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1.
Int J Mol Med ; 44(6): 2256-2264, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31638172

ABSTRACT

The Wnt/ß­catenin pathway confers a chain of molecular events in livers affected by non­alcoholic steatohepatitis (NASH). Namodenoson, a selective agonist of the A3 adenosine receptor (A3AR), which is highly expressed in pathological liver cells, induces a robust anti­inflammatory effect in the liver, mediated via the de­regulation of the Wnt/ß­catenin pathway. Namodenoson also acts as a liver protective agent by inhibiting ischemia/reperfusion injury. Based on these unique characteristics, we investigated the anti­NASH effect of Namodenoson in murine models of steatohepatitis and in the LX2 human hepatic stellate cell line (HSC). In the STAM model, Namodenoson significantly decreased the non­alcoholic fatty liver disease (NAFLD) activity score, NAS, demonstrating anti­inflammatory and anti­steatotic effects. In the carbon tetrachloride (CCl4) model, Namodenoson reversed alanine aminotransferase (ALT) to normal values and significantly improved liver inflammation and fibrosis, as well as the adiponectin and leptin levels. Namodenoson de­regulated the Wnt/ß­catenin pathway in the liver extracts of the CCl4 model mice and in the LX2 HSCs, manifested by a decrease in the expression of phosphoinositide 3­kinase (PI3K), nuclear factor κ­light­chain­enhancer of activated B cells (NF­κB), ß­catenin, lymphoid enhancer­binding factor 1 (Lef­1) and cyclin D1, and an increase in the expression level of glycogen synthase kinase 3ß (GSK­3ß). The fibrosis marker, α­smooth muscle actin (α­SMA) was also de­regulated, supporting the anti­fibrotic effect of Namodenoson. On the whole, the findings of this study demonstrate that Namodenoson exerts an anti­NASH effect mediated via the de­regulation of the PI3K/NF­κB/Wnt/ß­catenin signaling pathway. Thus, targeting A3AR may prove to be a novel direction in the pharmacotherapy of NAFLD/NASH.


Subject(s)
Adenosine A3 Receptor Agonists/pharmacology , Liver/drug effects , Non-alcoholic Fatty Liver Disease/drug therapy , Receptor, Adenosine A3/genetics , Actins/genetics , Adiponectin/genetics , Animals , Carbon Tetrachloride/toxicity , Disease Models, Animal , Gene Expression Regulation/drug effects , Humans , Leptin/genetics , Liver/metabolism , Liver/pathology , Mice , NF-kappa B/genetics , Non-alcoholic Fatty Liver Disease/chemically induced , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/pathology , Phosphatidylinositol 3-Kinases/genetics , Wnt Signaling Pathway/drug effects
2.
J Immunol Res ; 2018: 2310970, 2018.
Article in English | MEDLINE | ID: mdl-29862305

ABSTRACT

Interleukin-17 and interleukin-23 play major roles in the inflammatory process in psoriasis. The Gi protein-associated A3 adenosine receptor (A3AR) is known to be overexpressed in inflammatory cells and in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory conditions. Piclidenoson, a selective agonist at the A3AR, induces robust anti-inflammatory effect in psoriasis patients. In this study, we aimed to explore A3AR expression levels in psoriasis patients and its role in mediating the anti-inflammatory effect of piclidenoson in human keratinocyte cells. A3AR expression levels were evaluated in skin tissue and PBMCs derived from psoriasis patients and healthy subjects. Proliferation assay and the expression of signaling proteins were used to evaluate piclidenoson effect on human keratinocytes (HaCat). High A3AR expression levels were found in a skin biopsy and in PBMCs from psoriasis patients in comparison to healthy subjects. Piclidenoson inhibited the proliferation of HaCat cells through deregulation of the NF-κB signaling pathway, leading to a decrease in interleukin-17 and interleukin-23 expression levels. This effect was counteracted by the specific antagonist MRS 1523. A3AR overexpression in skin and PBMCs of psoriasis patients may be used as a target to inhibit pathological cell proliferation and the production of interleukin-17 and interleukin-23.


Subject(s)
Adenosine A3 Receptor Agonists/pharmacology , Interleukin-17/biosynthesis , Interleukin-23/biosynthesis , Keratinocytes/drug effects , Keratinocytes/metabolism , Receptor, Adenosine A3/metabolism , Case-Control Studies , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Gene Expression , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Psoriasis/genetics , Receptor, Adenosine A3/genetics , Skin/metabolism
3.
Mediators Inflamm ; 2014: 708746, 2014.
Article in English | MEDLINE | ID: mdl-25374446

ABSTRACT

The A3 adenosine receptor (A3AR) is overexpressed in inflammatory cells and in the peripheral blood mononuclear cells of individuals with inflammatory conditions. Agonists to the A3AR are known to induce specific anti-inflammatory effects upon chronic treatment. LUF6000 is an allosteric compound known to modulate the A3AR and render the endogenous ligand adenosine to bind to the receptor with higher affinity. The advantage of allosteric modulators is their capability to target specifically areas where adenosine levels are increased such as inflammatory and tumor sites, whereas normal body cells and tissues are refractory to the allosteric modulators due to low adenosine levels. LUF6000 administration induced anti-inflammatory effect in 3 experimental animal models of rat adjuvant induced arthritis, monoiodoacetate induced osteoarthritis, and concanavalin A induced liver inflammation in mice. The molecular mechanism of action points to deregulation of signaling proteins including PI3K, IKK, IκB, Jak-2, and STAT-1, resulting in decreased levels of NF-κB, known to mediate inflammatory effects. Moreover, LUF6000 induced a slight stimulatory effect on the number of normal white blood cells and neutrophils. The anti-inflammatory effect of LUF6000, mechanism of action, and the differential effects on inflammatory and normal cells position this allosteric modulator as an attractive and unique drug candidate.


Subject(s)
Adenosine A3 Receptor Agonists/pharmacology , Aminoquinolines/pharmacology , Anti-Inflammatory Agents/pharmacology , Imidazoles/pharmacology , Receptor, Adenosine A3/metabolism , Administration, Oral , Allosteric Regulation/drug effects , Aminoquinolines/administration & dosage , Animals , Anti-Inflammatory Agents/administration & dosage , Arthritis, Experimental/metabolism , Arthritis, Experimental/prevention & control , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/prevention & control , Female , Imidazoles/administration & dosage , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , NF-kappa B/metabolism , Osteoarthritis/metabolism , Osteoarthritis/prevention & control , Rats , Rats, Inbred Lew , Rats, Wistar , Signal Transduction/drug effects
4.
J Rheumatol ; 34(1): 20-6, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17216675

ABSTRACT

OBJECTIVE: A3 adenosine receptor (A3AR) upregulation has been found in cells of synovial tissue and in peripheral blood mononuclear cells (PBMC) of rats with adjuvant-induced arthritis. We investigated A3AR levels in PBMC of patients with rheumatoid arthritis (RA) and in mitogen-activated PBMC from healthy subjects. We examined the role of nuclear factor-kappaB (NF-kappaB), a transcription factor present in the A3AR promoter, in mediating receptor upregulation. METHODS: A3AR and NF-kappaB protein levels were evaluated in PBMC of RA patients (n = 23) and healthy subjects by Western blot. A3AR and NF-kappaB levels were also analyzed in phytohemagglutinin (PHA) and lipopolysaccharide (LPS)-stimulated PBMC in the presence and absence of antibodies against interleukin 2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha). Reverse transcription-polymerase chain reaction was performed in PHA-stimulated PBMC of healthy subjects to determine A3AR expression. RESULTS: A3AR was overexpressed in PBMC of RA patients compared to healthy subjects and was directly correlated to an increase in NF-kappaB. Similar findings were observed in PHA and LPS-stimulated PBMC from healthy subjects. Antibodies against IL-2 or TNF-alpha prevented the increase in A3AR and NF-kappaB expression. CONCLUSION: Overexpression of A3AR was found in PBMC of RA patients. Receptor upregulation was induced by inflammatory cytokines controlling the expression of the A3AR transcription factor NF-kappaB.


Subject(s)
Arthritis, Rheumatoid/blood , Leukocytes, Mononuclear/metabolism , Receptor, Adenosine A3/blood , Adenosine/analogs & derivatives , Adenosine/pharmacology , Arthritis, Rheumatoid/metabolism , Gene Expression Regulation , Humans , Interleukin-2/antagonists & inhibitors , Leukocytes, Mononuclear/pathology , Middle Aged , Mitogens/physiology , NF-kappa B/physiology , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology , Receptor, Adenosine A3/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors
5.
Arthritis Res Ther ; 8(6): R169, 2006.
Article in English | MEDLINE | ID: mdl-17101059

ABSTRACT

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.


