Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Leukocyte-Mediated Combined Targeted Chemo and Gene Therapy for Esophageal Cancer.

Poor prognosis of esophageal cancer is associated with limited clinical treatment efficacy and lack of targeted therapies. With advances in nanomedicine, nanoparticle drug delivery systems play increasingly important roles in tumor treatment by enabling the simultaneous delivery of multiple therapeutic agents. We here propose a novel nanovector for targeted combination gene therapy and chemotherapy in esophageal cancer. A novel lipid nanovector (EYLN) was designed to carry the chemotherapy drug doxorubicin (Dox) and small interfering RNA against the lipid anabolic metabolism gene LPCAT1, which we previously showed to be significantly overexpressed in esophageal cancer tissues, and its interference inhibited the proliferation, invasion, and metastasis of esophageal cancer cells. This vector, EYLN-Dox/siLPCAT1, was further coated with leukocyte membranes to obtain mEYLNs-Dox/siLPCAT1. The particle size of the coated nanovector was approximately 136 nm, and the surface zeta potential was -21.18 mV. Compared with EYLNs-Dox/siLPCAT1, mEYLNs-Dox/siLPCAT1 were more easily internalized by esophageal cancer cells due to the LFA-1 highly expressed leukocyte membrane coating and showed significant inhibition of the proliferation, migration, and metastasis of esophageal cancer cells, along with their LPCAT1 expression, through more effective delivery of the drugs. Moreover, the nanovectors showed improved blood circulation time, tissue distribution, tumor targeting, and tumor suppression in a mouse model. Thus, combining chemo and gene therapy with this new nanodelivery system achieved greater therapeutic efficacy, providing a new strategy for the treatment of esophageal cancer.

A novel assay for measuring recombinant human lysophosphatidylcholine acyltransferase 3 activity.

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme Km values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 µmol·L-1 , respectively, and the Vmax was 39.76 ± 1.86 pmol·min-1 ·U-1 . Moreover, we used our new method to determine the IC50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.

Lysophospholipid acyltransferases and eicosanoid biosynthesis in zebrafish myeloid cells.

Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.

Lysophosphatidylcholine acyltransferase 1 altered phospholipid composition and regulated hepatoma progression.

BACKGROUND & AIMS: Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS: Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines. RESULTS: IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa. CONCLUSIONS: Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.

Lysophospholipid metabolism facilitates Toll-like receptor 4 membrane translocation to regulate the inflammatory response.

Sepsis, an overwhelming inflammatory response to infection, is a major cause of morbidity and mortality worldwide and has no specific therapy. Phospholipid metabolites, such as lysophospholipids, have been shown to regulate inflammatory responses in sepsis, although their mechanism of action is not well understood. The phospholipid-metabolizing enzymes, lysophospholipid acyltransferases, control membrane phospholipid composition, function, and the inflammatory responses of innate immune cells. Here, we show that lysophosphatidylcholine acyltransferase (LPCAT) regulates inflammatory responses to LPS and other microbial stimuli. Specific inhibition of LPCAT down-regulated inflammatory cytokine production in monocytes and epithelial cells by preventing translocation of TLR4 into membrane lipid raft domains. Our observations demonstrate a new regulatory mechanism that facilitates the innate immune responses to microbial molecular patterns and provide a basis for the anti-inflammatory activity observed in many phospholipid metabolites. This provides the possibility of the development of new classes of anti-inflammatory and antisepsis agents.

The lysophospholipid acyltransferase antagonist CI-976 inhibits a late step in COPII vesicle budding.

The mechanism of coat protein (COP)II vesicle fission from the endoplasmic reticulum (ER) remains unclear. Lysophospholipid acyltransferases (LPATs) catalyze the conversion of various lysophospholipids to phospholipids, a process that can promote spontaneous changes in membrane curvature. Here, we show that 2,2-methyl-N-(2,4,6,-trimethoxyphenyl)dodecanamide (CI-976), a potent LPAT inhibitor, reversibly inhibited export from the ER in vivo and the formation of COPII vesicles in vitro. Moreover, CI-976 caused the rapid and reversible accumulation of cargo at ER exit sites (ERESs) containing the COPII coat components Sec23/24 and Sec13/31 and a marked enhancement of Sar1p-mediated tubule formation from ERESs, suggesting that CI-976 inhibits the fission of assembled COPII budding elements. These results identify a small molecule inhibitor of a very late step in COPII vesicle formation, consistent with fission inhibition, and demonstrate that this step is likely facilitated by an ER-associated LPAT.

Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide.

Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor-alpha (TNF-alpha) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-gamma (IFN-gamma), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF-alpha and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-gamma than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phosphonate) and YM 50201 (3-hydroxyethyl 5,3'-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-alpha and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-gamma. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-alpha production by decreasing TNF-alpha mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus.

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.

Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons.

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-([(4-azidosalicyl)-12-amino)]dodecanoyl-sn-glycerol-3-phospho cholin e (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.

Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs.

Synthesis of 32P-labeled 12-azidooleoyl-CoA and 125I-labeled 12-[(azidosalicyl)amino]dodecanoyl-CoA (ASD-CoA) was achieved. The synthesized radioactive, photoreactive reagents were tested as photoaffinity labels for acyl-CoA:lysophosphatidylcholine acyltransferase from the microsomal membranes of developing soybean cotyledons. When a mixture of microsomal membranes and the azidooleoyl-CoA or ASD-CoA were incubated in the dark, the analogs were recognized as substrate and competitive inhibitor, respectively. The enzyme preferentially utilizes unsaturated acyl-CoAs rather than saturated acyl-CoAs. Incubation of microsomal membranes with acyl-CoA analogs and immediately followed by photolysis resulted in an irreversible inhibition of lysophosphatidylcholine acyltransferase activity. Analysis of photolyzed microsomal membranes by SDS/PAGE and autoradiography revealed that azidooleoyl-CoA preferentially labeled eight acyl-CoA binding proteins, but ASD-CoA labeled only three polypeptides with molecular masses of 110, 90 and 32 kDa that are commonly labeled by both the analogs. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme against photoinactivation by azidooleoyl-CoA and ASD-CoA, respectively. The protection was profound in 110-kDa polypeptide indicating that this protein could be lysophosphatidylcholine acyltransferase. These results demonstrate that the photoaffinity of acyl-CoA analogs makes them potential probes to identify and characterize lipid biosynthetic enzymes.

Enhanced production of platelet-activating factor in stimulated rat leukocytes caused by the blockade of lysophospholipid acylation.

We have previously reported that triacsin C, an acyl-CoA synthetase inhibitor, enhanced the production of platelet-activating factor (PAF) in calcium ionophore-activated rat polymorphonuclear leukocytes (PMNs). In this report, we further demonstrated that the production of PAF by PMNs in response to opsonized zymosan was significantly enhanced by pretreatment with triacsin C and also by the pretreatment with merthiolate, which was reported to be an inhibitor of acyl-CoA/lysolecithin acyltransferase. Pretreatment with triacsin C or merthiolate also enhanced the lyso-PAF content in the stimulated PMNs. Addition of lyso-PAF in the incubation mixture of PMNs in the presence of opsonized zymosan augmented the production of PAF. The enhancement of PAF production by lyso-PAF has been reported by several authors, and the importance of lyso-PAF in the remodeling pathway of PAF synthesis has been generally recognized. Therefore, from the above findings, it is assumed that blockades of the reacylation of lyso-phospholipids, by inhibitors such as triacsin C and merthiolate, might lead to accumulation of lyso-PAF and might result in the enhancement of PAF production when the remodeling pathway is active.

Directed shift of fatty acids from phospholipids to triacylglycerols in HL-60 cells induced by nanomolar concentrations of triethyl lead chloride: involvement of a pertussis toxin-sensitive pathway.

Triethyl lead chloride (Et3PbCl) was found to induce a shift of fatty acids from membrane phospholipids to triacylglycerols in the human promyelocytic leukemia cell line HL-60. High concentrations of Et3PbCl (greater than 10 microM) caused a substantial liberation of [14C]arachidonic acid within 10 to 20 min in dimethyl sulfoxide-differentiated cells, comparable to the effect of the calcium ionophore A23187 (10 microM). Following liberation of arachidonic acid, its metabolites could be detected. Prolongation of the incubation time and reduction of Et3PbCl concentration resulted in a shift of fatty acids from phospholipids to triacylglycerols. Deacylation of phospholipids and reacylation into phospholipids and triacylglycerols were in equilibrium when the cells were treated with Et3PbCl at concentrations of less than or equal to 10 microM for 5 hr or less than or equal to 1 microM for 24 hr; no increase of free fatty acids could be observed, and the loss of fatty acids within the phospholipids was equivalent to the increase of fatty acid content within the triacylglycerols. Moreover, under these conditions, no loss of viability was seen after 24 hr, as compared with untreated differentiated cells. This concentration- and time-dependent effect of Et3PbCl might be due to a stimulated liberation of fatty acids via phospholipase A2, because this stimulation could be totally prevented by the phospholipase inhibitors quinacrine and p-bromophenacylbromide. Additionally, pretreatment of differentiated HL-60 cells with pertussis toxin resulted in a drastic reduction of [14C]arachidonic acid liberation when cells were stimulated with Et3PbCl. These results suggest the involvement of a pertussis toxin-sensitive GTP-binding protein and of a signal transduction mechanism during stimulated fatty acid release; release does not seem to be via a direct stimulation of phospholipase activity by the lead compound.

Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification.

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.

Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion.

Depletion of membrane phospholipids is known to be associated with myocardial ischemia, but its relationship to the injury involved with the reperfusion of ischemic myocardium is not known. The present study was designed to relate phospholipid degradation with reperfusion injury. The isolated in situ pig heart was subjected to 60 min of regional ischemia induced by occluding the left anterior descending (LAD) coronary artery and 60 min of global ischemia by hypothermic cardioplegic arrest followed by 60 min of reperfusion. The pigs were divided into two groups. In the treatment group, the heart was preperfused with mepacrine (0.05 mM), a known phospholipase inhibitor, for 15 min prior to LAD occlusion. In the control group, the total phospholipid content was not significantly decreased during LAD occlusion and arrest, but was reduced appreciably after reperfusion. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol followed a similar pattern. The lowering of these phospholipids during reperfusion was accompanied by enhancement of lysophosphatidylcholine. Mepacrine restored the normal levels of these phospholipids. During reperfusion, fatty acyl CoA synthetase, lysophospholipase, and lysophosphatidylcholine acyltransferase were depressed, whereas phospholipase A2 was enhanced. Mepacrine inhibited phospholipase A2, but had no effects on the other enzymes. Mepacrine also provided significant protection against reperfusion injury, as documented by the preservation of high-energy phosphate compounds and inhibition of the appearance of creatine kinase activity in the perfusate. These results suggest that membrane phospholipids play an important role in myocardial injury associated with ischemia and reperfusion, primarily because the deacylation-reacylation cycle of phospholipid biosynthesis becomes defective.

Ether phospholipids as inhibitors of the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase in macrophages.

1-Alkylglycerophosphatide analogs which are known to activate macrophages to enhanced tumor cytotoxicity are structurally closely related to 1-acyl-sn-glycero-3-phosphocholine. In this study we have examined the influence of some of these compounds and of platelet-activating factor (PAF-acether, 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine) on the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) in homogenate of bone-marrow-derived murine macrophages. This enzyme is suggested to be involved in the control of the availability of the icosanoid precursor, arachidonic acid. Kinetic experiments revealed apparent Km and V values for 1-palmitoyl-sn-glycero-3-phosphocholine of 6.0 microM and 16.10 nmol/mg protein per min, respectively. When the 1-palmitoyl-sn-glycero-3-phosphocholine concentration was equal to Km, the enzyme was dose-dependently inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine with a 50% inhibition at 30 microM. The kinetic parameters in the presence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (K'm = 10.0 microM, V' = 11.40 nmol X mg-1 X min-1) suggest that this alkyl phospholipid is a mixed-type inhibitor. All other alkyl analogs tested (1-O-methyl-2-O-octadecyl-rac-glycerol-3-phosphocholine, racemic PAF-acether, L-PAF-acether, D-1-O-hexadecyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-rac-glycero-3-phosphocholine) inhibited the enzyme to various degrees. Arachidonic acid transfer to the 1-alkylglycerophosphatide analogs themselves could be ruled out under the assay conditions used. Therefore, we conclude that the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase can be inhibited by synthetic and naturally occurring ether phospholipids in homogenate of bone-marrow-derived murine macrophages.

The acylation of lysophosphatidylcholine in the rat testicular tissue: the combined activity of acyl-CoA synthetase and lysolecithin acyltransferase.

The activity of lysophosphatidylcholine acyltransferase (EC 2.3.1.23) in combination with acyl-CoA synthetase (EC 6.2.1.3) has been determined in the homogenates and subcellular fractions of rat testis. The enzyme activity was found to be maximal at pH 7.4 ATP and CoASH were required for optimal incorporation of [1-14C] oleic acid into phosphatidylcholine. The sulfhydryl-binding reagents showed inhibitory effect on the acyltransferase activity. Dibutyryl cyclic AMP and beta-mercaptoethanol did not affect the enzyme activity. Subcellular distribution patterns of markers, marker enzymes and lysolecithin acyltransferase have shown that the acyltransferase activity was found to be predominantly localized in the microsomal fraction, though significant activity was also present in the mitochondrial fraction. These findings, together with our previous studies on testicular phospholipases A, suggest that the deacylation-reacylation cycle is operative in rat testicular tissue.