Analogues Of Lysophosphatidylcholine Research Articles

Synthetic alkyl-ether-lipid promotes TRPV2 channel trafficking trough PI3K/Akt-girdin axis in cancer cells and increases mammary tumour volume

The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

Differential impact of synthetic antitumor lipid drugs on the membrane organization of phosphatidic acid and diacylglycerol monolayers

Anti-tumour lipids are synthetic analogues of lysophosphatidylcholine. These drugs are both cytotoxic and cytostatic, and more interestingly, exert these effects preferentially in tumour cells. While the exact mechanism of action isn’t fully elucidated, these drugs appear to preferentially partition into rigid lipid domains in cell membranes. Upon insertion, the compounds alter membrane domain organization, disrupt normal signal transduction, and cause cell death. Recently, it has been reported that these drugs induce accumulation of diacylglycerol in yeast cells which in turn sensitizes cells to the drugs. Conversely, phosphatidic acid accumulation appears to protect cells against the drugs. In the current work, the aim was to compare the biophysical effects of the drugs edelfosine, miltefosine and perifosine on monolayers of dimyristoyl phosphatidic acid, dimyristoyl glycerol and an equimolar mixture, to understand how these lipids modulate the mode of action. Surface pressure – area isotherms, compression moduli and Brewster angle microscopy were used to compare drug effects on lipid packing, monolayer compressibility and lateral domain organization of these films. Results suggest that edelfosine and miltefosine have stabilizing effects on all of the monolayers, while perifosine destabilizes dimyristoyl glycerol and the equimolar mixture. Additionally, all three drugs change the morphology of the domains observed. Based on these results the stabilization of diacylgylcerol by edelfosine and miltefosine may contribute to the mode of action as diacylglycerol is a known disruptor of bilayers. Perifosine however does not stabilize diacylglycerol, and therefore cell death may occur through a more direct inhibition of specific signal transduction. These results suggest that perifosine may illicit cytotoxicity through a different mechanism compared to the other antitumor lipid drugs.

Synthesis of oxidized phospholipids by sn-1 acyltransferase using 2–15-HETE lysophospholipids

Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2α (cPLA2α) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.

Type II NKT cells directed immune regulatory mechanism(s) control spontaneous lupus nephritis in mice

Abstract NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer+) and type II (sulfatide/CD1d-tetramer+) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4+/CD8+T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.

Lysophosphatidylcholine Analogues Alter Yeast Nuclear Envelope Architecture and Function

The lysophosphatidylcholine analog edelfosine is an anticancer lipid drug that targets cellular membranes. Therefore, it is alleged that in contrast to other chemotherapeutic agents edelfosine does not impact DNA. Despite this claim, a genetic screen in Saccharomyces cerevisiae found that lack of silent information regulator (Sir) proteins, alleviated the toxic effect of edelfosine. Since Sir proteins are key architects and regulators of the genome, we decided to investigate if this unusual connection with edelfosine could be related to a potential effect of the lysophosphatidylcholine analog on the nuclear envelope (NE) structure and function. Indeed, we found that edelfosine alters NE morphology, leading to an abnormal elongation of the membrane. Since it is known that Sir proteins mediate tethering of subtelomeric chromatin to the NE, we investigated if this was affected by treatment of wild type cells with the drug. Results from chromatin immunoprecipitation of Sir4 showed a significant reduction in the association of the protein with telomeres when cells were treated with edelfosine. Current evidence also supports alterations of telomere clustering in the presence of the lysophosphatidylcholine analogue. Therefore, our results suggest that edelfosine alters NE morphology and function. We speculate that mislocalization of Sir proteins in the nucleus probably results in abnormal silencing of genes that may be implicated in pathways that confer resistance to this lysophosphatidylcholine analog.Support or Funding InformationThis work has been financially supported by the Natural Sciences and Engineering Research Council of Canada to V Zaremberg and JA Cobb.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysophosphatidylcholine and its phosphorothioate analogues potentiate insulin secretion via GPR40 (FFAR1), GPR55 and GPR119 receptors in a different manner.

Lysophosphatidylcholine (LPC) is an endogenous ligand for GPR119 receptor, mediating glucose-stimulated insulin secretion (GSIS). We demonstrate that LPC facilitates GSIS in MIN6 pancreatic β-cell line and murine islets of Langerhans by recognizing not only GPR119 but also GPR40 (free fatty acid receptor 1) and GPR55 activated by lysophosphatidylinositol. Natural LPCs are unstable when administered invivo limiting their therapeutic value and therefore, we present phosphorothioate LPC analogues with increased stability. All the modified LPCs under study (12:0, 14:0, 16:0, 18:0, and 18:1) significantly enhanced GSIS. The 16:0 sulfur analogue was the most potent, evoking 2-fold accentuated GSIS compared to the native counterpart. Interestingly, LPC analogues evoked GPR40-, GPR55-and GPR119-dependent [Ca2+]i signaling, but did not stimulate cAMP accumulation as in the case of unmodified molecules. Thus, introduction of a phosphorothioate function not only increases LPC stability but also modulates affinity towards receptor targets and evokes different signaling pathways.

