RESUMO
Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.
RESUMO
Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A1R (DPCPX) or A2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [3H]GUO binding became saturable at 100-300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [3H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [3H]GUO binding, but the sum of the two effects was able to displace [3H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A1R and A2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.
Assuntos
Ansiedade/etiologia , Ansiedade/metabolismo , Guanosina/efeitos adversos , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Ansiedade/psicologia , Comportamento Animal , Membrana Celular/metabolismo , Escuridão , Relação Dose-Resposta a Droga , Guanosina/metabolismo , Hipocampo/metabolismo , Luz , Ratos , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genéticaRESUMO
Guanine-based purines (GBPs) have been recently proposed to be not only metabolic agents but also extracellular signaling molecules that regulate important functions in the central nervous system. In such way, GBPs-mediated neuroprotection, behavioral responses and neuronal plasticity have been broadly described in the literature. However, while a number of these functions (i.e., GBPs neurothophic effects) have been well-established, the molecular mechanisms behind these GBPs-dependent effects are still unknown. Furthermore, no plasma membrane receptors for GBPs have been described so far, thus GBPs are still considered orphan neuromodulators. Interestingly, an intricate and controversial functional interplay between GBPs effects and adenosine receptors activity has been recently described, thus triggering the hypothesis that GBPs mechanism of action might somehow involve adenosine receptors. Here, we review recent data describing the GBPs role in the brain. We focus on the involvement of GBPs regulating neuronal plasticity, and on the new hypothesis based on putative GBPs receptors. Overall, we expect to shed some light on the GBPs world since although these molecules might represent excellent candidates for certain neurological diseases management, the lack of putative GBPs receptors precludes any high throughput screening intent for the search of effective GBPs-based drugs.
RESUMO
Guanine (GUA), guanosine and GMP exert a marked growth inhibition on the U87 glioma cell line that is not seen with other tested nucleotides, nucleosides and nucleobases. This effect could be replicated in several different human tumoral cell lines. Guanine shows a higher potency than guanosine or GMP, and co-treatments with adenosine or adenine are able to antagonize or revert the antiproliferative effect of guanine. The loss of the guanine effect in a cell line bearing a mutated inactive hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and the decreased potency of GUA in U87 cells silenced for HGPRT transcripts, demonstrates the central role of the intracellular metabolism of GUA for growth-inhibitory effects. Considering the potential application of growth-inhibitory substances in anticancer therapy, knowledge of the molecular mechanism underlying GUA-induced effects encourages studies aimed at defining possible tumoral targets for experimental therapies.
Assuntos
Guanina/química , Hipoxantina Fosforribosiltransferase/metabolismo , Purinas/química , Purinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Colorimetria , Humanos , Células Tumorais CultivadasRESUMO
In the present study we analysed the gene expression database provided by the National Cancer Institute in an attempt to correlate activity profiles of geldanamycin, 17AAG and 11 other analogues in 60 human tumor cell lines with their gene expression profiles determined by the cDNA microarray technique. On the basis of the activity profiles two classes of geldanamycin analogues could be distinguished, having geldanamycin and 17AAG, respectively, as prototype compounds (denominated as gelda-like and 17AAG-like classes). Application of the "soft" statistical methodology of PLS (partial least squares modelling in latent variables or projections to latent structures) allowed us to evaluate the influence of each gene expression target in determining the therapeutical responses. The transcript encoding the translocating chain-associated membrane protein (TRAM) showed a significant statistical correlation with activity profiles of 17AAG. In order to validate the role of TRAM in determining sensitivity to 17AAG we induced a selective knocking-down of this transcript by the RNA interference methodology in H226 non-small cell lung carcinoma cell line. The efficiency of double-stranded RNA oligonucleotides (short-interfering RNAs, siRNAs) was determined by measuring TRAM mRNA levels by quantitative real-time RT-PCR at different times (24-72 hours) after siRNA lipotransfection. A significant increase in chemosensitivity to 17AAG was observed in siRNA-silenced cells. Although a number of factors may affect tumour sensitivity to 17AAG the present methodology allowed us to dissect out a single parameter which may be partly responsible for its activity.