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Authors
Abstract(s)
O glioblastoma é um tumor de grau IV do sistema nervoso central e é caracterizado
pelo seu crescimento infiltrativo. A falta de oxigenação, também conhecida por hipóxia,
promove a invasão tumoral nos tecidos saudáveis do cérebro, como forma de mecanismo
de escape a este ambiente adverso. Nestas condições, há um aumento na concentração de
nucleótidos e nucleósidos extracelulares, particularmente a adenosina (Ado), sendo este
considerado um mecanismo de proteção endógeno durante a hipóxia. A concentração
extracelular basal de Ado encontra-se normalmente entre 30 – 200 nM, no entanto esta
pode aumentar por dois mecanismos: hidrólise enzimática do ATP extracelular a AMP e
posterior hidrólise deste a Ado através da ação da ecto-5’-nucleotidase (NT5E), ou efluxo da
Ado pelos transportadores de nucleósidos bidirecionais (ENTs) após a sua produção
intracelular. Intracelularmente, a Ado pode ser formada a partir do AMP através da acção
da 5’-ectonucleotidase, ou hidrólise da S-adenosil-L-homocisteína catalisada pela Sadenosil-homocisteinase (AHCY). A adenosina pode ser consumida intracelularmente por
fosforilação a AMP catalisada pela cinase da adenosina (ADK), no entanto, também pode
ocorrer a hidrólise a inosina através da desaminase da adenosina (ADA). As concentrações
intra- e extracelulares da Ado são mantidas em equilíbrio pelos ENTs – ENT1 e ENT2, que
transportam este metabolito de acordo com o gradiente de concentração. Resultados
anteriores indicam que a produção de adenosina está aumentada no glioblastoma (GBM),
onde pode desempenhar um papel na progressão do tumor. O principal objetivo deste
trabalho é avaliar a expressão das enzimas envolvidas no metabolismo da Ado, quer em
situação de concentrações de adenosina basais quer aumentadas, em linhas celulares de
glioblastoma e compará-la com a expressão observada em astrócitos humanos (HA). O
efeito do aumento da concentração de adenosina na proliferação/viabilidade celular será
também avaliado. Foram testadas 3 linhas de GBM: U87, SNB19 e U373. A expressão
enzimática foi analisada por qPCR e a viabilidade/proliferação celular foi avaliada pelo
método do MTT.
Os resultados obtidos mostram que a Ado (30 µM) adicionada exogenamente
diminui a proliferação/viabilidade celular em astrócitos apenas quando os recetores da
adenosina são bloqueados pela presença de um cocktail de antagonistas (CA), enquanto o
aumento da concentração de adenosina por inibição do seu consumo utilizando um inibidor
da ADK, o ABT-702 (30 µM), diminui a viabilidade/proliferação celular tanto nos astrócitos
como nas linhas celulares de GBM, quer na presença quer na ausência de CA, sugerindo um
efeito não mediado por recetor. Relativamente à expressão do mRNA, comparativamente a HA, a expressão de ADK
está aumentada nas linhas U373 e SNB19 enquanto a ADA está aumentada apenas na linha
SNB19. Relativamente à expressão dos ENTs, o ENT1 está aumentado em células U373 e
U87 enquanto o aumento da expressão do ENT2 foi observado apenas em U373,
comparativamente a HA. Observou-se ainda uma diminuição da expressão de NT5E em
U373 e SNB19 e um aumento em U87, relativamente a HA. Não foi observada diferenças de
expressão da AHCY em qualquer das linhas comparativamente a HA.
Comparativamente com o controle, o aumento da concentração de adenosina
produziu uma diminuição na expressão da ENT2, nas linhas celulares U373 e U87, e na
ENT1, nas células U373. Em HA e em células U373 a expressão de NT5E foi aumentada,
enquanto nas células U87 foi reduzida pelos níveis elevados de adenosina. Um aumento da
concentração de adenosina produziu aumento da expressão de AHCY em HA. Não foi
observada modificação na expressão de nenhum dos genes analisados na linha celular
SNB19 por aumento da concentração de adenosina.
Em conclusão, observam-se diferenças significativas entre os astrócitos e as linhas celulares
de glioblastoma, quer nos níveis de expressão das enzimas envolvidas no metabolismo da
adenosina quer na resposta dessa expressão face ao aumento da concentração de adenosina.