Primary Metabolism and Medium-Chain Fatty Acid Alterations Precede Long-Chain Fatty Acid Changes Impacting Neutral Lipid Metabolism in Response to an Anticancer Lysophosphatidylcholine Analogue in Yeast.

The nonmetabolizable lysophosphatidylcholine (LysoPC) analogue edelfosine is the prototype of a class of compounds being investigated for their potential as selective chemotherapeutic agents. Edelfosine targets membranes, disturbing cellular homeostasis. Is not clear at this point how membrane alterations are communicated between intracellular compartments leading to growth inhibition and eventual cell death. In the present study, a combined metabolomics/lipidomics approach for the unbiased identification of metabolic pathways altered in yeast treated with sublethal concentrations of the LysoPC analogue was employed. Mass spectrometry of polar metabolites, fatty acids, and lipidomic profiling was used to study the effects of edelfosine on yeast metabolism. Amino acid and sugar metabolism, the Krebs cycle, and fatty acid profiles were most disrupted, with polar metabolites and short-medium chain fatty acid changes preceding long and very long-chain fatty acid variations. Initial increases in metabolites such as trehalose, proline, and γ-amino butyric acid with a concomitant decrease in metabolites of the Krebs cycle, citrate and fumarate, are interpreted as a cellular attempt to offset oxidative stress in response to mitochondrial dysfunction induced by the treatment. Notably, alanine, inositol, and myristoleic acid showed a steady increase during the period analyzed (2, 4, and 6 h after treatment). Of importance was the finding that edelfosine induced significant alterations in neutral glycerolipid metabolism resulting in a significant increase in the signaling lipid diacylglycerol.

Phosphorothioate analogues of lysophosphatidylcholine as signaling molecules for type 2 diabetes-related membrane receptors

Phosphorothioate analogues of lysophosphatidylcholine as signaling molecules for type 2 diabetes-related membrane receptors

Tracking Diacylglycerol Pools in Budding Yeast

Many diacylglycerol (DAG) sensing probes have been developed to monitor DAG signalling in mammalian cells. Several C1 domains have been fused to fluorescent proteins that can sense DAG in cellular membranes. The C1 domain of PKCδ is very well characterized and known to bind DAG with high affinity, and has been successfully used as a probe fused to GFP in order to monitor DAG in live cells. We have cloned the C1 domain of PKCδ (C1δ) into a centromeric yeast expression vector under a constitutive promoter for in vivo visualization of DAG pools. Localization of C1δ‐GFP was first monitored in different wild type strains grown under standard conditions. The main localization of this DAG‐binding reporter in actively growing cells was the vacuolar membrane, further confirmed by co‐localization with FM4‐64. This localization is consistent with studies done using similar probes to show that DAG localizes to the membrane of isolated vacuoles from yeast cells. Importantly, no changes in vacuolar morphology of wild type cells due to expression of C1δ‐GFP were detected. In addition to vacuolar membrane localization, the C1δ probe also associated with the plasma membrane of daughter cells and at the bud neck. In contrast to the phosphatidic acid (PA)‐binding reporter GFP‐Spo2051–91, C1δ‐GFP did not show nuclear localization as determined by co‐localization analysis using the nucleolar marker Sik1‐RFP. We then tested if the localization would change in conditions where lipid homeostasis and levels of DAG are known to be altered. For this, we used cells lacking the four enzymes responsible for the final step in the synthesis of triglycerides in yeast, namely DAG acyltransferase Dga1, the transacylase Lro1, and the steryl acyltransferases Are1 and Are2 (4Δ quadruple mutant), which accumulate DAG and have defects in both nuclear and peripheral ER membrane organization. Localization of the C1δ‐GFP probe drastically changed when expressed in 4Δ quadruple mutant cells, clearly revealing its association with the expanded nuclear and cortical ER in addition to the vacuolar membrane. Additionally, the polarized distribution of C1δ‐GFP to the plasma membrane of buds and daughter cells significantly decreased in the quadruple mutant. After validating the use of this probe in yeast, we investigated the localization of DAG pools in response to lysophosphatidylcholine (LysoPC) analogues, as we have determined they induce a 5 times increase in DAG levels. Interestingly, incubation with LysoPC analogues induced a drastic change in the localization of DAG while no differences were observed regarding the distribution of PA. These results further support the use of C1δ‐GFP as a valuable tool to track cytosolic accessible pools of DAG in yeast.Support or Funding InformationThis work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ

Disruption of lipid domain organization in monolayers of complex yeast lipid extracts induced by the lysophosphatidylcholine analogue edelfosine in vivo

The lysophosphatidylcholine analogue edelfosine is a potent antitumor and antiparasitic drug that targets cell membranes. Previous studies have shown that edelfosine alters membrane domain organization inducing internalization of sterols and endocytosis of plasma membrane transporters. These early events affect signaling pathways that result in cell death. It has been shown that edelfosine preferentially partitions into more rigid lipid domains in mammalian as well as in yeast cells. In this work we aimed at investigating the effect of edelfosine on membrane domain organization using monolayers prepared from whole cell lipid extracts of cells treated with edelfosine compared to control conditions. In Langmuir monolayers we were able to detect important differences to the lipid packing of the membrane monofilm. Domain formation visualized by means of Brewster angle microscopy also showed major morphological changes between edelfosine treated versus control samples. Importantly, edelfosine resistant cells defective in drug uptake did not display the same differences. In addition, co-spread samples of control lipid extracts with edelfosine added post extraction did not fully mimic the results obtained with lipid extracts from treated cells. Altogether these results indicate that edelfosine induces changes in membrane domain organization and that these changes depend on drug uptake. Our work also validates the use of monolayers derived from complex cell lipid extracts combined with Brewster angle microscopy, as a sensitive approach to distinguish between conditions associated with susceptibility or resistance to lysophosphatidylcholine analogues.

Sulfur- and acyl chain-dependent influence of 2-methoxy-lysophosphatidylcholine analogues on β pancreatic cells.

Nutrient-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is one of the key mechanisms responsible for insulin release from pancreatic islet β cells. Lysophosphatidylcholine (LPC) was demonstrated to induce insulin secretion from β cells, activate glucose uptake and effectively lower blood glucose levels in mouse models of type 1 and 2 diabetes mellitus. The article hereby presents the results of a characterization of 2-OMe-LPC sulfur analogues with defined acyl residues in terms of their effect on intracellular Ca(2+) concentration and cellular membrane integrity in the murine βTC-3 cell model. Active LPC series that could induce calcium flux in βTC-3 cell model include unmodified LPC 12:0, 14:0, 16:0, and 18:0 as well as phosphorothioate analogues of LPC 12:0, 14:0 and 16:0. However, in the case of species bearing mirystoyl and palmitoyl residues [Ca(2+)]i was associated with membrane permeabilization as demonstrated by propidium iodide incorporation and lactate dehydrogenase release. LPC 12:0 (both unmodified and a sulfurcontaining counterpart) and unmodified LPC 18:0 did not demonstrate membrane disruption but acted as calcium inducers. Interestingly, no stimulation of calcium flux or membrane disruption was observed in the case of LPC analogues with two sulfur atoms introduced into a phosphate group. Experiments with nitrendipine and NiCl2 blocking voltage-dependent calcium channels and the general calcium influx, respectively, revealed remarkably that the compounds studied were involved in different signaling mechanisms while administered to the cell culture, which is clearly related to their chemical structure, both acyl chain and modification dependently.

Abstract B35: Vinyl sulfone analogues of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma

Abstract Autotaxin is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. Autotaxin is a unique ectonucleotide pyrophosphatase and phosphodiesterase that facilitates the removal of a choline headgroup from lysophosphatidylcholine to yield lysophosphatidic acid, which is a potent lipid stimulator of tumorigenesis. Thus, autotaxin has received renewed attention because it has a prominent role in malignant progression and represents a promising area of research with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Our goal was to discover agents with therapeutic efficacy in melanoma. For this purpose, we synthesized a set of lysophosphatidylcholine analogs that function as irreversible inhibitors of autotaxin and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 – 0.39 μM, consistent with a Ki of 3.50 μM for inhibition of autotaxin by the analog CVS-16 (10b). A control compound, GWJ-A-10, which is missing the critical moiety, had little effect on cell viability and did not inhibit autotaxin. In addition, CVS-16 (10b) significantly inhibited melanoma cell wound closure and pore migration (data not presented herein). Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent autotaxin inhibitor with significant biological activity, both in vitro and in vivo. Citation Format: Mandi Murph, Guowei Jiang, Molly Altman, Jia Wei, Sterling Tran, Duy Nguyen, Ali Alshamrani, William Hardman, Jianxing Zhang, Glenn Prestwich. Vinyl sulfone analogues of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B35.

Protective role of phosphatidic acid in the cellular response to lysophosphatidylcholine analogues

The synthetic lysophosphatidylcholine (LPC) analogue edelfosine and the naturally occurring platelet activating factor (PAF) are structurally related lipids. Edelfosine is a potent antitumor drug, while PAF is a neurotoxic lipid that has been implicated in Alzheimer's disease. These LPC analogues are also toxic to budding yeast, which can be used as an excellent model system to investigate their mode of action.LPC analogues induce alterations in membrane organization, lipid metabolism and signaling in yeast. Phosphatidic acid (PA) produced by the activity of PLD alleviates sensitivity of yeast cells to PAF and edelfosine. PA is a key lipid signalling and precursor of all glycerolipids. Recent lipid analysis indicates that diacylglycerol (DAG) accumulates in cells treated with LPC analogues and this could contribute to their toxicity. In fact, sensitivity assays in yeast genetically manipulated to alter PA and DAG levels clearly support a protective role of PA in the response to these lipid drugs. Altogether, the evidence suggests that cells are able to sense altered PA: DAG ratios induced by these metabolically stable lipids. While DAG binding proteins have not been clearly identified in yeast, several yeast proteins recognize PA with high specificity. One such protein is the transcriptional repressor Opi1, which regulates expression of phospholipid biosynthetic genes through its interaction with PA. We provide evidence supporting a role of Opi1 in sensing the membrane changes induced by edelfosine, including i) altered sensitivity of cells lacking Opi1, ii) prevalent nuclear localization and iii) Opi1‐dependent UASINO gene repression induced by edelfosine.

How perifosine affects liposome-encapsulated drug delivery across a cell barrier.

The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells. To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured. Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general.

Abstract 2672: Vinyl sulfone analogues of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma

Abstract Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression and represents a promising area of research with significant translational potential. Since ATX inhibitors may have broad implications for therapy, we sought to create a novel series of inhibitors for use against cancer, especially melanoma. Herein we report on the synthesis and biological activity of a targeted affinity label for the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of vinyl sulfone analogs of ATX that function as irreversible inhibitors of ATX and inactivate the enzyme. We commenced biological testing of this compound with viability assays against multiple cancer cell lines whereby the IC50 values ranged from 6.74 - 0.39 μM, which was consistent with the Ki of the vinyl sulfone (3.50 μM). In addition, this compound was also able to significantly inhibit melanoma cell wound closure and pore migration. Most importantly, the vinyl sulfone significantly inhibited melanoma progression using an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that the vinyl sulfone is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo. Note: This abstract was not presented at the meeting. Citation Format: Mandi M. Murph, Molly Altman, Wei Jia, Duy Nguyen, Jada Fambrough, William J. Hardman, Guowei Jiang, Damian Madan, Jianxing Zhang, Glenn D. Prestwich. Vinyl sulfone analogues of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2014-2672

Antitumor lipids in biomembranes modeled with the Langmuir monolayer technique

Studies on interactions between physiologically active compounds and cell membrane components in Langmuir monolayers, which provide suitable models of biomembranes, can be considered as one of the crucial factors leading to the understanding of the mode of action of many drugs that act on membrane level. A good example of membrane-targeted drugs are synthetic analogues of lysophosphatidylcholine, generally named as antitumor lipids (ATLs), due to their anticancer properties. They have advantages over traditional cytostatics as they do not target DNA but cellular membrane. Apart from many studies on cell lines, little is known about the transport of ATLs to the cells, and it is still unclear which membrane components target drug molecules to the tumor cell membrane, sparing the normal cells. This review provides examples of the successful application of the Langmuir monolayer technique in getting insight into biological action and selectivity of representative ATL – edelfosine.

Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate

Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

Abstract 3918: Validation of ATX-Red imaging as a potential companion diagnostic for autotaxin-expressing cancers.

Abstract Autotaxin (ATX), an autocrine motility factor highly upregulated in metastatic cancer, is a lysophospholipase D that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (ATX-Red) that enables visualization of ATX activity in vivo. ATX-Red exhibits minimal fluorescence until it is activated by ATX, which significantly increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with ATX-expressing breast cancer orthotopic tumors, ATX activated ATX-Red fluorescence. Injection of ATX-Red into the tail vein of tumor-bearing mice resulted in high fluorescence in the tumor and low fluorescence in healthy tissues. Indeed, tumor fluorescence directly correlated with ATX activity and expression levels. Pretreatment of mice with a known ATX inhibitor markedly and selectively decreased tumor fluorescence. Taken together, the data show that ATX-Red is an effective, non-invasive, and selective tool that enables intravital visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo in real time. ATX-Red has the potential to (1) identify pathological levels of ATX in specific tissues, (2) monitor the time course of therapeutic efficacy of candidate ATX inhibitors in living animal models of human disease, (3) serve as a companion diagnostic for following treatment regimens that target ATX in human patients and (4) serve as a companion diagnostic to select patients that would benefit from ATX-directed therapy. Citation Format: Damian Madan, Colin Ferguson, Won Yong Lee, Charles Testa, Glenn Prestwich. Validation of ATX-Red imaging as a potential companion diagnostic for autotaxin-expressing cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2013-3918

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.