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1.
Eur J Pharmacol ; 560(1): 17-22, 2007 Mar 29.
Article in English | MEDLINE | ID: mdl-17303109

ABSTRACT

We have reported that valproic acid upregulates melatonin MT1 receptor expression in rat C6 glioma cells. In addition to its anticonvulsant and mood stabilizing properties, valproic acid can also inhibit the growth of cancer cells. Since the melatonin MT1 receptor has been implicated in the oncostatic action of melatonin on human MCF-7 breast cancer cells, the effect of valproic acid on its expression was examined in this cell line. Treatment of MCF-7 cells with valproic acid (0.5 or 1 mM) for 24 or 72 h caused a significant increase in melatonin MT1 receptor mRNA or protein expression, as shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis and western blotting, respectively. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays revealed a concentration-dependent inhibition of MCF-7 cell proliferation by valproic acid (0.5, 1.0 or 5 mM), but melatonin (1 or 10 nM) was ineffective alone or in combination with valproic acid, in the first (MCF-7A) subline examined. However, in subsequent experiments using a different (MCF-7B) subline, which expressed higher levels of MT1 receptor mRNA and showed modest sensitivity to melatonin, a combination of this hormone with valproic acid produced a significant synergistic inhibition of cell proliferation. These findings indicate that clinically relevant concentrations of valproic acid upregulate melatonin MT1 receptor expression in human breast cancer cells. Moreover, the enhanced antiproliferative effect observed with a combination of valproic acid and melatonin suggests that a similar therapeutic approach may be beneficial in breast cancer.


Subject(s)
Anticonvulsants/pharmacology , Breast Neoplasms/metabolism , Melatonin/pharmacology , Receptor, Melatonin, MT1/metabolism , Valproic Acid/pharmacology , Animals , Anticonvulsants/administration & dosage , Apoptosis/drug effects , Blotting, Western , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Therapy, Combination , Gene Expression , Humans , Melatonin/administration & dosage , Rats , Receptor, Melatonin, MT1/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/drug effects , Valproic Acid/administration & dosage
2.
Purinergic Signal ; 3(4): 399-409, 2007 Sep.
Article in English | MEDLINE | ID: mdl-18404453

ABSTRACT

Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson's disease (PD). MPP(+), a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson's disease. We investigated if extracellular guanosine affected MPP(+)-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP(+) (10 muM-5 mM for 24-72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 muM) before, concomitantly with or, importantly, after the addition of MPP(+) abolished MPP(+)-induced DNA fragmentation. Addition of MPP(+) (500 muM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP(+) eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP(+) nor guanosine had any significant effect on alpha-synuclein expression. Thus, guanosine antagonizes and reverses MPP(+)-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.

3.
Purinergic Signal ; 2(4): 651-61, 2006 Nov.
Article in English | MEDLINE | ID: mdl-18404468

ABSTRACT

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5-2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8-3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.

4.
Purinergic Signal ; 1(2): 161-72, 2005 Jun.
Article in English | MEDLINE | ID: mdl-18404501

ABSTRACT

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3',5'-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). [Formula: see text]-Nitro-L-: arginine methyl ester (L-: NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.

5.
Exp Brain Res ; 162(1): 56-62, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15599730

ABSTRACT

Enteric glia share morphological, biochemical, and functional properties with astrocytes. Thus, like astrocytes, transplantation of enteric glia into the central nervous system (CNS) might facilitate the development of the characteristics of the blood brain barrier (BBB) in endothelial cells. This study explored this possibility by examining barrier formation after implantation into the spinal cord of rats. Phaseolus vulgaris leucoagglutin (PHAL)-treated enteric glia suspensions were injected into the spinal cord at the T11-T12 level of adult Wistar female rats. Control animals were injected with either 3T3 fibroblast, glioma C6 cells, or culture medium. Evan's blue, a dye excluded by the BBB, was injected intravenously from 1 week to 2 months after implantation. Leakage of dye was determined macroscopically and the ultrastructure of the capillaries was examined. During the first week leakage of dye correlated ultrastructurally with predominantly non-overlapping endothelial cell junctions, even with clefts between adjacent cells. Tight junctions were fully formed by 2 months and no dye leaked. Electron microscopic analysis showed that enteric glia had end-feet in close contact with endothelial cells. In contrast, the injection sites in all control animals leaked dye until 2 months, and most of the tight junctions that did form were incomplete. Furthermore, most 3T3 or C6 control cells had died at 2 months and those that survived, unlike enteric glia, had no anatomical relationship to blood vessels. These data demonstrate that implantation of enteric glia accelerates the formation of the characteristics of the BBB in spinal cord capillaries.


