Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Pachymic Acid Induces Apoptosis of EJ Bladder Cancer Cells by DR5 Up-Regulation, ROS Generation, Modulation of Bcl-2 and IAP Family Members.

Pachymic acid (PA) is a lanostane-type triterpenoid derived from Poria cocos mushroom that possess various biological effects such as anti-cancer, antiinflammatory and anti-metastasis effects. In this study, we investigated the anti-cancer effects of PA in EJ bladder cancer cells. The results showed that PA significantly inhibited proliferation of EJ cells in a dose-dependent manner. PA induced accumulation of sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in EJ cells in a dose-dependent manner. PA also induces activation of caspase-3, -8 and -9, and subsequent cleavage of poly (ADP-ribose) polymerase, and significantly suppressed the inhibitor of apoptosis protein family proteins in a dose-dependent manner. Furthermore, PA activates Bid and induced the loss of mitochondrial membrane potential (ΔΨm ) with up-regulated pro-apoptotic proteins (Bax and Bad), down-regulated anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. In turn, PA increased the generation of reactive oxygen species (ROS); also, the ROS production was blocked by N-acetyl-L-cysteine. The expressions of TNF-related apoptosis inducing ligand and death receptor 5 were up-regulated by PA in a dose-dependent manner, suggesting extrinsic pathway also involved in PA-induced apoptosis. This study provides evidence that PA might be useful in the treatment of human bladder cancer.

Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.

Anandamide and other bioactive N-acylethanolamines (NAEs) are a class of lipid mediators and are produced from glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs). Although the generation of NAPE by N-acylation of phosphatidylethanolamine is thought to be the rate-limiting step of NAE biosynthesis, the enzyme responsible, N-acyltransferase, remains poorly characterized. Recently, we found that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess phospholipid-metabolizing activities including NAPE-forming N-acyltransferase activity, and proposed to call HRASLS1-5 phospholipase A/acyltransferase (PLA/AT)-1-5, respectively. Among the five members, PLA/AT-1 attracts attention because of its relatively high N-acyltransferase activity and predominant expression in testis, skeletal muscle, brain and heart of human, mouse and rat. Here, we examined the formation of NAPE by PLA/AT-1 in living cells. As analyzed by metabolic labeling with [(14)C]ethanolamine or [(14)C]palmitic acid, the transient expression of human, mouse and rat PLA/AT-1s in COS-7 cells as well as the stable expression of human PLA/AT-1 in HEK293 cells significantly increased the generation of NAPE and NAE. Liquid chromatography-tandem mass spectrometry also exhibited that the stable expression of PLA/AT-1 enhanced endogenous levels of NAPE, N-acylplasmenylethanolamine, NAE and glycerophospho-NAE. Furthermore, the knockdown of endogenous PLA/AT-1 in mouse ATDC5 cells lowered NAPE levels. Interestingly, the dysfunction of peroxisomes, which was caused by PLA/AT-2 and -3, was not observed in the PLA/AT-1-expressing HEK293 cells. Altogether, these results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A2 and their effect on the Ca²âº-induced permeabilization of the bacterial membrane.

Phospholipase A of the bacterial outer membrane (OMPLA) is a ß-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2--palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²âº-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca²âº-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²âº and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial.

IMPORTANCE: Secretory phospholipase A2 (sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2 inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2 inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measure was a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95% CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95% CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2 inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246.

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine.

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).

Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice.

While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.

Modulation of proteasome machinery by natural and synthetic analogues of the marine bioactive compound petrosaspongiolide M.

Natural or synthetic? Several petrosaspongiolide M natural and synthetic analogues have been tested as proteasome inhibitors and apoptosis modulators. The natural petrosaspongiolide M congeners gave a consistent decrease in activity. Among the synthetic analogues, the introduction of the benzothiophene ring resulted in a bioequivalent alternative of the petrosaspongiolide M terpenoid system.

Essential role of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis.

Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca(2+). The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP(3) receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP(2) levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca(2+).

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

Potential therapeutic efficacy of pachymic acid in chronic kidney disease induced in rats: role of Wnt/ß-catenin/renin-angiotensin axis.

OBJECTIVES: Chronic kidney disease (CKD) is a major public health problem associated with high mortality. The therapeutic effects of pachymic in CKD management and its underlying mechanisms have not been studied. Therefore, we aimed to investigate the possible inhibitory effect of PA on renal Wnt/ß-catenin signalling in CKD. METHODS: CKD was induced in rats by doxorubicin (DOX; 3.5 mg/kg i.p., twice weekly for 3 weeks). Rats were treated orally with PA (10 mg/kg/day), LOS (10 mg/kg/day) or their combination (PA + LOS) for 4 weeks starting after the last dose of DOX. KEY FINDINGS: DOX-induced renal injury was characterized by high serum cystatin-C, and urine albumin/creatinine ratio, renal content of podocin and klotho were decreased. Tumour necrosis factor-α, interleukin-6, interleukin-1ß, Wnt1, active ß-catenin/total ß-catenin ratio and fibronectin along with mRNA expression of RENIN, ACE and AT1 were increased in renal tissues. Treatment with either PA or LOS ameliorated all DOX-induced changes. The combined treatment was more effective in improving all changes than monotherapy. CONCLUSIONS: These results suggest a new therapeutic benefit of PA in ameliorating CKD in rats through its up-regulatory effect on renal klotho thereby preventing Wnt/ß-catenin reactivation and RAS gene expression. PA/LOS combination provided an additional inhibition of Wnt/ß-catenin signalling and its downstream targets.

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella.

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A(2) to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.

A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement.

Platelets are potent antibody- and complement-dependent cytotoxic effector cells. We showed previously that a single platelet can lyse a target cell sensitized with immunoglobulin G (IgG) and complement components up to C3 (C integral of 3b denotes the target cell-bound fragment of complement up to C3; the precise nature of the bound C3 fragment has not been established), and that the complete cytotoxic system capable of specific recognition and lysis resides in platelet membranes. To define the components of platelet membranes required for cytotoxicity, a set of inhibitors of phospholipase A2 (PLA2) that act by different chemical mechanisms was tested. The lytic reaction is blocked at appropriate concentrations of bromophenacylbromide, mepacrine, and manoalide. When platelets are treated with bromophenacylbromide, inhibition of cytolytic activity and that of PLA2 enzymatic activity occur in parallel. Platelets release arachidonate when incubated with target cells bearing IgG and C integral of 3b, confirming that Fc gamma R and complement receptor trigger both PLA2 action and efficient lysis. Inhibition by thimerosal of a reverse reaction, i.e., reacylation catalyzed by acyltransferase, causes increased target cell lysis, presumably by increasing the products of PLA2 action. Platelet cytotoxicity is increased when platelets are pretreated with some products of PLA2: exogenous lysophospholipids and not free arachidonic acid increase cytotoxicity. Electron microscopy suggests that platelets and target cells may fuse, possibly as a result of the formation of lysophospholipids which are well-known membrane fusogens. Fixation with paraformaldehyde does not affect platelet cytotoxicity, suggesting that the complete cytotoxic system resides as a preformed complex in platelet membranes. The results indicate that platelet membrane-associated PLA2, together with receptors for Fc and complement, are required for platelet cytotoxicity.

An essential role of cytosolic phospholipase A2alpha in prostaglandin E2-mediated bone resorption associated with inflammation.

Prostaglandin E (PGE)2 produced by osteoblasts acts as a potent stimulator of bone resorption. Inflammatory bone loss is accompanied by osteoclast formation induced by bone-resorbing cytokines, but the mechanism of PGE2 production and bone resorption in vivo is not fully understood. Using cytosolic phospholipase A2alpha (cPLA2alpha)-null mice, we examined the role of cPLA2alpha in PGE2 synthesis and bone resorption. In bone marrow cultures, interleukin (IL)-1 markedly stimulated PGE2 production and osteoclast formation in wild-type mice, but not in cPLA2alpha-null mice. Osteoblastic bone marrow stromal cells induced the expression of cyclooxygenase (COX)-2 and membrane-bound PGE2 synthase (mPGES) in response to IL-1 and lipopolysaccharide (LPS) to produce PGE2. Osteoblastic stromal cells collected from cPLA2alpha-null mice also induced the expression of COX-2 and mPGES by IL-1 and LPS, but could not produce PGE2 due to the lack of arachidonic acid release. LPS administration to wild-type mice reduced femoral bone mineral density by increased bone resorption. In cPLA2alpha-null mice, however, LPS-induced bone loss could not be observed at all. Here, we show that cPLA2alpha plays a key role in PGE production by osteoblasts and in osteoclastic bone resorption, and suggest a new approach to inflammatory bone disease by inhibiting cPLA2alpha.

Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor.

Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.

Antiinflammatory peptides (antiflammins) inhibit synthesis of platelet-activating factor, neutrophil aggregation and chemotaxis, and intradermal inflammatory reactions.

