adenosine-kinase and Glioma

The aim of the present study was to investigate adenosine deaminase (ADA) and adenosine kinase (ADK) expression in human glioma and to explore its correlation with glioma‑associated epilepsy. Tumor tissues (n=45) and peritumoral tissues (n=14) were obtained from glioma patients undergoing surgery. Normal control tissues (n=8) were obtained from brain trauma patients. The disease grade was determined by histological evaluation and the degree of tumor invasion was evaluated using immunofluorescence analyses. mRNA and protein expression of ADA and ADK were evaluated using reverse transcription quantitative polymerase chain reaction or western blot analysis, respectively. Based on histological evaluations, four cases were classified as Grade I gliomas, 18 cases as Grade II, 17 cases as Grade III and six cases were considered Grade IV. Increased ADA and ADK expression was observed in tumor tissues. ADA was predominantly distributed in the cytoplasm of tumor cells, whereas ADK was detected in the cytoplasm as well as in the nuclei. ADA and ADK levels were upregulated in patients with Grade II and Grade III gliomas compared to those in control subjects (p<0.05). In addition, tumor invasion was detected in peritumoral tissues. The number of ADA‑positive or ADK‑positive cells in tumor tissues was similar between glioma patients with and without epilepsy (p>0.05). However, ADA and ADK expression was upregulated in peritumoral tissues derived from patients with epilepsy compared to that in glioma patients without epilepsy. The results of the present study suggested that ADA and ADK are involved in glioma progression, and that increased ADA and ADK levels in peritumoral tissues may be associated with epilepsy in glioma patients.

Topics: Adenosine Deaminase; Adenosine Kinase; Adolescent; Adult; Aged; Brain Neoplasms; Case-Control Studies; Cell Nucleus; Child; Child, Preschool; Cytoplasm; Disease Progression; Epilepsy; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Middle Aged; Neoplasm Grading

In C6 glioma cells, adenine nucleotides, especially AMP, and adenosine inhibited cell proliferation in time- and concentration-dependent manners. alpha,beta-methylene-ADP, an ecto-5'-nucleotidase inhibitor, suppressed the hydrolysis of AMP and reversed the inhibition of cell growth induced by AMP but not by adenosine. Adenosine deaminase eliminated both AMP- and adenosine-mediated growth inhibitions. 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, had little effect on the cell growth. Equilibrative nucleoside transporters, ENT-1 and ENT-2, were expressed in C6 cells by determining their mRNAs. ENT inhibitors, nitrobenzylthioinosine and dipyridamole, suppressed the uptake of [(3)H]adenosine into C6 cells, and attenuated AMP- or adenosine-mediated growth inhibition. Furthermore, an adenosine kinase inhibitor 5-iodotubercidin reversed the growth inhibition induced by AMP and adenosine. When uridine was added in the extracellular space, AMP- or adenosine-induced cell growth inhibition was completely reversed, suggesting that intracellular pyrimidine starvation would be involved in their cytostatic effects. These results indicate that extracellular adenine nucleotides inhibit C6 cell growth via adenosine, which is produced by ecto-nucleotidases including CD73 at the extracellular space and then incorporated into cells by ENT2. Intracellular AMP accumulation by adenosine kinase after adenosine uptake would induce C6 cell growth inhibition through pyrimidine starvation.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine; Adenosine Deaminase; Adenosine Diphosphate; Adenosine Kinase; Adenosine Monophosphate; Animals; Brain Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Glioma; Hydrolysis; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Thioinosine; Uridine

The present study demonstrates the anti-inflammatory effect of adenosine kinase inhibitor (ADKI) in glial cells. Treatment of glial cells with IC51, an ADKI, stimulated the extracellular adenosine release and reduced the LPS/IFNgamma-mediated production of NO, and induction of iNOS and TNF-alpha gene expression. The recovery of IC51-mediated inhibition of iNOS expression by adenosine transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and the inhibition of LPS/IFNgamma-induced iNOS gene expression by exogenous adenosine indicate a role for adenosine release in IC51-mediated iNOS expression. The rescue of IC51-mediated inhibition of iNOS expression by adenosine receptor antagonist for A2A, 8-(3-chlorostyryl)caffeine (CSC) and alloxazine for A2B, further supports a role for interaction of adenosine and its receptors in anti-inflammatory activity. The IC51-mediated induction of cAMP levels, downstream target of A2A and A2B, and inhibition of LPS/IFNgamma-induced expression of iNOS by forskolin, a cAMP activator, document a role for cAMP mediated pathway in anti-inflammatory activity of IC51. Taken together, these studies document that IC51-mediated inhibition of iNOS expression is through activation of adenosine receptors, which activates A2A and A2B resulting in increased cAMP levels following LPS/IFNgamma stimulation. Moreover, the lack of effect of IC51 or adenosine on NFkappaB DNA binding activity and its transactivity indicates that the inhibition of iNOS expression mediated by IC51 may be through an NFkappaB independent pathway.

Topics: Adenosine; Adenosine Kinase; Analysis of Variance; Animals; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Enzymologic; Glioma; Interferon-gamma; L-Lactate Dehydrogenase; Leupeptins; Lipopolysaccharides; Neuroglia; NF-kappaB-Inducing Kinase; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Rats; Thiocarbamates; Transfection; Tumor Necrosis Factor-alpha

To investigate the effect of morphine on catabolism and anabolism of purine nucleotide in c6 glioma cells.. C6 glioma cells were cultured and divided into 3 groups: 1) morphine group: morphine (10 micro g/ml culture) was added for 6 h, 12 h, 24 h, 48 h, and 72 h; 2) morphine + naloxone group: naloxone (1 micro mol/L) was added for 1 hour and then morphine (10 micro g/ml) was added for 24 hours; and 3) control group: normal saline was used for 6, 12, 24, 48, and 72 hours. The C6 glioma cells were centrifuged. RT-PCR was used to examine the gene transcripts of key enzymes of purine salvage way, hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) and adenylate kinase (AK). RT-PCR-Southern blotting was used to examine the gene transcripts of key enzymes of purine salvage way, xanthine dehydrogenase (XD)/xanthine oxidase (XO) mRNA.. Compared with that in the control group, the transcript of AK mRNA was significantly lower in the C6 cells treated with morphine for 24 hours, and began to re-increase 48 hours after morphine treatment. The transcript of AK mRNA remained at a low level after treatment of naloxone for 1 hour and treatment of morphine for 24 hours. The levels of transcript of HGPRT mRNA were lower in the morphine group than in the control group at all time points after treatment. However, the level of transcript of HGPRT mRNA 72 hours after treatment was higher in the morphine group than in the control group. The level of transcript of HGPRT mRNA 24 hours after exposure to morphine in the naloxon2 + morphine group was still lower than in the control group. The levels of transcripts of XD/XO mRNA were significantly higher after exposure to morphine in comparison with those in the control group at all time points after treatment. However, the levels of XD/XO mRNA 24 hours after exposure to morphine in the naloxone + morphine group recovered to the normal level.. The downregulation effect of morphine on the gene expression of AK and HGPRT may not be mediated by mu receptor. The upregulation effect of morphine on the gene expression of XD/XO may be mediated by mu receptor. Naloxone reverses the effect of morphine on enhancement of XD/XO gene expression and cannot reverse the inhibitory effect of morphine on HGPRT and AK gene expression.

Topics: Adenosine Kinase; Analgesics, Opioid; Animals; Drug Interactions; Gene Expression; Glioma; Hypoxanthine Phosphoribosyltransferase; Morphine; Naloxone; Narcotic Antagonists; Purine Nucleotides; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured