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1.
Biol Pharm Bull ; 47(5): 1058-1065, 2024.
Article in English | MEDLINE | ID: mdl-38825533

ABSTRACT

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.


Subject(s)
Chemokine CCL2 , Diet, High-Fat , Group IV Phospholipases A2 , Hepatic Stellate Cells , Liver , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease , Up-Regulation , Animals , Diet, High-Fat/adverse effects , Chemokine CCL2/metabolism , Chemokine CCL2/genetics , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/drug effects , Liver/pathology , Up-Regulation/drug effects , Male , Mice , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/metabolism , Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , Group IV Phospholipases A2/antagonists & inhibitors , Hepatocytes/metabolism , Hepatocytes/drug effects , Humans , Mice, Knockout , Collagen/metabolism , Collagen/biosynthesis , Macrophages/metabolism , Macrophages/drug effects , Endothelial Cells/metabolism , Endothelial Cells/drug effects , Cells, Cultured
2.
Bioorg Med Chem Lett ; 107: 129792, 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38734389

ABSTRACT

Ceramide 1-phosphate (C1P) is a lipid mediator that specifically binds and activates cytosolic phospholipase A2α (cPLA2α). To elucidate the structure-activity relationship of the affinity of C1P for cPLA2α in lipid environments, we prepared a series of C1P analogs containing structural modifications in the hydrophilic parts and subjected them to surface plasmon resonance (SPR). The results suggested the presence of a specific binding site for cPLA2α on the amide, 3-OH and phosphate groups in C1P structure. Especially, dihydro-C1P exhibited enhanced affinity for cPLA2α, suggesting the hydrogen bonding ability of 3-hydroxy group is important for interactions with cPLA2α. This study helps to understand the influence of specific structural moieties of C1P on the interaction with cPLA2α at the atomistic level and may lead to the design of drugs that regulate cPLA2α activation.


Subject(s)
Ceramides , Drug Design , Surface Plasmon Resonance , Ceramides/chemistry , Ceramides/chemical synthesis , Ceramides/metabolism , Structure-Activity Relationship , Group IV Phospholipases A2/metabolism , Group IV Phospholipases A2/antagonists & inhibitors , Humans , Molecular Structure , Binding Sites
3.
Front Immunol ; 13: 1094375, 2022.
Article in English | MEDLINE | ID: mdl-36700214

ABSTRACT

Introduction: Psoriasis is a common inflammatory skin disease recognized by the World Health Organization as "an incurable chronic, noninfectious, painful, disfiguring and disabling disease." The fact that metabolic syndrome (MetS) is the most common and important comorbidities of psoriasis suggests an important role of lipid metabolism in the pathogenesis of psoriasis. Narciclasine (Ncs) is an alkaloid isolated from the Amaryllidaceae plants. Its biological activities include antitumor, antibacterial, antiinflammatory, anti-angiogenic and promoting energy expenditure to improve dietinduced obesity. Here, we report that Ncs may be a potential candidate for psoriasis, acting at both the organismal and cellular levels. Methods: The therapeutic effect of Ncs was assessed in IMQ-induced psoriasis-like mouse model. Then, through in vitro experiments, we explored the inhibitory effect of Ncs on HaCaT cell proliferation and Th17 cell polarization; Transcriptomics and lipidomics were used to analyze the major targets of Ncs; Single-cell sequencing data was used to identify the target cells of Ncs action. Results: Ncs can inhibit keratinocyte proliferation and reduce the recruitment of immune cells in the skin by inhibiting psoriasis-associated inflammatory mediators. In addition, it showed a direct repression effect on Th17 cell polarization. Transcriptomic and lipidomic data further revealed that Ncs extensively regulated lipid metabolismrelated genes, especially the Phospholipase A2 (PLA2) family, and increased antiinflammatory lipid molecules. Combined with single-cell data analysis, we confirmed that keratinocytes are the main cells in which Ncs functions. Discussion: Taken together, our findings indicate that Ncs alleviates psoriasiform skin inflammation in mice, which is associated with inhibition of PLA2 in keratinocytes and improved phospholipid metabolism. Ncs has the potential for further development as a novel anti-psoriasis drug.


