Lea Madi

The A3 adenosine receptor is highly expressed in tumor versus normal cells: potential target for tumor growth inhibition.

Purpose: A3 adenosine receptor (A3AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor κB and cyclin D1. To additionally elucidate whether A3AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted. Experimental Design: A3AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A3AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A3AR 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 A3AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A3AR mRNA than the primary tumor tissue. Protein analysis of A3AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A3AR expression in the tumor versus normal adjacent tissue, respectively. The high A3AR expression level in the tumor tissues was associated with elevated nuclear factor κB and cyclin D1 levels. High A3AR mRNA expression was also demonstrated in other solid tumor types. Conclusions: Primary and metastatic tumor tissues highly express A3AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A3AR as a potential target for tumor growth inhibition.

A3 adenosine receptor as a target for cancer therapy

Targeting the A3 adenosine receptor (A3AR) by adenosine or a synthetic agonist to this receptor (IB-MECA and Cl-IB-MECA) results in a differential effect on tumor and on normal cells. Both the adenosine and the agonists inhibit the growth of various tumor cell types such as melanoma, colon or prostate carcinoma and lymphoma. This effect is specific and is exerted on tumor cells only. Moreover, exposure of peripheral blood mononuclear cells to adenosine or the agonists leads to the induction of granulocyte colony stimulating factor (G-CSF) production. When given orally to mice, the agonists suppress the growth of melanoma, colon and prostate carcinoma in these animals, while inducing a myeloprotective effect via the induction of G-CSF production. The de-regulation of the Wnt signaling pathway was found to be involved in the anticancer effect. Receptor activation induces inhibition of adenylyl cyclase with a subsequent decrease in the level of protein kinase A and protein kinase B/Akt leading to activation of glycogen synthase kinase-3beta, a key element in the Wnt pathway. The oral bioavailability of the synthetic A3AR agonists, and their induced systemic anticancer and myeloprotective effect, renders them potentially useful in three different modes of treatment: as a stand-alone anticancer treatment, in combination with chemotherapy to enhance its therapeutic index and myelprotection. It is evident that use of the A3AR agonist for increasing the therapeutic index of chemotherapy may also invariably give rise to myeloprotection and vice versa. The A3AR agonists are thus a promising new class of agents for cancer therapy.

An agonist to the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3 beta and NF-kappa B.

A3 adenosine receptor (A3AR) 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-3β (GSK-3β) was observed. This gave rise to downregulation of β-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 A3AR antagonist MRS 1523 and by the GSK-3β inhibitors lithium and SB216763, confirming that the observed effects were A3AR and GSK-3β mediated. CF101 downregulated PKB/Akt expression level, resulting in a decrease in the level and DNA-binding capacity of NF-κB, 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 A3AR activation induces colon carcinoma growth inhibition via the modulation of the key proteins GSK-3β and NF-κB.

Evidence for involvement of Wnt signaling pathway in IB-MECA mediated suppression of melanoma cells

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 3β, GSK-3β, 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-3β levels in IB-MECA treated melanoma cells. This observation suggests that the treatment of cells with IB-MECA augments the activity of GSK-3β in the cells. Evaluation of β-catenin, a key component of Wnt signaling pathway which, upon phosphorylation by GSK-3β rapidly ubiquitinates, showed a substantial decrease in its level after IB-MECA treatment. Accordingly, the level of β-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-3β and β-catenin in the anti-tumor activity of A3AR agonists.

The PI3K–NF-κB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis.The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated.The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with naïve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-κB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K–NF-κB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A 3 adenosine receptor expression

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.

Enhancing pigmentation via activation of A3 adenosine receptors in B16 melanoma cells and in human skin explants

A3 adenosine receptor, A3AR, belongs to the Gi proteins coupled receptors. Activation of A3AR by its agonist, IB‐MECA, decreases cAMP and was expected to reduce melanin level. Unexpectedly, B16 melanoma cells exposed to IB‐MECA increased melanin levels in a dose‐dependent manner. Human skin explants exposed to IB‐MECA showed an increase in DOPA positive cells and in melanin deposition in keratinocytes. The agonist induced AKT phosphorylation, leading to a rapid translocation of the transcription factor MiTF towards the nucleus. DOPA oxidase activity and melanin levels induced by IB‐MECA were further enhanced by PD98509, an inhibitor ERK signalling pathway. Our study shows that IB‐MECA decreases cAMP while inducing melanogenesis. The proposed mechanism involves activation of PI3K/AKT signalling pathway by β/γ subunits of the G protein coupled to A3AR. The increase in melanin level in human skin explants suggests that IB‐MECA may be a potential candidate to the treatment of hypopigmentation of skin.

CF101 enhances the apoptotic effect of chemotherapy on colon and pancreatic carcinoma cell lines: Molecular mechanisms involved

3173 Background: The A3 adenosine Gi protein-coupled receptor is highly expressed in tumor as compared to normal cells. Targeting the A3 adenosine receptor (A3AR) with the highly selective agonist CF101 induces growth inhibition of colon carcinoma and other tumor cell types via down-regulation of PKB/Akt and NF-kB. High expression level of these two signaling proteins is associated with the development of chemo-resistance in various tumor cell types, thus it prompted us to explore whether CF101 would enhance the anti-tumor effect of chemotherapy. METHODS HCT-116 human colon and Bx-PC3 human pancreatic carcinoma cell lines were used. Cells were cultured for 48h in the presence of 5-FU (0.625 μM), oxaliplatin (0.5 μM) or gemcitabine (0.02 mM) and CF101 (10nM) was introduced for an additional 24h. Proliferation assay (MTT) and protein expression profile were conducted at 72h. In vivo studies included nude mice that were inoculated with the tumor cells. Treatment with chemotherapy alone or in combination with CF101 was initiated when tumor reached a size of 150 mm3. Tumor size was monitored each 4 days and study was terminated after 35 days. RESULTS In HCT-116 human colon and Bx-PC3 human pancreatic carcinoma cells, CF101 enhanced the cytotoxic effect of 5-FU, oxaliplatin and gemcitabine in vitro by 20% 25% and 32% respectively (P<0.01). The molecular mechanism involved a marked decrease in the level of PKB/Akt and NF-kB followed by an increase in caspase-3 level resulting in apoptosis, as observed by DNA ladder analysis. In xenograft models of HCT-116 colon carcinoma, CF101 doubled the time to tumor progression (p<0.002) when given in combination with 5-FU. CONCLUSIONS CF101 enhanced the chemotherapeutic effect in vitro and doubled the time to tumor progression in xenograft models. It is thus suggested that CF101 may be further developed as an add-on treatment to chemotherapy for colon and pancreatic carcinoma. [Table: see text].