The Effect of Various Combinations of Peripheral Nerve Blocks on Postoperative Pain in Laparoscopic Cholecystectomy: A Comparative Prospective Study.

Objectives: Most patients who undergo laparoscopic cholecystectomy (LC) experience moderate to severe pain in the first 24 hours after surgery. The transversus abdominal plane (TAP) is currently used for post-LC analgesia. Posterior, subcostal, or rectus sheath TAP blocks are the conventional approaches used. The aim of the current study was to compare the efficacy of combinations of various peripheral blocks on pain intensity and the use of pain killers, shortly after LC. Methods: This was a prospective, double-blind study, in which 200 patients who were about to undergo a LC procedure were recruited and randomized into 4 groups: patients receiving one of the following: TAP block alone, subcostal Tap block alone, subcostal TAP block with a TAP block, or subcostal TAP with a rectus sheath block. The intensity of pain (VAS score) and the use of painkillers were monitored in the recovery unit and in the department for up to 24 hours after surgery. Results: Pain levels decreased with time from 3.6 ± 3.2 at 30 minutes to 0.9 ± 2.0 at 24 hours after the surgery. Nevertheless, no difference between the various block types groups was noted. The percentage of patients who consumed analgesic medications decreased over time, from 83% at 30 to 21% at 24 hours after surgery. The mean/median number of medications consumed by each of the patients was lower among the patients who received a combination of 2 blocks compared to those who received a single one (mean/median of 2.7/3 and 2.8/3 for the TAP or subcostal TAP blocks, respectively; 2.5/2 and 2.3/2 for the subcostal TAP + TAP or subcostal TAP + rectus sheath blocks, respectively). Conclusion: A combination of peripheral nerve blocks reduced the use of analgesic consumption during the 24 hours after LC surgery, compared to standalone blocks.

Hospital staff perspectives towards health technology assessment: data from a multidisciplinary survey.

BACKGROUND: Technology adoption in hospitals is usually based on cost-effectiveness analysis, feasibility and potential success. Different countries have embraced a range of principles to accomplish an effective comprehensive process of health technology assessment (HTA). The aim of the study was to analyse the viewpoints and relative weight of technology-oriented hospital staff members toward the clinical, social, technological and economic aspects of HTA. METHODS: Using a structured questionnaire, a survey was conducted among different professionals in an 850-bed hospital. RESULTS: We revealed a range of viewpoints among hospital staff members according to their personal characteristics and professional standpoints. The clinical aspects of HTA were considered 'highly important' (HI) by most participants, especially the 'lifesaving' parameter. Similarly, the 'lack of effective alternative technology' was ranked HI by a high percentage of participants, independent of their profession. Economic aspects were ranked HI only by half of the participants, while social and technological aspects were ranked HI only by a relatively low percentage. Nurses added 'improving quality of life', 'increasing teamwork efficiency' and 'improving medical standards'. Allied health professionals focused on 'lack of effective alternative technologies' as a main argument for adoption of HTA, alongside increasing efficiency, budget savings and contribution to hospital reputation. Engineers emphasised the requirement of significant investment in infrastructure and increasing efficiency. Administrators ranked patient experience as HI. Interestingly, the high ranking of social aspects correlated with older responders, while junior staff ranked safety significantly higher. CONCLUSIONS: A multi-perspective multidisciplinary approach would be beneficial for policy-makers at hospitals and even on a national scale in Israel.

Antithrombotic Treatment Prior to Intracerebral Hemorrhage: Analysis in the National Acute Stroke Israeli Registry.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is the most disastrous stroke subtype. Prognosis is considered worse with prior antithrombotic treatment. Our aim was to evaluate the association of prior antithrombotic treatment on the radiological and clinical outcome after ICH in a subgroup of patients included in a national registry. METHODS: Based on the National Acute Stroke Israeli (NASIS) registry during 2004, 2007, 2010, and 2013 (2-month periods), characteristics, volumetric parameters, and prognosis of a subgroup of patients with ICH were analyzed. RESULTS: Among the 634 patients with ICH in the NASIS registry, 310 (49%) were not treated previously with antithrombotic medications, 232 (37%) were treated with an antiplatelet agent, and 92 (14.5%) patients were on oral anticoagulant therapy, of them 30 patients (33%) with an international normalised ratio (INR) value below 2, 33 (36%) patients with an INR value of 2-3, and 29 patients (31%) with an INR value above 3 upon admission. Patients with deep hemorrhage on prior anticoagulants treatment had the highest probability for poor outcome at hospital discharge. Patients with low bleeding volume (0-30 cm3), were likely to have admission National Institute of Health Stroke Scale < 10 (62%), while those with higher volumes (30-59 cm3 and > 60 cm3), had only 16.7% and 14.3% chance, respectively. We did not observe a significant difference between prior antithrombotic treatment and functional outcome at discharge, yet prior anticoagulant treatment was associated with higher long-term mortality rates. CONCLUSIONS: Our findings, based on a national registry, support the high mortality and poor outcome of anticoagulant related ICH.

A3 adenosine receptor allosteric modulator induces an anti-inflammatory effect: in vivo studies and molecular mechanism of action.

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.

Targeting the A3 adenosine receptor for glaucoma treatment (review).

Glaucoma is a worldwide disease and the second leading cause of blindness. Current treatments are associated with a number of side-effects and poor compliance, due to the requirement for treatment administration several times a day. These treatments typically aim to lower intraocular pressure (IOP); however, they are unable to protect retinal ganglion cells (RGCs) from undergoing apoptosis, which is the main cause of vision loss. A3 adenosine receptor (A3AR) agonists have been found to protect normal cells from undergoing apoptosis via the downregulation of death signals. Furthermore, A3AR agonists have been reported to have several ophthalmological effects, including the prevention of ganglion cell apoptosis in vitro and in vivo and anti­inflammatory effects in experimental models of autoimmune uveitis. CF101, an orally bioavailable A3AR agonist, has been analyzed in dry eye syndrome phase II clinical trials and was identified to be safe and well tolerated. The anti­inflammatory effect of CF101 was shown to significantly improve corneal staining, tear meniscus and tear break­up time in dry eye patients. In addition, CF101 was found to decrease IOP in patients. The safety and efficacy of CF101, together with its suitability for oral administration, indicates that it has potential as a candidate drug for the treatment of glaucoma.

CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.

BACKGROUND: The A(3) adenosine receptor (A(3)AR) is overexpressed in the tumor and in the peripheral blood mononuclear cells of patients with hepatocellular carcinoma (HCC). The orally active drug candidate CF102, an A(3)AR agonist, induces apoptosis of HCC cells via deregulation of the Wnt signaling pathway. In this open label phase I/II trial, the safety and clinical effects of CF102 were assessed in patients with advanced unresectable HCC. METHODS: The primary objectives of this trial were to examine the safety and pharmacokinetic (PK) behavior of CF102 given orally (1, 5, and 25 mg BID) in 28-day cycles. Evaluation of anti-tumor effects and the utilization of A(3)AR as a biological predictive marker of response to CF102 were the secondary objectives. RESULTS: Eighteen patients received CF102-six at each dose level. No serious drug-related adverse events or dose-limiting toxicities were observed. CF102 demonstrated good oral bioavailability and linear PK behavior. Median overall survival in the study population, 67% of whom had received prior sorafenib, was 7.8 months, and for Child Pugh B patients (28%) it was 8.1 months. Stable disease by RECIST was observed in four patients for at least 4 months. CF102 maintained liver function over a 6-month period. A correlation between receptor overexpression levels at baseline and patients' overall survival was found. One of the patients who presented with skin nodules that were biopsy-proven to be HCC metastases prior to the trial showed complete metastasis regression during three months of treatment with CF102. CONCLUSIONS: CF102 is safe and well-tolerated, showing favorable PK characteristics in Child Pugh A and B HCC patients, justifying further clinical development.

Pharmacological and therapeutic effects of A3 adenosine receptor agonists.

The A(3) adenosine receptor (A(3)AR) coupled to G(i) (inhibitory regulative guanine nucleotide-binding protein) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A(3)AR as a potential therapeutic target. Highly selective A(3)AR agonists have been synthesized and molecular recognition in the binding site has been characterized. In this article, we summarize preclinical and clinical human studies that demonstrate that A(3)AR agonists induce specific anti-inflammatory and anticancer effects through a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. At present, A(3)AR agonists are being developed for the treatment of inflammatory diseases, including rheumatoid arthritis (RA) and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis.

Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101.

Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.

Putative role of the adenosine A(3) receptor in the antiproliferative action of N (6)-(2-isopentenyl)adenosine.

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N (6)-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A(3) receptor (hA(3)R) ligands with affinities in the high nanomolar range (K (i) values of 159 and 649 nM, respectively). These values were comparable to the observed K (i) value of adenosine on hA(3)R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A(3)R. In a functional assay in Chinese hamster ovary cells transfected with hA(3)R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A(3)R antagonist VUF5574. Both IPA and reference A(3)R agonist 2-chloro-N (6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A(3)R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A(3)R-independent mechanism, as was previously reported for other A(3)R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A(3)R.

The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis.

Targeting the A(3) adenosine receptor (A(3)AR) to combat inflammation is a new concept based on two findings. First, A(3)AR is highly expressed in inflammatory cells, whereas low expression is found in normal tissues. This receptor was also found to be overexpressed in peripheral blood mononuclear cells, reflecting receptor status in the remote inflammatory process. Second, A(3)AR activation with a specific agonist induces de-regulation of the NF-kappaB signaling pathway in inflammatory cells, as well as initiation of immunomodulatory effects. The A(3)AR agonist CF-101 (known generically as IB-MECA) induces anti-inflammatory effects in experimental animal models of collagen- and adjuvant-induced arthritis. Combined therapy with CF-101 and methotrexate in adjuvant-induced arthritis rats yielded an additive anti-inflammatory effect. Methotrexate induced upregulation of A(3)AR, rendering the inflammatory cells more susceptible to CF-101. In Phase I and in Phase IIa human studies, CF-101 was safe, well tolerated and showed strong evidence of an anti-inflammatory effect in rheumatoid arthritis patients. In peripheral blood mononuclear cells withdrawn from the patients at base line, a statistically significant correlation between A(3)AR expression level and response to the drug was noted. It is suggested that A(3)AR may serve as a biologic marker to predict patient response to the drug. Taken together, this information suggests that A(3)AR agonists may be a new family of orally bioavailable drugs to be developed as potent inhibitors of autoimmune-inflammatory diseases.

Overexpression of A3 adenosine receptor in peripheral blood mononuclear cells in rheumatoid arthritis: involvement of nuclear factor-kappaB in mediating receptor level.

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.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 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.

The PI3K-NF-kappaB 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-kappaB 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-kappaB 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.

Antiinflammatory effect of A3 adenosine receptor agonists in murine autoimmune arthritis models.

OBJECTIVE: CF101, an A3 adenosine receptor (A3AR) agonist, is a small orally bioavailable molecule known to suppress in vitro the production of tumor necrosis factor-alpha (TNF-alpha). We evaluated its therapeutic potential and antiinflammatory effects in 3 murine models of adjuvant induced arthritis (AIA). METHODS: The antiinflammatory effect of CF101 was examined in rat AIA, in mouse collagen induced arthritis, and in tropomyosin induced arthritis. The clinical effect of another A3AR agonist, Cl-IB-MECA, was examined in rat AIA. The effect of low dose (10 or 100 mg/kg/day) A3AR agonists administered orally once daily on arthritis severity was assessed clinically and histologically. The effect of CF101 on the protein expression level of TNF-alpha in the synovial tissue, draining lymph nodes, and spleen cells was determined by Western blot. RESULTS: CF101 and Cl-IB-MECA markedly ameliorated the clinical and histological features of arthritis in the 3 models when administered orally at a low dose of 10 mg/kg body weight in the 3 autoimmune arthritis models. The lower dose of 10 mg/kg of either CF101 or Cl-IB-MECA had better antiinflammatory effect than the higher 100 mg/kg dose. Decreased expression of TNF-alpha was noted in protein extracts of synovia, draining lymph nodes, and spleen tissues. CONCLUSION: The results provide evidence that A3AR agonists exert significant antirheumatic effects in different autoimmune arthritis models by suppression of TNF-alpha production. The beneficial activity of the drugs at the low dose demonstrates that the effect is A3AR mediated.

CF101, an agonist to the A3 adenosine receptor, enhances the chemotherapeutic effect of 5-fluorouracil in a colon carcinoma murine model.

NF-kappaB and the upstream kinase PKB/Akt are highly expressed in chemoresistance tumor cells and may hamper the apoptotic pathway. CF101, a specific agonist to the A3 adenosine receptor (A3AR), inhibits the development of colon carcinoma growth in cell cultures and xenograft murine models. Because CF101 has been shown to downregulate PKB/Akt and NF-kappaB protein expression level, we presumed that its combination with chemotherapy will enhance the antitumor effect of the cytotoxic drug. In this study, we utilized 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and a colon carcinoma xenograft model. It has been shown that a combined treatment of CF101 and 5-fluorouracil (5-FU) enhanced the cytotoxic effect of the latter on HCT-116 human colon carcinoma cell proliferation and tumor growth. Downregulation of PKB/Akt, NF-kappaB, and cyclin D1, and upregulation of caspase-3 protein expression level were observed in cells and tumor lesions on treatment with a combination of CF101 and 5-FU. Moreover, in mice treated with the combined therapy, myelotoxicity was prevented as was evidenced by normal white blood cell and neutrophil counts. These results show that CF101 potentiates the cytotoxic effect of 5-FU, thus preventing drug resistance. The myeloprotective effect of CF101 suggests its development as an add-on treatment to 5-FU.

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

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.

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

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.

A3 adenosine receptor agonist potentiates natural killer cell activity.

Activation of the Gi-protein-coupled A3 adenosine receptor (A3AR) has been reported to be involved in the inhibition of tumor cell growth. A3AR is highly expressed in tumor cells whereas lower expression was noted in a variety of normal cells. Recently we showed that A3AR activation in melanoma cells resulted in growth inhibition via a direct anti-proliferative effect which entailed cell cycle arrest in the G0/G1 and down-regulation of cyclin D1 and c-Myc. In the present study we present an additional mechanism demonstrating that A3AR agonists activate natural killer (NK) cells which further enhance the anti-tumor effect of this group of molecules. NK cells mediate the natural cytotoxicity and their number and function is reduced in cancer patients. We show Cl-IB-MECA to inhibit tumor development via the activation of NK cells is an additional mechanism which accounts for the anti-tumor effect of A3AR agonists. This effect was noted at a low dose of 10 micro g/kg, demonstrating that the response is exclusively A3AR mediated. Treatment of naïve mice for four days yielded the highest effect on NK cell potentiation. In mice inoculated with B16-F10 melanoma cells and treated each orally with Cl-IB-MECA, melanoma growth inhibition correlated with higher serum level of IL-12 and potentiation of NK cells. Moreover, in adoptive transfer experiments in melanoma bearing mice, marked inhibition of lung metastatic foci was noted upon engraftment with splenocytes derived from Cl-IB-MECA treated mice. Taken together, the ability of Cl-IB-MECA to inhibit tumor development via the activation of NK cells is an additional mechanism which accounts for the anti-tumor effect of A3AR agonists.

Targeting the A3 adenosine receptor for cancer therapy: inhibition of prostate carcinoma cell growth by A3AR agonist.

BACKGROUND: Agonists to A3 adenosine receptor (A3AR) were shown to inhibit the growth of various tumor cell types. The present study demonstrates that a synthetic A3AR agonist, 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purine- 9-yl]-N-methyl-beta-D-ribofura-nuronamide (IB-MECA), inhibits the growth of androgen-independent PC-3 prostate human carcinoma cells and illustrates the molecular mechanism involved. MATERIALS AND METHODS: PC-3 prostate carcinoma cells were used. Cell growth was examined in vitro by the thymidine incorporation assay and in vivo by inoculating the tumor cells subcutaneously into nude mice and monitoring tumor size. The protein expression level in cells and tumor extracts was tested by Western blot analysis. RESULTS: A decrease in the protein expression level of A3AR and the downstream effector PKAc was observed. Consequently, the GSK-3 beta protein level increased, resulting in the destabilization of beta-catenin and the subsequent suppression of cyclin D1 and c-myc expression. IB-MECA treatment also induced down-modulation of the expression of NF-kappa B/p65, known to regulate the transcription of cyclin D1 and c-Myc. This chain of events occurred both in vitro and in vivo and suggests the use of the above-mentioned signaling proteins as markers to predict tumor cell response to A3AR activation. CONCLUSION: Taken together, we demonstrated that A3AR activation deregulates the Wnt and the NF-kappa B signaling pathways resulting in the inhibition of prostate carcinoma cell growth.

A3 adenosine receptor activation in melanoma cells: association between receptor fate and tumor growth inhibition.

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.