Glioblastoma is a grade IV type tumor of the central nervous system and is characterized by its infiltrative growth. The lack of oxygenation, also known as hypoxia, promotes the tumor cell invasiveness in the healthy tissues of the brain as an escape mechanism to this adverse environment. Under these conditions, there is an extracellular nucleotides and nucleosides increase in concentration, particularly adenosine (Ado), which is considered an endogenous protection mechanism during hypoxia. Ado basal concentration is usually between 30 – 200 nM, however it can be increased by two mechanisms: extracellular ATP enzymatic hydrolysis into AMP followed by its hydrolysis to Ado by ecto-5’-nucleotidase (NT5E), or Ado export by equilibrative nucleoside transporters (ENTs) after its intracellular production. Intracellularly, Ado can be formed from AMP hydrolysis catalyzed by 5’-ectonucleotidase, or from S-adenosylhomocysteine hydrolysis by catalyzed by the S-adenosylhomocysteinase (AHCY) enzyme. Adenosine can be consumed intracellularly by phosphorylation into AMP catalysed by adenosine kinase (ADK), however it may also be hydrolyzed into inosine by adenosine deaminase (ADA). Intracellular and extracellular Ado concentrations are maintained in equilibrium by ENTs – ENT1 and ENT2, which carry this metabolite according to the concentration gradient. Previous reports indicate that adenosine production is increased in glioblastoma where it might play a role on tumor progression. The main goal of this work is to evaluate enzymes expression involved in Ado metabolism, either under basal and increased adenosine concentrations, in human glioblastoma cell lines and compare it with the expression observed in human astrocytes (HA). The effect of increasing adenosine concentration on cell proliferation/viability will also be assessed. Three lines of GBM were tested: U87, SNB19 and U373. Enzymatic expression was analyzed by qPCR and cell viability/proliferation was evaluated by the MTT method. The results obtained suggest that Ado (30 µM) added exogenously decreases cell proliferation/viability in astrocytes only when adenosine receptors are blocked by the presence of a cocktail of antagonists (CA), while increasing the concentration of adenosine by inhibiting their consumption using an ADK inhibitor, ABT-702 (30 µM), decreases cell viability/proliferation in both astrocytes and GBM cell lines, either in the presence or in the absence of CA, suggesting a non-receptor mediated effect. Regarding mRNA gene expression, compared to HA, ADK expression is increased in the U373 and SNB19 cell lines while ADA is increased only in the SNB19 line. Considering the expression of ENTs, ENT1 is increased in U373 and U87 cells while increased expression of ENT2 was observed only in U373, compared to HA. There was also a decrease in NT5E expression in U373 and SNB19 and an increase in U87, relative to HA. There were no differences in expression of AHCY in any of the lines compared to HA. Compared to the control, the increase in adenosine concentration produced a decrease in the expression of ENT2, in the U373 and U87 cell lines, and in ENT1, in the U373 cells. In HA and U373 cells, NT5E expression was increased, while in U87 cells it was reduced by increased levels of adenosine. An increase in the concentration of adenosine produced an increase in AHCY expression in HA. There was no change in the expression of any of the genes analyzed in the SNB19 cell line by increasing the concentration of adenosine. In conclusion, there are significant differences between astrocytes and glioblastoma cell lines, both in the levels of expression of the enzymes involved in the metabolism of adenosine and in the response of that expression to the increase in the concentration of adenosine.
Glioblastoma is a grade IV type tumor of the central nervous system and is characterized by its infiltrative growth. The lack of oxygenation, also known as hypoxia, promotes the tumor cell invasiveness in the healthy tissues of the brain as an escape mechanism to this adverse environment. Under these conditions, there is an extracellular nucleotides and nucleosides increase in concentration, particularly adenosine (Ado), which is considered an endogenous protection mechanism during hypoxia. Ado basal concentration is usually between 30 – 200 nM, however it can be increased by two mechanisms: extracellular ATP enzymatic hydrolysis into AMP followed by its hydrolysis to Ado by ecto-5’-nucleotidase (NT5E), or Ado export by equilibrative nucleoside transporters (ENTs) after its intracellular production. Intracellularly, Ado can be formed from AMP hydrolysis catalyzed by 5’-ectonucleotidase, or from S-adenosylhomocysteine hydrolysis by catalyzed by the S-adenosylhomocysteinase (AHCY) enzyme. Adenosine can be consumed intracellularly by phosphorylation into AMP catalysed by adenosine kinase (ADK), however it may also be hydrolyzed into inosine by adenosine deaminase (ADA). Intracellular and extracellular Ado concentrations are maintained in equilibrium by ENTs – ENT1 and ENT2, which carry this metabolite according to the concentration gradient. Previous reports indicate that adenosine production is increased in glioblastoma where it might play a role on tumor progression. The main goal of this work is to evaluate enzymes expression involved in Ado metabolism, either under basal and increased adenosine concentrations, in human glioblastoma cell lines and compare it with the expression observed in human astrocytes (HA). The effect of increasing adenosine concentration on cell proliferation/viability will also be assessed. Three lines of GBM were tested: U87, SNB19 and U373. Enzymatic expression was analyzed by qPCR and cell viability/proliferation was evaluated by the MTT method. The results obtained suggest that Ado (30 µM) added exogenously decreases cell proliferation/viability in astrocytes only when adenosine receptors are blocked by the presence of a cocktail of antagonists (CA), while increasing the concentration of adenosine by inhibiting their consumption using an ADK inhibitor, ABT-702 (30 µM), decreases cell viability/proliferation in both astrocytes and GBM cell lines, either in the presence or in the absence of CA, suggesting a non-receptor mediated effect. Regarding mRNA gene expression, compared to HA, ADK expression is increased in the U373 and SNB19 cell lines while ADA is increased only in the SNB19 line. Considering the expression of ENTs, ENT1 is increased in U373 and U87 cells while increased expression of ENT2 was observed only in U373, compared to HA. There was also a decrease in NT5E expression in U373 and SNB19 and an increase in U87, relative to HA. There were no differences in expression of AHCY in any of the lines compared to HA. Compared to the control, the increase in adenosine concentration produced a decrease in the expression of ENT2, in the U373 and U87 cell lines, and in ENT1, in the U373 cells. In HA and U373 cells, NT5E expression was increased, while in U87 cells it was reduced by increased levels of adenosine. An increase in the concentration of adenosine produced an increase in AHCY expression in HA. There was no change in the expression of any of the genes analyzed in the SNB19 cell line by increasing the concentration of adenosine. In conclusion, there are significant differences between astrocytes and glioblastoma cell lines, both in the levels of expression of the enzymes involved in the metabolism of adenosine and in the response of that expression to the increase in the concentration of adenosine.
Description
Keywords
Adenosina:metabolismo Enzimas Glioblastoma