Subject(s)
Dermatitis , Group IV Phospholipases A2 , Psoriasis , Animals , Mice , Anti-Inflammatory Agents/pharmacology , Dermatitis/drug therapy , Group IV Phospholipases A2/antagonists & inhibitors , Imiquimod/adverse effects , Lipid Metabolism , Phospholipids , Psoriasis/drug therapy
4.
Molecules ; 26(24)2021 Dec 09.
Article in English | MEDLINE | ID: mdl-34946532

ABSTRACT

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.


Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Fatty Acids, Omega-3/pharmacology , Group IV Phospholipases A2 , Multiple Myeloma , Neoplasm Proteins , Cell Line, Tumor , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/metabolism , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/enzymology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism
6.
Int J Mol Sci ; 22(17)2021 Aug 30.
Article in English | MEDLINE | ID: mdl-34502319

ABSTRACT

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.


Subject(s)
Biomarkers, Tumor/metabolism , CRISPR-Cas Systems , Gene Expression Regulation, Neoplastic , Group IV Phospholipases A2/antagonists & inhibitors , Homeodomain Proteins/metabolism , Leukemia, Myeloid, Acute/pathology , Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Group IV Phospholipases A2/genetics , High-Throughput Screening Assays , Homeodomain Proteins/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Tumor Cells, Cultured
7.
Nat Commun ; 12(1): 2330, 2021 04 20.
Article in English | MEDLINE | ID: mdl-33879780

ABSTRACT

The hypothalamus plays a central role in monitoring and regulating systemic glucose metabolism. The brain is enriched with phospholipids containing poly-unsaturated fatty acids, which are biologically active in physiological regulation. Here, we show that intraperitoneal glucose injection induces changes in hypothalamic distribution and amounts of phospholipids, especially arachidonic-acid-containing phospholipids, that are then metabolized to produce prostaglandins. Knockdown of cytosolic phospholipase A2 (cPLA2), a key enzyme for generating arachidonic acid from phospholipids, in the hypothalamic ventromedial nucleus (VMH), lowers insulin sensitivity in muscles during regular chow diet (RCD) feeding. Conversely, the down-regulation of glucose metabolism by high fat diet (HFD) feeding is improved by knockdown of cPLA2 in the VMH through changing hepatic insulin sensitivity and hypothalamic inflammation. Our data suggest that cPLA2-mediated hypothalamic phospholipid metabolism is critical for controlling systemic glucose metabolism during RCD, while continuous activation of the same pathway to produce prostaglandins during HFD deteriorates glucose metabolism.


Subject(s)
Glucose/metabolism , Phospholipases A2, Cytosolic/metabolism , Prostaglandins/biosynthesis , Ventromedial Hypothalamic Nucleus/metabolism , Animals , Arachidonic Acid/metabolism , Biosynthetic Pathways , Diet, High-Fat/adverse effects , Disease Models, Animal , Gene Knockdown Techniques , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , Hyperglycemia/metabolism , Insulin Resistance , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phospholipases A2, Cytosolic/antagonists & inhibitors , Phospholipases A2, Cytosolic/genetics , Phospholipids/metabolism
8.
Exp Biol Med (Maywood) ; 246(11): 1253-1262, 2021 06.
Article in English | MEDLINE | ID: mdl-33641447

ABSTRACT

Abnormal lipid metabolism is regarded as a crucial cause of psoriasis. The specific mechanism of how phospholipase PLA2G4B mediates local immune dysfunction and skin lesions remains unclear. The aim of this study was to explore the mechanisms of anti-psoriasis and immune suppression effect by inhibiting PLA2G4B in psoriasis progression. We successfully transfected si-PLA2G4B in a murine keratinocyte cell-line PAM212 to verify the effect of progression by PLA2G4B. The Imiquimod psoriasis mouse model was then successfully constructed, followed by emulsion wrapped PLA2G4B-siRNA applied to the skin lesions. The phenotype, pathology, immunofluorescence staining of PLA2G4B, IL17, CD3, and CD1b, and bulk transcriptome analysis were performed to decipher the effect and mechanism of si-PLA2G4B. Interfering with PLA2G4B significantly inhibited the proliferation and migration of PAM212. The interference of PLA2G4B in vivo showed a therapeutic effect on psoriasis, comparable to that of betamethasone. The phenotype and pathology revealed reduced keratinocytes in the si-PLA2G4B group compared to the model mice. Immunofluorescence showed that CD1b, CD3+ T cells, and IL17 were suppressed in the skin lesions. RNA-seq and deconvolution revealed that immune cells such as myeloid dendritic cell and T cell CD8+ naive were inactivated. Th17 reduce the release of inflammatory factors such as IL17 and IL36. Pathway analysis revealed the potential therapeutic mechanism involved in the inhibition of sphingolipid or ceramide secretion. This study verified the anti-psoriatic effect of using si-PLA2G4B. The immune response was alleviated after administration. This phospholipase inhibition-based therapy sheds light on the pharmaceutical potential against psoriasis.


Subject(s)
Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , Psoriasis/etiology , Psoriasis/therapy , Animals , Antigens, CD1/metabolism , Cell Line , Disease Models, Animal , Gene Expression Regulation , Group IV Phospholipases A2/antagonists & inhibitors , Humans , Imiquimod/toxicity , Inflammation/pathology , Interleukin-17/metabolism , Keratinocytes , Male , Mice, Inbred BALB C , Psoriasis/chemically induced , Psoriasis/pathology , RNA, Small Interfering
9.
Biomolecules ; 10(10)2020 10 02.
Article in English | MEDLINE | ID: mdl-33023184

ABSTRACT

As a regulator of cellular inflammation and proliferation, cytosolic phospholipase A2 α (cPLA2α) is a promising therapeutic target for psoriasis; indeed, the cPLA2α inhibitor AVX001 has shown efficacy against plaque psoriasis in a phase I/IIa clinical trial. To improve our understanding of the anti-psoriatic properties of AVX001, we sought to determine how the compound modulates inflammation and keratinocyte hyperproliferation, key characteristics of the psoriatic epidermis. We measured eicosanoid release from human peripheral blood mononuclear cells (PBMC) and immortalized keratinocytes (HaCaT) and studied proliferation in HaCaT grown as monolayers and stratified cultures. We demonstrated that inhibition of cPLA2α using AVX001 produced a balanced reduction of prostaglandins and leukotrienes; significantly limited prostaglandin E2 (PGE2) release from both PBMC and HaCaT in response to pro-inflammatory stimuli; attenuated growth factor-induced arachidonic acid and PGE2 release from HaCaT; and inhibited keratinocyte proliferation in the absence and presence of exogenous growth factors, as well as in stratified cultures. These data suggest that the anti-psoriatic properties of AVX001 could result from a combination of anti-inflammatory and anti-proliferative effects, probably due to reduced local eicosanoid availability.


Subject(s)
Dinoprostone/genetics , Group IV Phospholipases A2/genetics , Inflammation/drug therapy , Psoriasis/drug therapy , Celecoxib/pharmacology , Cell Proliferation/drug effects , Eicosanoids/pharmacology , Fatty Acids, Omega-3/genetics , Fatty Acids, Omega-3/pharmacology , Group IV Phospholipases A2/antagonists & inhibitors , Humans , Inflammation/chemically induced , Inflammation/genetics , Inflammation/pathology , Keratinocytes/drug effects , Leukocytes, Mononuclear/drug effects , Lipopolysaccharides/toxicity , Naproxen/pharmacology , Psoriasis/genetics , Psoriasis/pathology
10.
JCI Insight ; 5(16)2020 08 20.
Article in English | MEDLINE | ID: mdl-32814707

ABSTRACT

Type 1 diabetes (T1D) is a consequence of autoimmune ß cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2ß (iPLA2ß) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2ß can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering ß cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2ß-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2ß reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.


Subject(s)
Biomarkers/metabolism , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/etiology , Lipid Metabolism , Macrophages, Peritoneal/metabolism , Adolescent , Animals , Child , Diabetes Mellitus, Type 1/therapy , Eicosanoids/metabolism , Fatty Acids/metabolism , Female , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/metabolism , Humans , Ketones/pharmacology , Lipid Metabolism/drug effects , Lipids/blood , Macrophages, Peritoneal/pathology , Macrophages, Peritoneal/transplantation , Male , Mice, Inbred C57BL , Mice, Inbred NOD , Naphthalenes/pharmacology
11.
PLoS One ; 15(2): e0229657, 2020.
Article in English | MEDLINE | ID: mdl-32106235

ABSTRACT

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.


Subject(s)
Boidae/blood , Group IV Phospholipases A2/antagonists & inhibitors , Phospholipase A2 Inhibitors/blood , Amino Acid Sequence , Animals , Bothrops/metabolism , Brazil , Crotalid Venoms/antagonists & inhibitors , Crotalid Venoms/chemistry , Group IV Phospholipases A2/chemistry , Molecular Weight , Phospholipase A2 Inhibitors/chemistry , Protein Interaction Domains and Motifs , Snake Venoms/antagonists & inhibitors , Snake Venoms/chemistry , Tandem Mass Spectrometry
12.
Yakugaku Zasshi ; 139(9): 1155-1162, 2019.
Article in Japanese | MEDLINE | ID: mdl-31474631

ABSTRACT

Nonalcoholic steatohepatitis (NASH) is a lifestyle-related disease characterized by hepatic fibrosis with the accumulation of fat and inflammation and can progress to cirrhosis or hepatocellular carcinoma. However, effective pharmacotherapeutic strategies for hepatic fibrosis in NASH remain to be established. Among the initiators of inflammation, we have been investigating the possible involvement of group IVA phospholipase A2 (IVA-PLA2), which catalyzes the initial step in the generation of lipid mediators, including eicosanoids and lysophospholipids, in the progression of hepatic fibrosis. We have recently demonstrated that a lack of IVA-PLA2 alleviates hepatic fibrosis in NASH model mice fed a high-fat and high-cholesterol diet and in CCl4-treated mice. CCl4-induced hepatic fibrosis was also prevented by the administration of an orally active, specific IVA-PLA2 inhibitor even after hepatic fibrosis had developed. Based on these findings suggesting that IVA-PLA2 mediates the cellular responses contributing to the progression of hepatic fibrosis, we have been exploring which types of cells in the liver are involved in IVA-PLA2-mediated hepatic fibrosis using cell-specific IVA-PLA2 knockout mice. The preliminary experimental results suggest that IVA-PLA2 in endothelial cells, but not monocyte-derived cells, plays a role, in part, in the hepatic stellate cell-mediated progression of hepatic fibrosis. In this paper, we discuss the possibility that IVA-PLA2 and/or its related molecules are candidate pharmacotherapeutic targets for NASH treatment.


Subject(s)
Group IV Phospholipases A2/antagonists & inhibitors , Indoles/therapeutic use , Molecular Targeted Therapy , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/etiology , Propionates/therapeutic use , Animals , Disease Models, Animal , Group IV Phospholipases A2/physiology , Humans , Indoles/administration & dosage , Indoles/pharmacology , Mice , Mice, Knockout , Non-alcoholic Fatty Liver Disease/enzymology , Propionates/administration & dosage , Propionates/pharmacology
13.
Curr Top Med Chem ; 19(22): 1952-1961, 2019.
Article in English | MEDLINE | ID: mdl-31345152

ABSTRACT

BACKGROUND: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. OBJECTIVE: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. METHODS: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. RESULTS: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. CONCLUSION: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.


Subject(s)
Group IV Phospholipases A2/antagonists & inhibitors , Myoblasts/drug effects , Peptides/pharmacology , Phospholipase A2 Inhibitors/pharmacology , Animals , Bothrops , Cells, Cultured , Drug Evaluation, Preclinical , Group IV Phospholipases A2/isolation & purification , Group IV Phospholipases A2/metabolism , Mice , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Phospholipase A2 Inhibitors/chemical synthesis , Phospholipase A2 Inhibitors/chemistry
14.
J Pharmacol Exp Ther ; 369(3): 511-522, 2019 06.
Article in English | MEDLINE | ID: mdl-30971478

ABSTRACT

3-[3-Amino-4-(indan-2-yloxy)-5-(1-methyl-1H-indazol-5-yl)-phenyl]-propionic acid (AK106-001616) is a novel, potent, and selective inhibitor of the cytosolic phospholipase A2 (cPLA2) enzyme. Unlike traditional nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors, AK106-001616 reduced prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production by stimulated cells. The suppression of PGE2 and LTB4 production was also confirmed using an air pouch model in rats administered a single oral dose of AK106-001616. AK106-001616 alleviated paw swelling in a rat adjuvant-induced arthritis (AIA) model. The maximum effect of the inhibitory effect of AK106-001616 was comparable with that of naproxen on paw swelling in a rat AIA model. Meanwhile, the inhibitory effect of AK106-001616 was more effective than that of naproxen in the mouse collagen antibody-induced arthritis model with leukotrienes contributing to the pathogenesis. AK106-001616 dose dependently reversed the decrease in paw withdrawal threshold not only in rat carrageenan-induced hyperalgesia, but also in a rat neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI). However, naproxen and celecoxib did not reverse the decrease in the paw withdrawal threshold in the CCI model. Furthermore, AK106-001616 reduced the disease score of bleomycin-induced lung fibrosis in rats. In addition, AK106-001616 did not enhance aspirin-induced gastric damage in fasted rats, increase blood pressure, or increase the thromboxane A2/ prostaglandin I2 ratio that is thought to be an underlying mechanism of thrombotic cardiovascular events increased by selective cyclooxygenase-2 inhibitors. Taken together, these data demonstrate that oral AK106-001616 may provide valuable effects for wide indications without attendant gastrointestinal and cardiovascular risks.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Enzyme Inhibitors/pharmacology , Group IV Phospholipases A2/antagonists & inhibitors , Indans/pharmacology , Indazoles/pharmacology , Neuralgia/drug therapy , Propionates/pharmacology , Pulmonary Fibrosis/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Aspirin/adverse effects , Blood Pressure/drug effects , Cell Line, Tumor , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/therapeutic use , Humans , Indans/adverse effects , Indans/therapeutic use , Indazoles/adverse effects , Indazoles/therapeutic use , Inflammation/drug therapy , Male , Propionates/adverse effects , Propionates/therapeutic use , Rats , Rats, Sprague-Dawley , Receptors, Epoprostenol/metabolism , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , Stomach/drug effects , Stomach/pathology
15.
Cancer Biol Ther ; 20(6): 912-921, 2019.
Article in English | MEDLINE | ID: mdl-30829552

ABSTRACT

Cytosolic phospholipase A2alpha (cPLA2α) is a key mediator of tumorigenesis. In this study, by using a combination of pharmacological and genetic approaches in cell models and patient samples, we identify cPLA2α as a selective target to increase chemosensitivity in cervical cancer. We found that transcript and protein levels of cPLA2α but not other forms of cPLA2 (e.g., cPLA2ß and cPLA2αδ) were consistently increased in all tested malignant cervical cancer cells and tissues compared to normal counterparts, suggesting that cPLA2α upregulation is a common feature in cervical cancer. We further found that promoting growth and survival rather than invasion were the predominant roles of cPLA2α on cervical cancer. In addition, chemotherapeutic agents achieved ~100% inhibition efficacy in cPLA2α-depleted cervical cancer cells, demonstrating the important role of cPLA2α in chemoresistance. Importantly, we identify that ß-catenin is critically involved in the molecular mechanism of cPLA2α's action in cervical cancer. In summary, our work demonstrates the multiple essential roles of cPLA2α in cervical cancer, particularly in chemoresistance, via a ß-catenin-dependent manner. Our work also suggests that targeting cPLA2α has a therapeutic value in overcoming chemoresistance in cervical cancer or other cPLA2α-regulated cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/metabolism , Signal Transduction/drug effects , Uterine Cervical Neoplasms/metabolism , beta Catenin/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Group IV Phospholipases A2/genetics , Humans , Immunohistochemistry
16.
Arthritis Res Ther ; 21(1): 29, 2019 01 21.
Article in English | MEDLINE | ID: mdl-30665457

ABSTRACT

BACKGROUND: Cytosolic phospholipase A2 group IVA (cPLA2α)-deficient mice are resistant to collagen-induced arthritis, suggesting that cPLA2α is an important therapeutic target. Here, the anti-inflammatory effects of the AVX001 and AVX002 cPLA2α inhibitors were investigated. METHODS: In vitro enzyme activity was assessed by a modified Dole assay. Effects on inhibiting IL-1ß-induced release of arachidonic acid (AA) and prostaglandin E2 (PGE2) were measured using SW982 synoviocyte cells. In vivo effects were studied in prophylactic and therapetic murine collagen-induced arthritis models and compared to methotrexate (MTX) and Enbrel, commonly used anti-rheumatic drugs. The in vivo response to treatment was evaluated in terms of the arthritis index (AI), histopathology scores and by plasma levels of PGE2 following 14 and 21 days of treatment. RESULTS: Both cPLA2α inhibitors are potent inhibitors of cPLA2α in vitro. In synoviocytes, AVX001 and AVX002 reduce, but do not block, release of AA or PGE2 synthesis. In both CIA models, the AI and progression of arthritis were significantly lower in the mice treated with AVX001, AVX002, Enbrel and MTX than in non- treated mice. Several histopathology parameters of joint damage were found to be significantly reduced by AVX001 and AVX002 in both prophylactic and therapeutic study modes; namely articular cavity and peripheral tissue inflammatory cell infiltration; capillary and synovial hyperplasia; articular cartilage surface damage; and periostal and endochondral ossification. In comparison, MTX did not significantly improve any histopathology parameters and Enbrel only improved ossification. Finally, as a biomarker of inflammation and as an indication that AVX001 and AVX002 blocked the cPLA2α target, we determined that plasma levels of PGE2 were significantly reduced in response to the AVX inhibitors and MTX, but not Enbrel. CONCLUSIONS: AVX001 and AVX002 display potent anti-inflammatory activity and disease-modifying properties in cellular and in vivo models. The in vivo effects of AVX001 and AVX002 were comparable to, or superior, to those of MTX and Enbrel. Taken together, this study suggests that cPLA2α inhibitors AVX001 and AVX002 are promising small molecule disease-modifying anti-rheumatic therapies.


Subject(s)
Arthritis, Experimental/prevention & control , Fatty Acids, Omega-3/pharmacology , Group IV Phospholipases A2/antagonists & inhibitors , Animals , Antirheumatic Agents/pharmacology , Arachidonic Acid/metabolism , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Cell Line, Tumor , Dinoprostone/metabolism , Etanercept/pharmacology , Fatty Acids, Omega-3/chemistry , Group IV Phospholipases A2/metabolism , Humans , Male , Methotrexate/pharmacology , Mice, Inbred DBA , Molecular Structure , Synovial Membrane/cytology , Synovial Membrane/drug effects , Synovial Membrane/metabolism
17.
Toxicol In Vitro ; 54: 215-223, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30253184

ABSTRACT

Cigarette smoke is responsible for multiple disorders and causes almost 10 million annual deaths globally but underlying mechanisms are still underexplored. Continuous exposure of Cigarette smoke condensate (CSC) leads to cytosolic phospholipase A2 (cPLA2) mediated high free radicals where cPLA2s seems to play crucial role in generated various patho-physiological conditions such as chronic inflammation, oxidative stress and cancer. In this view, we assessed the therapeutic potential of arachidonyl trifluromethyl ketone (ATK), a cPLA2 inhibitor, via pharmacological inhibition of most expressible CSC-induced cPLA2 group IVA in type-I and type-II alveolar epithelial cells. The In Vitro inhibitory effect of ATK on CSC-induced PLA2 activity and its cellular role were assessed in terms of cell viability, fluorescein diacetate (FDA) dye uptake assay for membrane integrity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels and pro apoptotic as well as anti apoptosis markers via flow cytometry, along with extracellular signal-regulated kinases (ERK) levels using enzyme-linked immunosorbent assay (ELISA). The experimental findings demonstrated that ATK acts as potent inhibitor of cPLA2 activity and shown its effectiveness as therapeutic agent by significantly mimicking CSC-induced levels of free radicals, primary apoptosis, ratio of pro-apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Thus, this study is an important step towards the opening up of avenues for the applicability of the cPLA2 isoform specific inhibitors such as ATK for pre-clinical and clinical studies and could be beneficial during smoking-induced lung pathological conditions.


Subject(s)
Alveolar Epithelial Cells/drug effects , Arachidonic Acids/pharmacology , Group IV Phospholipases A2/antagonists & inhibitors , Nicotiana , Phospholipase A2 Inhibitors/pharmacology , Smoke/adverse effects , Cell Line , Group IV Phospholipases A2/genetics , Group IV Phospholipases A2/metabolism , RNA, Messenger/metabolism
18.
Cell Physiol Biochem ; 48(4): 1595-1604, 2018.
Article in English | MEDLINE | ID: mdl-30071514

