Efficacy and safety of piclidenoson in plaque psoriasis: Results from a randomized phase 3 clinical trial (COMFORT-1).

OBJECTIVE: A3 adenosine receptor (A3AR) is overexpressed in the skin and peripheral blood mononuclear cells of psoriasis patients. We investigated the efficacy/safety of piclidenoson (CF101), an orally bioavailable A3AR agonist that inhibits IL-17 and IL-23 production in keratinocytes, in moderate-to-severe plaque psoriasis. METHODS: The randomized, placebo- and active-controlled, double-blind phase 3 COMFORT-1 trial randomized patients (3:3:3:2) to piclidenoson 2 mg BID, piclidenoson 3 mg BID, apremilast 30 mg BID or placebo. At Week 16, patients in the placebo arm were re-randomized (1:1:1) to piclidenoson 2 mg BID, piclidenoson 3 mg BID or apremilast 30 mg BID. The primary end point was the proportion of patients achieving ≥75% improvement in Psoriasis Area and Severity Index (PASI) from baseline (PASI-75) at Week 16 versus placebo. RESULTS: A total of 529 patients were randomized and received ≥1 dose of study medication (safety population). The efficacy analysis population for the primary end point included 426 patients (piclidenoson 2 mg BID, 127; piclidenoson 3 mg BID, 103; apremilast, 118; placebo, 78). Piclidenoson at 2 and 3 mg BID exhibited similar efficacy. The primary end point was met with the 3 mg BID dose: PASI 75 rate of 9.7% versus 2.6% for piclidenoson versus placebo, p = 0.037. The PASI responses with piclidenoson continued to increase throughout the study period in a linear manner. At week 32, analysis in the per-protocol population showed that a greater proportion of patients in the piclidenoson 3 mg BID arm (51/88, 58.0%) achieved improvement from baseline in Psoriasis Disability Index (PDI) compared to apremilast (59/108, 55.1%), and the test for noninferiority trended towards significance (p = 0.072). The safety/tolerability profile of piclidenoson was excellent and superior to apremilast. CONCLUSIONS: Piclidenoson demonstrated efficacy responses that increased over time alongside a favourable safety profile. These findings support its continued clinical development as a psoriasis treatment (ClinicalTrials.gov identifier: NCT03168256).

Treatment of plaque-type psoriasis with oral CF101: data from an exploratory randomized phase 2 clinical trial.

AIMS: CF101 demonstrated a marked anti-inflammatory effect in Phase 2 studies conducted in patients with rheumatoid arthritis and dry eye syndrome. The aim of this study was to evaluate the safety and efficacy of CF101 for the treatment of patients with moderate to severe plaque-type psoriasis. MATERIALS AND METHODS: This was a phase 2, multicentre, randomized, double-blind, dose-ranging, placebo-controlled study. Seventy five patients with moderate to severe plaque-type psoriasis were enrolled, randomized and treated with CF101 (1, 2, or 4 mg) or placebo administered orally twice daily for 12 weeks. Safety and change from base line of Psoriasis Area and Severity Index (PASI) score and physician's global assessment (PGA) score over 12 weeks. RESULTS: In the 2 mg CF101-treated group, a progressive improvement in the mean change from baseline in the PASI score vs. placebo throughout the study period was observed, with a statistically significant difference on weeks 8 and 12 (P = 0.047; P = 0.031, respectively). In this group, 35.3% of the patients achieved PASI ≥ 50 response, and 23.5% of the patients achieved a PGA score of 0 or 1. CF101 was safe and well tolerated. CONCLUSIONS: CF101 was well tolerated and demonstrated clear evidence of efficacy in patients with moderate to severe plaque psoriasis.

CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver.

The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3ß, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.

Induction of an antiinflammatory effect and prevention of cartilage damage in rat knee osteoarthritis by CF101 treatment.

