy-beta-D-arabino-hexopyranoside 2 was also an effective growth inhibitor of two drug-resistant leukemic cell lines, P388/Adr and L1210/vmdr.

Differentiation of HL-60 cells distinguishes between cytostatic and cytotoxic effects of the alkylphospholipid ET-18-OCH3.

The synthetic dialkylphospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) inhibits growth of the acute myelogenous leukemia cell line HL-60. Incubation of HL-60 cells with demethyl-sulfoxide causes the cells to differentiate to a granulocyte-like phenotype and become quiescent. Incubation of the DMSO-treated cells with ET-18-OCH3 results in a reduction in cell numbers due to cytotoxicity. In contrast, treatment of undifferentiated HL-60 cells with lower concentrations of ET-18-OCH3 leads to growth inhibition. These data indicate that the model of differentiated HL-60 cells currently used for the study of resistance to growth inhibition is inappropriate. HL-60 cells can be used to measure growth inhibition and at higher doses cytotoxicity. However, the differentiated, nonproliferative, cells can only be used to measure direct cytotoxicity. Therefore, the results of studies directly comparing the effects of ET-18-OCH3 in proliferative HL-60 cells and quiescent DMSO-treated HL-60 cells should be reevaluated. An evaluation of the effects of low concentrations of ET-18-OCH3 (0.5-1.5 microM) in proliferative HL-60 cells indicated that ET-18-OCH3 was an effective cytostatic agent at nontoxic concentrations. In summary, studies on the mechanism of action of ET-18-OCH3, or related ether lipids, should carefully investigate differences in the effects at cytostatic versus cytotoxic concentrations.

Influence of serum levels on leukemic cell destruction by the ether lipid ET-18-OCH3.

The effect of serum on the antineoplastic action of the alkyl-lysophospholipid 1-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3) was studied in two human leukemia cell lines, HL60 and K562, and in leukemic cells of patients. Decreasing amounts of serum in the culture medium enhanced the cytotoxic action of ET-18-OCH3 dramatically in both cell lines and in the leukemic cells, as measured by cell survival and proliferation. Uptake of ET-18-OCH3 was likewise increased at reduced serum levels. Similar effects were obtained when fetal calf serum (FCS) in the culture medium was substituted by bovine serum albumin (BSA, fatty acid free). Selectivity of the alkyl-lysophospholipid at reduced serum or BSA level was demonstrated by clonogenic assays of normal marrow progenitor cells. Our study provides an optimalization of the purging conditions in autologous bone marrow transplantation, by using a low concentration of BSA during ET-18-OCH3 treatment (20 micrograms/ml for 4 h) in serum-free culture medium.

Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine.

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.