Subject(s)
Blood-Brain Barrier/physiology , Enteric Nervous System/cytology , Nerve Regeneration/physiology , Neuroglia/transplantation , Spinal Cord/blood supply , Spinal Cord/physiology , Animals , Blood-Brain Barrier/ultrastructure , Cell Differentiation/physiology , Cells, Cultured , Endothelial Cells/physiology , Endothelial Cells/ultrastructure , Enteric Nervous System/physiology , Evans Blue , Female , Graft Survival/physiology , Microscopy, Electron, Transmission , Neuroglia/physiology , Neuroglia/ultrastructure , Phytohemagglutinins/pharmacology , Rats , Rats, Wistar , Spinal Cord/surgery , Tight Junctions/physiology , Tight Junctions/ultrastructure , Tumor Cells, Cultured
6.
Neuroreport ; 15(5): 833-6, 2004 Apr 09.
Article in English | MEDLINE | ID: mdl-15073525

ABSTRACT

Apoptosis is implicated in the pathophysiology of Alzheimer's disease. Extracellular guanosine inhibits staurosporine-induced apoptosis in astrocytes. We examined whether guanosine protects SH-SY5Y human neuroblastoma cells against beta-amyloid(betaA)-induced apoptosis. Addition of betaA (fragment 25-35, 5 microM for 24 h) to SH-SY5Y cells increased the number of apoptotic cells, as evaluated by oligonucleosome ELISA. Guanosine pre-treatment decreased betaA-induced apoptosis (maximal effect after 24 h, 300 microM, p<0.05). The anti-apoptotic effect of guanosine was reduced by LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) (p<0.05). Guanosine increased phosphorylation of Akt/PKB, and this was abolished by inhibiting PI3K or MEK, (p<0.001, 5 min). Thus, the protective effect of guanosine against betaA-induced apoptosis of SH-SY5Y cells is mediated via activation of the PI3K/Akt/PKB and MAPK pathways.


Subject(s)
Amyloid beta-Peptides/toxicity , Apoptosis , Guanosine/pharmacology , Neuroprotective Agents/pharmacology , Peptide Fragments/toxicity , 3-Phosphoinositide-Dependent Protein Kinases , Blotting, Western , Cell Line, Tumor , Chromones/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Flavonoids/pharmacology , Humans , Morpholines/pharmacology , Neuroblastoma , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Time Factors
7.
Glia ; 46(4): 356-68, 2004 May.
Article in English | MEDLINE | ID: mdl-15095366

ABSTRACT

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.


Subject(s)
Apoptosis/physiology , Astrocytes/metabolism , Guanosine/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Animals , Animals, Newborn , Apoptosis/drug effects , Astrocytes/drug effects , Caspase 3 , Caspases/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Guanosine/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Purinergic Antagonists , Purinergic P1 Receptor Antagonists , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Receptors, Purinergic/metabolism , Receptors, Purinergic P1/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
8.
Neuroreport ; 14(18): 2463-7, 2003 Dec 19.
Article in English | MEDLINE | ID: mdl-14663211

ABSTRACT

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Administration of guanosine (8 mg/kg/day, i.p.) for 7 consecutive days, starting 5 weeks after moderate SCI in rats, improved locomotor function and spinal cord remyelination. Myelinogenesis was associated with an increase in the number of mature oligodendrocytes detected in guanosine-treated spinal cord sections in comparison with controls. These data indicate that guanosine-induced remyelination resulted, at least in part, from activation of endogenous oligodendrocyte lineage cells. These findings may have significant implications for chronic demyelinating diseases.


Subject(s)
Guanosine/pharmacology , Guanosine/therapeutic use , Myelin Sheath/physiology , Recovery of Function/drug effects , Spinal Cord Injuries/drug therapy , Animals , Chronic Disease , Female , Motor Activity/drug effects , Motor Activity/physiology , Myelin Basic Protein/metabolism , Myelin Sheath/drug effects , Myelin Sheath/metabolism , Nerve Fibers, Myelinated/drug effects , Nerve Fibers, Myelinated/metabolism , Rats , Rats, Wistar , Recovery of Function/physiology , Spinal Cord Injuries/metabolism
9.
Neuroreport ; 14(10): 1301-4, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12876461

ABSTRACT

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.