OBJECTIVE: Studies have suggested that rheumatoid arthritis (RA) and osteoarthritis (OA) share common characteristics. The highly selective A(3) adenosine receptor agonist CF101 was recently defined as a potent antiinflammatory agent for the treatment of RA. The purpose of this study was to examine the effects of CF101 on the clinical and pathologic manifestations of OA in an experimental animal model. METHODS: OA was induced in rats by monosodium iodoacetate, and upon disease onset, oral treatment with CF101 (100 microg/kg given twice daily) was initiated. The A(3) adenosine receptor antagonist MRS1220 (100 microg/kg given twice daily) was administered orally, 30 minutes before CF101 treatment. The OA clinical score was monitored by knee diameter measurements and by radiographic analyses. Histologic analyses were performed following staining with hematoxylin and eosin, Safranin O-fast green, or toluidine blue, and histologic changes were scored according to a modified Mankin system. Signaling proteins were assayed by Western blotting; apoptosis was detected via immunohistochemistry and TUNEL analyses. RESULTS: CF101 induced a marked decrease in knee diameter and improved the changes noted on radiographs. Administration of MRS1220 counteracted the effects of CF101. CF101 prevented cartilage damage, osteoclast/osteophyte formation, and bone destruction. In addition, CF101 markedly reduced pannus formation and lymphocyte infiltration. Mechanistically, CF101 induced deregulation of the NF-kappaB signaling pathway, resulting in down-regulation of tumor necrosis factor alpha. Consequently, CF101 induced apoptosis of inflammatory cells that had infiltrated the knee joints; however, it prevented apoptosis of chondrocytes. CONCLUSION: CF101 deregulated the NF-kappaB signaling pathway involved in the pathogenesis of OA. CF101 induced apoptosis of inflammatory cells and acted as a cartilage protective agent, which suggests that it would be a suitable candidate drug for the treatment of OA.

A3 adenosine receptor: pharmacology and role in disease.

The study of the A(3) adenosine receptor (A(3)AR) represents a rapidly growing and intense area of research in the adenosine field. The present chapter will provide an overview of the expression patterns, molecular pharmacology and functional role of this A(3)AR subtype under pathophysiological conditions. Through studies utilizing selective A(3)AR agonists and antagonists, or A(3)AR knockout mice, it is now clear that this receptor plays a critical role in the modulation of ischemic diseases as well as in inflammatory and autoimmune pathologies. Therefore, the potential therapeutic use of agonists and antagonists will also be described. The discussion will principally address the use of such compounds in the treatment of brain and heart ischemia, asthma, sepsis and glaucoma. The final part concentrates on the molecular basis of A(3)ARs in autoimmune diseases such as rheumatoid arthritis, and includes a description of clinical trials with the selective agonist CF101. Based on this chapter, it is evident that continued research to discover agonists and antagonists for the A(3)AR subtype is warranted.

Adenosine receptors and cancer.

The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.

The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation.

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

The A3 adenosine receptor agonist CF102 induces apoptosis of hepatocellular carcinoma via de-regulation of the Wnt and NF-kappaB signal transduction pathways.

The A3 adenosine receptor (A(3)AR) is highly expressed in tumors and was suggested as a target for cancer treatment. In this study, we show that A(3)AR is highly expressed in tumor tissues and in peripheral blood mononuclear cells (PBMCs) derived from patients with HCC, as well as from HCC tumor-bearing rats. The high expression level of the receptor was directly correlated to overexpression of NF-kappaB, known as a transcription factor of A(3)AR. CF102, a synthetic highly selective agonist to A(3)AR induced a marked dose response inhibition of tumor growth in N1S1 HCC tumor rats, via de-regulation of the NF-kappaB and the Wnt signal transduction pathways, resulting in apoptosis of tumor cells. Taken together, A(3)AR is highly expressed in tumors and PBMCs of HCC patients and tumor-bearing rats. CF102 induced apoptosis and tumor growth inhibition. These data suggest A(3)AR as a novel targeted therapy to treat HCC.

The Anti-Cancer Effect of A3 Adenosine Receptor Agonists: A Novel, Targeted Therapy.

The A3 adenosine receptor (A3AR) is highly expressed in various human solid tumor cells whereas low expression is found in the adjacent normal tissues. Activation of the A3AR with synthetic highly selective agonists, such as IB-MECA, Cl-IB-MECA or LJ529, induces tumor growth inhibition of melanoma, lymphoma, breast, hepatoma, prostate and colon carcinoma cells both in vitro and in vivo. Two molecular events take place upon receptor activation and include: a. receptor internalization and subsequent degradation, followed by decreased receptor mRNA and protein expression level. b. modulation of down-stream signal transduction pathways, including those related to Wnt and NF-κB. Subsequently, the levels of cyclin D1 and c-Myc are decreased leading to tumor growth inhibition. IB-MECA synergizes with chemotherapeutic agents to yield an additive anti-tumor effect and protects against myelotoxicity induced by chemotherapy. Taken together, A3AR agonists may be suggested as a new family of orally bioavailable compounds to be developed as potent inhibitors of malignant diseases.

IB-MECA, an A3 adenosine receptor agonist prevents bone resorption in rats with adjuvant induced arthritis.

OBJECTIVES: The anti-inflammatory effect of adenosine is partially mediated via the A3 adenosine receptor (A3AR), a Gi protein associated cell surface receptor. The highly selective A3AR agonist, IB-MECA was earlier shown to prevent the clinical and pathological manifestations of arthritis in experimental animal models of collagen and adjuvant induced arthritis (AIA). In this study we tested the effect of IB-MECA on the prevention of bone resorption in AIA rats and looked at the molecular mechanism of action. METHODS: Rats with AIA were treated orally twice daily with IB-MECA starting upon onset of disease and the clinical score was evaluated every other day. At study termination the foot, knee and hip region of both vehicle and IB-MECA treated animals were subjected to histomorphometric analysis. Western blot analysis was carried out on paw protein extracts. RESULTS: IB-MECA ameliorated the clinical manifestations of the disease and reduced pannus and fibrosis formation, attenuated cartilage and bone destruction and decreased the number of osteoclasts. In cell protein extracts derived from paw of AIA rats, A3AR was highly expressed in comparison to naïve animals. In paw extracts derived from IB-MECA treated AIA rats, down-regulation of the A3AR protein expression level was noted. PI3K, PKB/Akt, IKK, NF-kappaB, TNF-alpha and RANKL were down-regulated whereas caspase 3 was up-regulated. CONCLUSION: IB-MECA, a small highly bioavailable molecule, induces modulation of proteins which control survival and apoptosis resulting in the amelioration of the inflammatory process and the preservation of bone mass in AIA rats.

Tolerability, pharmacokinetics and concentration-dependent hemodynamic effects of oral CF101, an A3 adenosine receptor agonist, in healthy young men.

OBJECTIVES: To assess safety, tolerability, pharmacokinetics and hemodynamic effects of oral CF 101, an A3 adenosine receptor (A3AR) agonist, in healthy men. METHODS: One single and 1 repeated dose, parallel-group, ascending dose, double-blind and placebo-controlled study in normal volunteers. In the single dose study, n = 15 subjects received 1, 5 or 10 mg oral CF101; in each group 1 subject received placebo, the remainder active CF101. In the repeat-dose study, n = 28 subjects received repeated 12-hourly oral doses of CF 101 (2, 3, 4 or 5 mg) for 7 days, in each group 2 subjects received placebo, the remainder active CF101. TEST MATERIALS: Single-dose study: CF101 in 30% Cremophor RH40. Multiple-dose sudy: CF101 in 0.5% methylcellulose suspension. Both studies: the corresponding vehicles were used as placebos. Galenicals were prepared remotely from the clinical study site to ensure double-blind nature of the study. RESULTS TOLERABILITY: Single doses up to 5 mg CF101 were safe and well-tolerated. However, the single dose of 10 mg CF101 was associated with flushing, tachycardia, nausea and vomiting, which were viewed as dose-limiting in normal volunteers. Single doses of CF101 (as well as the first of the multiple doses) were associated with increases in heart rate (8 - 24 beats/min after 5 mg and 18 - 55 beats/min after 10 mg). Multiple doses up to 4 mg 12-hourly for 7 days were safe and well-tolerated. However, the 5 mg multiple-dose group reported headache, drowsiness, hot flushes and dizziness on standing; this declined with dosing duration and was not dose-limiting in this study. Adverse events were commonest near t(max). RESULTS PHARMACOKINETICS: For oral CF101, the t(max) was always 1 - 2 h post-dose and t 1/2 about 9 h, in both the single- and multiple-dose studies. For a single 5 mg dose (mean +/- SD) C(max) = 81.6 +/- 23.6 ng/ml in the single dose study, and 63.6 +/- 22.0 ng/ml after the first of the multiple doses; AUC if was 904.0 +/- 221.9 ng.h/ml and 596.1 +/- 196.6 ng.h/ml for the 2 studies, respectively. After 7 days of multiple dosing there was little change, and AUC(0-24h) = 601.0 +/- 163.6 ng.h/ml. These pharmacokinetic parameters were linearly proportional to dose in the other treatment groups. RESULTS PHARMACODYNAMICS: Increases in heart rate were related to plasma concentration and evident only in the upper range of concentrations observed. There were no changes on ECG monitoring beyond sinus tachycardia, and, in particular, no evidence of PR prolongation in any subject (n = 43). In comparison with single doses, this response was almost absent after 7 days of dosing. Leucocytosis (increases up to about 1.5 x 10(9)/l after 5 and 10 mg) was similarly transient and reversible after multiple dosing. CONCLUSIONS: Single oral doses up to 5 mg CF101 and repeated doses up to 4 mg 12-hourly for 7 days were safe and well-tolerated. Multiple-dose CF101 pharmacokinetics were unchanged and predictable from single-dose estimates, and were linearly proportional to dose. Increases in heart rate and neutrophil count were reversible during multiple dosing and were not dose-limiting in the repeat dose study. CF101 warrants further study for its efficacy in treating human disease.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS.

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

Inhibition of primary colon carcinoma growth and liver metastasis by the A3 adenosine receptor agonist CF101.

Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A(2A), A(2B), and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, P<0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.

The A3 adenosine receptor as a new target for cancer therapy and chemoprotection.

Adenosine, a purine nucleoside, acts as a regulatory molecule, by binding to specific G-protein-coupled A(1), A(2A), A(2B), and A(3) cell surface receptors. We have recently demonstrated that adenosine induces a differential effect on tumor and normal cells. While inhibiting in vitro tumor cell growth, it stimulates bone marrow cell proliferation. This dual activity was mediated through the A3 adenosine receptor. This study showed that a synthetic agonist to the A3 adenosine receptor, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide (Cl-IB-MECA), at nanomolar concentrations, inhibited tumor cell growth through a cytostatic pathway, i.e., induced an increase number of cells in the G0/G1 phase of the cell cycle and decreased the telomeric signal. Interestingly, Cl-IB-MECA stimulates murine bone marrow cell proliferation through the induction of granulocyte-colony-stimulating factor. Oral administration of Cl-IB-MECA to melanoma-bearing mice suppressed the development of melanoma lung metastases (60.8 +/- 6.5% inhibition). In combination with cyclophosphamide, a synergistic anti-tumor effect was achieved (78.5 +/- 9.1% inhibition). Furthermore, Cl-IB-MECA prevented the cyclophosphamide-induced myelotoxic effects by increasing the number of white blood cells and the percentage of neutrophils, demonstrating its efficacy as a chemoprotective agent. We conclude that A3 adenosine receptor agonist, Cl-IB-MECA, exhibits systemic anticancer and chemoprotective effects.

Involvement of Wnt signaling pathway in murine medulloblastoma induced by human neurotropic JC virus.

By using the early genome of the human neurotropic polyomavirus, JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors which model human medulloblastoma. Expression of T-antigen was found in some, but not all, tumor cells, and examination of the clonal cell lines derived from the tumor population showed enhanced tumorigenicity of cells expressing T-antigen in comparison to T-antigen negative cells. Considering the earlier notion on the potential involvement of beta-catenin with human medulloblastoma, we investigated various components of the Wnt signaling pathway including beta-catenin, its partner transcription factor, LEF-1, and their downstream target gene c-myc in these two cell populations. Immunohistochemical staining of the cells revealed enhanced nuclear appearance of beta-catenin in T-antigen positive cells. Results from Western blot showed higher levels of beta-catenin and LEF-1 in T-antigen positive cells in comparison to those in T-antigen negative cells. The enhanced level of LEF-1 expression correlated with the increase in DNA binding activity of this protein in nuclear extracts of T-antigen positive cells. Results from Northern and Western blot analyses revealed that the level of c-myc expression is augmented both at the RNA and protein levels in T-antigen positive cells. These observations corroborated results from transfection studies indicating the ability of JCV T-antigen to stimulate c-myc promoter activity. Further, co-transfection experiments revealed that the amount of c-myc and T-antigen protein in tumor cells may dictate the activity of JCV early promoter in these cells. These observations are interesting in light of recent discoveries on the association of JCV with human medulloblastoma and suggest that communication between JCV and the Wnt pathway may be an important event in the genesis of these tumors.

Resistance of muscle to tumor metastases: a role for a3 adenosine receptor agonists.

Tumor metastases are extremely rare in striated muscles. Lately, we have found that muscle cell conditioned medium (MCM) inhibits the proliferation of various tumor cells while maintaining the growth of normal murine bone marrow cells. This dual activity was confirmed in vivo when the MCM was administered orally, i.e., it inhibited the development of tumor growth in mice and prevented the myelotoxic effects of chemotherapy. Adenosine was found to be one of the active components of MCM, inhibiting tumor cell growth while maintaining bone marrow cell proliferation in vitro. Adenosine is known to act as an important regulatory molecule through its binding to specific G-protein-associated A1, A(2a), A(2b) and A3 cell surface receptors. In distinction from MCM, adenosine did not suppress tumor development in mice and was not active as a chemoprotective agent when administered orally or intravenously. Thus, the in vivo activity of MCM could not be attributed to adenosine. In this study, MCM from which adenosine was enzymatically removed still retained its dual activity that was also found to be mediated through the A3 adenosine receptor (A3AR). This result led to the conclusion that natural agonists to A3AR were responsible for the activity of MCM. We further tested synthetic agonist to the A3AR and demonstrated that it possessed the same in vitro and in vivo activity profile as MCM. Taken together, muscle cells, in addition to adenosine, secrete natural agonists to A3AR. These agonists are stable nondegradable molecules and may contribute to the systemic anticancer and chemoprotective activity exerted by MCM. This group of molecules may account for the rarity of tumor metastases in muscle.

Protein kinase C-mediated down-regulation of beta(2)-adrenergic receptor and gene expression in rat C6 glioma cells.

We investigated the regulation of beta(2)-adrenergic receptors (beta(2)AR) by protein kinase C (PKC) in rat C6 glioma cells at the levels of receptor activity, protein expression and gene expression. Cells exposed to 4beta-phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, exhibited a time- and concentration-dependent decrease in beta(2)AR binding activity. Maximum down-regulation was approximately 50% by 24 h and western blot analysis revealed a parallel decrease in beta(2)AR protein. In addition, PMA treatment resulted in an acute desensitization of beta(2)AR-stimulated cyclic AMP response prior to any reduction in receptor levels. PMA exposure also affected steady-state beta(2)AR mRNA levels in a time-dependent, biphasic manner. During the first 4 h, levels decreased by approximately 60% and then slowly recovered to approximately 75% of control by 24 h. As the reduction in receptor mRNA was not due to a decrease in its stability, we examined beta(2)AR gene transcription by nuclear run-on assays. Transcriptional activity in nuclei from C6 cells treated with PMA for 2 h was reduced by 70% compared to controls. Thus PKC can regulate beta(2)AR at least two levels: the first being an acute desensitization of receptor function, and the second being a more prolonged repression of receptor gene transcription that in turn results in decreased receptor expression.