Subject(s)
Adenosine/analogs & derivatives , Anti-Inflammatory Agents/pharmacology , Arthritis/drug therapy , Arthritis/metabolism , Methotrexate/pharmacology , Receptor, Adenosine A3/drug effects , Adenosine/pharmacology , Animals , Arthritis/pathology , Blotting, Western , Drug Therapy, Combination , Female , Humans , Immunohistochemistry , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Middle Aged , Rats , Rats, Inbred Lew , Receptor, Adenosine A3/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation
6.
Clin Cancer Res ; 10(13): 4472-9, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15240539

ABSTRACT

PURPOSE: A(3) adenosine receptor (A(3)AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor kappaB and cyclin D1. To additionally elucidate whether A(3)AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted. EXPERIMENTAL DESIGN: A(3)AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A(3)AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A(3)AR protein expression was studied in fresh tumors and was correlated with that of the adjacent normal tissue. RESULTS: Reverse transcription-PCR analysis of colon and breast carcinoma tissues showed higher A(3)AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A(3)AR mRNA than the primary tumor tissue. Protein analysis of A(3)AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A(3)AR expression in the tumor versus normal adjacent tissue, respectively. The high A(3)AR expression level in the tumor tissues was associated with elevated nuclear factor kappaB and cyclin D1 levels. High A(3)AR mRNA expression was also demonstrated in other solid tumor types. CONCLUSIONS: Primary and metastatic tumor tissues highly express A(3)AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A(3)AR as a potential target for tumor growth inhibition.


Subject(s)
Receptor, Adenosine A3/biosynthesis , Blotting, Western , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Cell Line, Tumor , Colonic Neoplasms/metabolism , Cyclin D1/biosynthesis , Down-Regulation , Humans , Lung Neoplasms/metabolism , Lymphatic Metastasis , Melanoma/metabolism , NF-kappa B/biosynthesis , Neoplasm Metastasis , Neoplasms/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
7.
Oncogene ; 23(14): 2465-71, 2004 Apr 01.
Article in English | MEDLINE | ID: mdl-14691449

ABSTRACT

A(3) adenosine receptor (A(3)AR) activation with the specific agonist CF101 has been shown to inhibit the development of colon carcinoma growth in syngeneic and xenograft murine models. In the present study, we looked into the effect of CF101 on the molecular mechanisms involved in the inhibition of HCT-116 colon carcinoma in mice. In tumor lesions derived from CF101-treated mice, a decrease in the expression level of protein kinase A (PKA) and an increase in glycogen synthase kinase-3 beta (GSK-3 beta) was observed. This gave rise to downregulation of beta-catenin and its transcriptional gene products cyclin D1 and c-Myc. Further mechanistic studies in vitro revealed that these responses were counteracted by the selective A(3)AR antagonist MRS 1523 and by the GSK-3 beta inhibitors lithium and SB216763, confirming that the observed effects were A(3)AR and GSK-3 beta mediated. CF101 downregulated PKB/Akt expression level, resulting in a decrease in the level and DNA-binding capacity of NF-kappa B, both in vivo and in vitro. Furthermore, the PKA and PKB/Akt inhibitors H89 and Worthmannin mimicked the effect of CF101, supporting their involvement in mediating the response to the agonist. This is the first demonstration that A(3)AR activation induces colon carcinoma growth inhibition via the modulation of the key proteins GSK-3 beta and NF-kappa B.


Subject(s)
Adenosine/agonists , Carcinoma/pathology , Colonic Neoplasms/pathology , Glycogen Synthase Kinase 3/metabolism , NF-kappa B/metabolism , Purinergic P1 Receptor Agonists , Adenosine/analogs & derivatives , Adenosine/therapeutic use , Animals , Cell Division/drug effects , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Cyclic AMP-Dependent Protein Kinases/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin D1/drug effects , Cytoskeletal Proteins/metabolism , Down-Regulation , Gene Expression Regulation, Neoplastic/drug effects , Glycogen Synthase Kinase 3/drug effects , Glycogen Synthase Kinase 3 beta , Growth Inhibitors/pharmacology , Humans , Indoles/pharmacology , Lithium/pharmacology , Maleimides/pharmacology , Mice , Mice, Nude , Proto-Oncogene Proteins c-myc/drug effects , Pyridines/pharmacology , Trans-Activators/metabolism , beta Catenin
8.
J Biol Chem ; 278(43): 42121-30, 2003 Oct 24.
Article in English | MEDLINE | ID: mdl-12865431