Subject(s)
Muscle Contraction/physiology , Neuroglia/physiology , Recovery of Function , Skin/innervation , Spinal Cord Injuries/physiopathology , 3T3 Cells/physiology , 3T3 Cells/transplantation , Animals , Carbocyanines/pharmacokinetics , Cells, Cultured , Female , Fluorescent Dyes/pharmacokinetics , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Intestines , Mice , Neuroglia/transplantation , Rats , Rats, Wistar , Spinal Nerve Roots/metabolism , Spinal Nerve Roots/pathology , Spinal Nerve Roots/physiopathology , Time Factors , Transplantation/methods , Tumor Cells, Cultured/physiology , Tumor Cells, Cultured/transplantation
10.
Exp Neurol ; 181(1): 79-83, 2003 May.
Article in English | MEDLINE | ID: mdl-12710936

ABSTRACT

After spinal cord injury axonal regeneration is poor, but may be enhanced by the implantation of olfactory ensheathing glia (OEG). Enteric glia (EG) share many properties of OEG. Transected dorsal root axons normally do not regenerate through the central nervous system myelin into the spinal cord. We tested whether EG, like OEG, could promote regeneration in this paradigm. Three weeks after EG implantation, numerous regenerating dorsal root axons reentered the spinal cord. Ingrowth of dorsal root axons was observed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal gray commissure. No axonal ingrowth was observed in control animals, indicating that transplanted EG enabled regeneration of the injured dorsal root axons into the adult spinal cord. Thus, EG implantation may be beneficial in promoting axonal growth after central nervous system injury.


Subject(s)
Enteric Nervous System/cytology , Nerve Regeneration , Neuroglia/transplantation , Spinal Cord Injuries/therapy , Spinal Cord , Spinal Nerve Roots/injuries , 3T3 Cells , Animals , Axons/pathology , Axons/physiology , Cell Culture Techniques , Cell Division , Cell Movement , Cell Separation , Coculture Techniques , Disease Models, Animal , Female , Graft Survival , Intestine, Small/innervation , Mice , Neuroglia/cytology , Neurons, Afferent/cytology , Neurons, Afferent/physiology , Rats , Rats, Wistar , Spinal Cord/pathology , Spinal Cord/physiopathology , Spinal Cord/surgery , Spinal Cord Injuries/pathology , Spinal Nerve Roots/pathology , Spinal Nerve Roots/physiopathology , Spinal Nerve Roots/surgery
11.
Glia ; 38(3): 179-90, 2002 May.
Article in English | MEDLINE | ID: mdl-11968056

ABSTRACT

Astrocytes release adenine-based and guanine-based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration-dependent manner up to 100 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2-3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine-based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 microM) or propentophylline (100 microM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S-adenosyl-L-homocysteine (SAH) and greatly reduced the ratio of S-adenosyl-L-methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.


Subject(s)
Adenine/analogs & derivatives , Apoptosis/physiology , Astrocytes/metabolism , Central Nervous System/metabolism , Purine Nucleosides/metabolism , Receptors, Purinergic P1/metabolism , Adenine/pharmacology , Adenosine/metabolism , Adenosine/pharmacology , Adenosine Deaminase/metabolism , Adenosine Deaminase Inhibitors , Animals , Apoptosis/drug effects , Astrocytes/cytology , Astrocytes/drug effects , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Cells, Cultured , Central Nervous System/cytology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Guanine/metabolism , Guanine/pharmacology , Guanosine/metabolism , Guanosine/pharmacology , Hypoxanthine/metabolism , Hypoxanthine/pharmacology , Inosine/metabolism , Inosine/pharmacology , Male , Purine Nucleosides/pharmacology , Purinergic P1 Receptor Antagonists , Pyridines/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A3
12.
Br J Pharmacol ; 135(4): 969-76, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11861325

ABSTRACT

1. Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine-based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) of 0.57 +/- 0.03 pmol mg(-1) protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [(3)H]-guanosine was: guanosine=6-thio-guanosine > inosine > 6-thio-guanine > guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. 5. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.


Subject(s)
Brain/metabolism , Guanosine/metabolism , Animals , Binding Sites , Binding, Competitive , Brain/ultrastructure , In Vitro Techniques , Kinetics , Male , Membranes , Radioligand Assay , Rats , Rats, Wistar
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