ABSTRACT

BACKGROUND/AIMS: The aim of this study was to investigate the roles of miR-543 and phospholipase A2 group IVA (PLA2G4A) in cell mobility and the invasiveness cascade in esophageal squamous cell carcinoma (ESCC) and to validate the interactive relationship between miR-543 and PLA2G4A. METHODS: Microarray analysis showed the different expression levels of PLA2G4A in two ESCC cell lines (KYSE30 and KYSE180). The expression levels of miR-543 and PLA2G4A in ESCC tissues were confirmed by qRT-PCR and Western blotting. The targeted relationship between miR-543 and PLA2G4A was studied and verified by a luciferase activity assay. Then, the invasion and metastasis ability of ESCC cell lines transfected with miR-543 mimics, miR-543 inhibitor, or PLA2G4A and miR-543 mimics were analyzed separately by Transwell migration and invasion assays. In addition, the roles of miR-543 and PLA2G4A in the expression of E-cadherin and vimentin were also investigated. RESULTS: PLA2G4A up-regulated the level of E-cadherin and down-regulated the level of vimentin, which curbed ESCC cell mobility and invasion. In ESCC cells, the expression of miR-543 was significantly higher, whereas the expression of PLA2G4A was markedly lower. MiR-543 facilitated ESCC cell mobility and invasion by repressing PLA2G4A. CONCLUSIONS: MiR-543 enhanced the cell mobility and the invasiveness cascade in ESCC cells via the down-regulation of PLA2G4A expression.


Subject(s)
Esophageal Neoplasms/pathology , Group IV Phospholipases A2/metabolism , MicroRNAs/metabolism , 3' Untranslated Regions , Animals , Antagomirs/metabolism , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Epithelial-Mesenchymal Transition , Esophageal Neoplasms/genetics , Female , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/genetics , Humans , Male , Mice , Mice, Nude , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Middle Aged , RNA Interference , RNA, Small Interfering/metabolism , Vimentin/metabolism
19.
J Biosci ; 43(2): 277-285, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29872016

ABSTRACT

Secretory phospholipase A2-IIA (sPLA2-IIA) is one of the key enzymes causing lipoprotein modification and vascular inflammation. Maslinic acid is a pentacyclic triterpene which has potential cardioprotective and anti-inflammatory properties. Recent research showed that maslinic acid interacts with sPLA2-IIA and inhibits sPLA2-IIA-mediated monocyte differentiation and migration. This study elucidates the potential of maslinic acid in modulating sPLA2-IIA-mediated inflammatory effects in THP-1 macrophages. We showed that maslinic acid inhibits sPLA2-IIA-mediated LDL modification and suppressed foam cell formation. Further analysis revealed that sPLA2-IIA only induced modest LDL oxidation and that inhibitory effect of maslinic acid on sPLA2-IIA-mediated foam cells formation occurred independently of its anti-oxidative properties. Interestingly, maslinic acid was also found to significantly reduce lipid accumulation observed in macrophages treated with sPLA2-IIA only. Flow cytometry analysis demonstrated that the effect observed in maslinic acid might be contributed in part by suppressing sPLA2-IIA-induced endocytic activity, thereby inhibiting LDL uptake. The study further showed that maslinic acid suppresses sPLA2-IIA-induced up-regulation of PGE2 levels while having no effects on COX-2 activity. Other pro-inflammatory mediators TNF-a and IL-6 were not induced in sPLA2-IIA-treated THP-1 macrophages. The findings of this study showed that maslinic acid inhibit inflammatory effects induced by sPLA2-IIA, including foam cells formation and PGE2 production.


Subject(s)
Cardiotonic Agents/administration & dosage , Group IV Phospholipases A2/genetics , Inflammation/drug therapy , Triterpenes/administration & dosage , Animals , Foam Cells/drug effects , Foam Cells/pathology , Gene Expression Regulation/drug effects , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/metabolism , Humans , Inflammation/genetics , Inflammation/pathology , Interleukin-6/genetics , Lipoproteins, LDL/genetics , Macrophages/drug effects , Macrophages/pathology , Tumor Necrosis Factor-alpha/genetics
20.
Am J Hypertens ; 31(5): 622-629, 2018 04 13.
Article in English | MEDLINE | ID: mdl-29342227

ABSTRACT

BACKGROUND: Recently, we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of prohypertensive eicosanoids. Since Ang II increases blood pressure (BP) via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. METHODS: Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α-/- male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and BP was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. RESULTS: Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α, Ang II-induced cPLA2α activity and oxidative stress in the SFO, BP, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α-/- mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, BP, cardiac, and renal fibrosis. CONCLUSIONS: These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.


Subject(s)
Angiotensin II/pharmacology , Brain/enzymology , Group IV Phospholipases A2/physiology , Hypertension/etiology , Reactive Oxygen Species/metabolism , Animals , Collagen/metabolism , Endoplasmic Reticulum Stress , Group IV Phospholipases A2/antagonists & inhibitors , Group IV Phospholipases A2/genetics , Hypertension/drug therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
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