ABSTRACT

Activation of the Gi protein-coupled A3 adenosine receptor (A3AR) has been implicated in the inhibition of melanoma cell growth by deregulating protein kinase A and key components of the Wnt signaling pathway. Receptor activation results in internalization/recycling events that play an important role in turning on/off receptor-mediated signal transduction pathways. Thus, we hereby examined the association between receptor fate, receptor functionality, and tumor growth inhibition upon activation with the agonist 1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purine-9-yl]-N-methyl-beta-D-ribofuranuronamide (IB-MECA). Results showed that melanoma cells highly expressed A3AR on the cell surface, which was rapidly internalized to the cytosol and "sorted" to the endosomes for recycling and to the lysosomes for degradation. Receptor distribution in the lysosomes was consistent with the down-regulation of receptor protein expression and was followed by mRNA and protein resynthesis. At each stage, receptor functionality was evidenced by the modulation in cAMP level and the downstream effectors protein kinase A, glycogen synthase kinase-3beta, c-Myc, and cyclin D1. The A3AR antagonist MRS 1523 counteracted the internalization process as well as the modulation in the expression of the signaling proteins, demonstrating that the responses are A3AR-mediated. Supporting this notion are the in vivo studies showing tumor growth inhibition upon IB-MECA treatment and reverse of this response when IB-MECA was given in combination with MRS 1523. In addition, in melanoma tumor lesions derived from IB-MECA-treated mice, the expression level A3AR and the downstream key signaling proteins were modulated in the same pattern as was seen in vitro. Altogether, our observations tie the fate of A3AR to modulation of downstream molecular mechanisms leading to tumor growth inhibition both in vitro and in vivo.


Subject(s)
Adenosine/analogs & derivatives , Melanoma/pathology , Neoplasm Proteins/metabolism , Receptor, Adenosine A3/metabolism , Adenosine/pharmacology , Animals , Cell Division , Cell Line, Tumor , Cyclin D1/biosynthesis , Down-Regulation , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/drug effects , Neoplasms, Experimental/drug therapy , Protein Transport , Proto-Oncogene Proteins c-myc/biosynthesis , Receptor, Adenosine A3/biosynthesis , Receptor, Adenosine A3/drug effects , Signal Transduction
9.
Oncogene ; 21(25): 4060-4, 2002 Jun 06.
Article in English | MEDLINE | ID: mdl-12037688

ABSTRACT

The A3 adenosine receptor, A3AR, belongs to the family of Gi proteins, which upon induction, suppresses the formation of cAMP and its downstream effectors. Recent studies have indicated that activation of A3AR by its agonist, IB-MECA, results in growth inhibition of malignant cells. Here we demonstrate the ability of IB-MECA to decrease the levels of protein kinase A, a downstream effector of cAMP, and protein kinase B/Akt in melanoma cells. Examination of glycogen synthase kinase 3beta, GSK-3beta, whose phosphorylation is controlled by protein kinase A and B, showed a substantial decrease in the levels of its phosphorylated form and an increase in total GSK-3beta levels in IB-MECA treated melanoma cells. This observation suggests that the treatment of cells with IB-MECA augments the activity of GSK-3beta in the cells. Evaluation of beta-catenin, a key component of Wnt signaling pathway which, upon phosphorylation by GSK-3beta rapidly ubiquitinates, showed a substantial decrease in its level after IB-MECA treatment. Accordingly, the level of beta-catenin responsive cell growth regulatory genes including c-myc and cyclin D1 was severely declined upon treatment of the cells with IB-MECA. These observations which link cAMP to the Wnt signaling pathway provide mechanistic evidence for the involvement of Wnt pathway via its key elements GSK-3beta and beta-catenin in the anti-tumor activity of A3AR agonists.


Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Cell Division/drug effects , Melanoma/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Purinergic P1 Receptor Agonists , Signal Transduction/physiology , Trans-Activators , Tumor Cells, Cultured/drug effects , Zebrafish Proteins , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Division/physiology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclins/metabolism , Cytoskeletal Proteins/metabolism , Down-Regulation , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Humans , Melanoma/drug therapy , Melanoma/enzymology , Proto-Oncogene Proteins c-akt , Ubiquitin , Wnt Proteins , beta Catenin
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