Synthetic peptides corresponding to the region of highest similarity between human lipocortin I and rabbit uteroglobin inhibit phospholipase A2 and show potent antiinflammatory activity on the carrageenan-induced rat footpad edema. The peptide HDMNKVLDL (antiflammin-2) inhibits the synthesis of platelet-activating factor (PAF) induced by TNF or phagocytosis in rat macrophages and human neutrophils, and by thrombin in vascular endothelial cells. The peptide MQMKKVLDS (antiflammin-1) is less inhibitory than antiflammin-2 for macrophages and not inhibitory for neutrophils after a 5-min preincubation. This finding suggests that antiflammin-1 is inactivated by neutrophils secretory products, possibly oxidizing agents. Synthesis of PAF is inhibited by antiflammin-2 without an appreciable lag, but this inhibition is reversed when neutrophils or macrophages are washed and incubated in fresh medium. Therefore, antiflammins must be continuously present to inhibit PAF synthesis. Antiflammins block activation of the acetyltransferase required for PAF synthesis, suggesting that this enzyme is another target for the inhibitory activity of antiflammins. These peptides inhibit neutrophil aggregation and chemotaxis induced by complement component C5a. Antiflammin-2 suppresses the increase in vascular permeability and the leukocyte infiltration induced in rats by an Arthus reaction or by intradermal injection of rTNF and C5a.

Pachymic acid inhibits cell growth and modulates arachidonic acid metabolism in nonsmall cell lung cancer A549 cells.

Aberrant arachidonic acid (AA) metabolism has been involved in inflammation and carcinogenesis. The key enzymes in AA metabolism such as cytosolic phospholipase A2 (cPLA(2)) and cyclooxygenase-2 (COX-2) have been implicated in the development and progression of many human cancers, including lung cancer. Hence, the blockade of these enzymes may suppress promotion and survival of human cancer cells. We and others have shown that a natural triterpenoid, pachymic acid (PA), can exhibit antiinflammatory and anticancer properties; however, its potential mechanism has not been fully clarified. In this study, we examined the effect of PA on the proliferation of human nonsmall cell lung cancer A549 cells. Furthermore, we investigated the influences of nontoxic levels of PA on AA metabolism. Additionally, the cellular events and signal transduction pathways influenced by PA were also examined. Our results showed that PA (1) inhibited anchorage-dependent and -independent A549 growth in a concentration-dependent manner, (2) induced apoptosis and disrupted mitochondrial membrane potential in A549 cells, and at nonlethal levels, (3) decreased IL-1 beta-induced activation of cPLA(2) and COX-2, (4) suppressed IL-1 beta-induced activation of mitogen-activated protein kinases (MAPKs), and (5) inhibited IL-1 beta-stimulated nuclear factor kappa B (NF-kappaB) signaling pathways. We speculate that inhibition of AA metabolism by PA is mediated in part by its inhibition of MAPKs and NF-kappaB signaling pathways. Our study reveals that, apart from its cytotoxic effect, PA has the chemopreventive potential by reducing production of eicosanoids from AA metabolism.

Design of new potent and selective secretory phospholipase A(2) inhibitors. 6-Synthesis, structure-activity relationships and molecular modelling of 1-substituted-4-[4,5-dihydro-1,2,4-(4H)-oxadiazol-5-one-3-yl(methyl)]-functionalized aryl piperazin/one/dione derivatives.

The group IIA human non-pancreatic secretory phospholipase A(2) (hnp-sPLA(2)) is one of the enzymes implied in the inflammatory process. In the course of our work on inhibitors of this enzyme we investigated the influence of rigidity of the piperazine region on the biological activity. Several modifications were explored. Various linkers, such as amide, urea, carbamate, or alkoxyphenyl were inserted between the piperazine and the lipophilic chain. Also, modification of the piperazine core to incorporate carbonyl groups was studied. In an in vitro fluorimetric assay using the human GIIA (HPLA(2)) and porcine pancreatic GIB enzymes, compound 60a (Y=phenoxy, R=C(18)H(37), Z=CH(2)) had the optimal activity with an IC(50)=30nM on HPLA(2). By means of molecular modelling we attempted to get informations towards comprehension of differences in activity.

Inhibitory effect of tea polyphenols on local tissue damage induced by snake venoms.

The methanolic extract of fresh tea leaves of Camellia sinensis L. (Theaceae) (CS) was assayed for its potential to inhibit enzymes with hydrolytic activity in Naja naja kaouthia Lesson (Elapidae) and Calloselasma rhodostoma Kuhl (Viperidae) venoms. These snake venom enzymes are responsible for the early effects of envenomation, such as local tissue damage and inflammation. The CS extract inhibited phospholipase A(2), proteases, hyaluronidase and L-amino acid oxidase in both venoms by in vitro neutralization and inhibited the hemorrhagic and the dermonecrotic activities of the venoms in vivo. It is suggested that the inhibitory potential of the CS extract against local tissue damage induced by snake venoms may be attributed to complexation and chelation between the venom proteins and the phenolic contents of the extract.

PLA2 activity is required for nuclear shrinkage in caspase-independent cell death.

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage.