Gil Ohana

Differential Effect of Adenosine on Tumor and Normal Cell Growth: Focus on the A3 Adenosine Receptor

Adenosine is an ubiquitous nucleoside present in all body cells. It is released from metabolically active or stressed cells and subsequently acts as a regulatory molecule through binding to specific A1, A2A, A2B and A3 cell surface receptors. The synthesis of agonists and antagonists to the adenosine receptors and their cloning enabled the exploration of their physiological functions. As nearly all cells express specific adenosine receptors, adenosine serves as a physiological regulator and acts as a cardioprotector, neuroprotector, chemoprotector, and as an immunomodulator. At the cellular level, activation of the receptors by adenosine initiates signal transduction mechanisms through G‐protein associated receptors. Adenosines unique characteristic is to differentially modulate normal and transformed cell growth, depending upon its extracellular concentration, the expression of adenosine cell surface receptors, and the physiological state of the target cell. Stimulatio n of cell proliferation following incubation with adenosine has been demonstr ated in a variety of normal cells in the range of low micromolar con centrations, including mesangial and thymocyte cells, Swiss mouse 3T3 fibroblasts, and bone marrow cells. Induction of apoptosis in tumor or normal cells was shown at higher adenosine concentrations (<100 μM) such as in leukemia HL‐60, lymphoma U‐937, A431 epidermoid cells, and GH3 tumor pituitary cell lines. It was further noted that the A3 adenosine receptor (A3AR) plays a key role in the inhibitory and stimulatory growth activities of adenosine. Modulation of the A3AR was found to affect cell growth either positively or negatively depending on the concentration of the agonist, similar to the effect described for adenosine. At nanomolar concentrations, the A3AR agonists possess dual activity, i.e., anti‐proliferative activity toward tumor cells and stimulator y effect on bone marrow cells. In vivo, these agonists exerted anti‐cancer effects, and when given in combination with chemotherapy, they enhanced the chemotherapeutic index and acted as chemoprotective agents. Taken together, activation of the A3AR, by minute concentrations of its natural ligand or synthetic agonists, may serve as a new approach for cancer therapy. J. Cell. Physiol. 186:19–23, 2001.

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.

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.

Adenosine acts as an inhibitor of lymphoma cell growth: a major role for the A3 adenosine receptor.

In this study, we demonstrated several mechanisms exploring the inhibitory effect of low-dose adenosine on lymphoma cell growth. Adenosine, a purine nucleoside present in plasma and other extracellular fluids, acts as a regulatory molecule, by binding to G-protein associated cell-surface receptors, A1, A2 and A3. Recently we showed that low-dose adenosine released by muscle cells, inhibits tumour cell growth and thus attributes to the rarity of muscle metastases. In the present work, a cytostatic effect of adenosine on the proliferation of the Nb2-11C rat lymphoma cell line was demonstrated. This effect was mediated through the induction of cell cycle arrest in the G0/G1 phase and by decreasing the telomeric signal in these cells. Adenosine was found to exert its antiproliferative effect mainly through binding to its A3 receptor. The cytostatic anticancer activity, mediated through the A3 adenosine receptor, turns it into a potential target for the development of anticancer therapies.

Adenosine Acts as a Chemoprotective Agent by Stimulating G-CSF Production: A Role for A1 and A3 Adenosine Receptors

Adenosine, a ubiquitous nucleoside, is released into the extracellular environment from metabolically active or stressed cells. It binds to cells through specific A1, A2A, A2B, and A3 G‐protein–associated cell‐surface receptors, thus acting as a signal‐transduction molecule by regulating the levels of adenylyl cyclase and phospholipase C. In this study, we showed that adenosine stimulates the proliferation of murine bone marrow cells in vitro. Pharmacological studies, using antagonists to the adenosine receptors, revealed that this activity was mediated through the binding of adenosine to its A1 and A3 receptors. This result was further corroborated by showing that the two selective A1 and A3 receptor agonists, N‐cyclopentyladenosine (CPA) and 1‐deoxy‐1‐[6‐[[(3‐iodophenyl)methyl]amino]‐9H‐purin‐9‐yl]‐N‐methyl‐β‐D‐ribofuranuronamide (IB‐MECA) respectively, induced bone marrow cell proliferation in a manner similar to adenosine. Adenosines interaction with its A1 and A3 receptors induced G‐CSF production, which led to its stimulatory effect on bone marrow cells. These results were confirmed in vivo when we demonstrated that low‐dose adenosine (0.25 mg/kg) acted as a chemoprotective agent. When administered after chemotherapy, it restored the number of leukocytes and neutrophils to normal levels, compared with the decline in these parameters after chemotherapy alone. It is suggested that low‐dose adenosine, already in clinical use, may also be applied as a chemoprotective agent. J. Cell. Physiol. 183:393–398, 2000.

Comparative study of the early postoperative course and complications in patients undergoing Billroth I and Billroth II gastrectomy.

Abstract. Distal gastric resection can be followed by reconstruction according to the Billroth I (BI) or Billroth II (BII) techniques. The aim of this study was to compare the early postoperative results and complications of patients undergoing BI and those undergoing BII resection. Eighty-eight patients operated during the years 1991 to 1994 underwent distal gastric resection (41 had BI, and 43 had BII resections). The indications for BI resections were gastric tumors in 39 patients (95%) and duodenal ulcer in 2 (5%). The indications for BII resection were malignancy in 28 patients (65%) and duodenal ulcer disease in 15 (35%). The average duration of the procedure was 147 ± 28 minutes for the BI resection and 175 ± 38 minutes for the BII resection (p < 0.05). No patient in the BI group developed anastomic leakage. Two patients who underwent BII resection developed duodenal stump leakage (4.7%). Relaparotomy was indicated in five patients, two from the BI group (malignant cells in the resection margins) and three from the BII group (one due to duodenal stump leakage and two for bleeding). There was no postoperative mortality in the BI group. The postoperative mortality in the BII group was 7.1% (p < 0.05). The average proximal gastric resection margins were significantly smaller in the BI group than in the BII group (3.65 ± 2.83 cm and 5.18 ± 2.57 cm, respectively; p < 0.05). The number of lymph nodes found in the resected specimen did not differ significantly between the two groups. Recurrent tumor at the gastric remnant developed in two patients in the BI group but not in the BII group. The results of our study revealed that the BI procedure is accompanied by significantly lower postoperative complication and mortality rates than the BII procedure in cases of gastric malignancy. BI resection performed for malignancy seems to achieve smaller proximal gastric resection margins, which may influence the recurrence rate.

Oral administration of muscle derived small molecules inhibits tumor spread while promoting normal cell growth in mice

Tumor metastases are extremely rare in striated muscles. This is surprising given the fact that this tissue constitutes 60% of body weight. The present study focuses on small molecules produced and secreted by muscle cells which possess anti-cancer activity in vivo. Recently we have shown that a low molecular weight fraction (<1000 Dalton) of skeletal muscle cell conditioned medium (muscle factor-MF), markedly inhibits the proliferation of carcinoma, sarcoma or melanoma cell lines in vitro. The MF exerts a cytostatic effect on tumor cell growth and arrests the cells in the G0/G1 of the cell cycle. However, normal cell proliferation, such as bone marrow and fibroblasts, was stimulated following incubation with MF. In this study, the effect of orally administered MF on melanoma and sarcoma growth was examined in mice. The administration of MF to mice inoculated intravenously with melanoma (B16–F10) or sarcoma (MCA-105) cells, resulted in a statistically significant inhibition of metastatic lung foci. In a different model, melanoma was induced in the foot pad and after development of a local lesion, the leg was amputated. A prolonged survival time was observed in the MF treated groups. Since the MF stimulated bone marrow cell proliferation in vitro, we decided to test its efficacy as an inhibitor of the myelotoxic effect exerted by chemotherapy, in vivo. MF, administered after chemotherapy, restored the number of white blood cells and yielded an increased percentage of neutrophils compared with the decline in these parameters after administration of chemotherapy alone. Thus, it is indicated that MF exerted a systemic anti tumor and chemoprotective effect when given orally. It can be concluded that it is bioavailable and is not biodegradable in the digestive system. MF may be considered as a potential therapy for the prevention of tumor spread.

Small Nonpolypoid Colorectal Carcinoma

The objective of this study was to characterize and assess the presence and frequency of small nonpolypoid colorectal adenocarcinomas among patients with colorectal cancer referred for surgery. The medical, endoscopic, and surgical reports and the histopathologic slides of all patients operated on for colorectal cancer were retrospectively reviewed. Small nonpolypoid colorectal cancer (SNPCC) was defined as a malignant, nonpolypoid lesion smaller than 15 mm. SNPCC was classified according to the Japanese macroscopic classification of colorectal carcinoma. The frequency of SNPCC among patients referred for operation was 1.8%. Most of these patients were asymptomatic and were diagnosed by the same endoscopist using a high-resolution video-endoscope without the assistance of enhancement techniques. These lesions had a mean size of 10.8 mm, were mainly of the flat or flat elevated type, and were located in the distal colon. Among patients with colorectal cancer referred for surgery, 1.8% had SNPCC. These lesions can be detected using high-resolution video-endoscopy equipment without the need for enhancement techniques, as reported in Japanese series. Increased awareness of the existence of such SNPCC lesions may help the average endoscopist detect such lesions. As SNPCC represents colorectal cancer, all the cases in our series were treated by typical oncologic surgical resection.

Development of Epidural Abscess Following Surgical Drainage of Perianal Abscess: Report of a Case

PURPOSEA case of epidural abscess originating from a perianal abscess is reported.METHODSThe history of the patient, erythrocyte sedimentation rate, magnetic resonance imaging, and bacteriological tests were used to reach a diagnosis and the possible mechanism.RESULTSEpidural abscess was suspected because the patient had a fever and intense low back pain following drainage of a perianal abscess. Magnetic resonance imaging was used to correctly diagnose the epidural abscess and bacteriologic studies disclosed the pathophysiologic mechanism.CONCLUSIONSEpidural abscess is an extremely rare complication of perianal abscess. It should always be suspected in a patient with acute onset of back pain, fever, history of recent infection, and an elevated erythrocyte sedimentation rate, because delay in diagnosis can cause neurologic compromise and even death.