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Authors
Abstract(s)
Os gliomas, tumores do sistema nervoso central (SNC), incluem a maioria dos tumores
cerebrais malignos, e constituem uma importante causa de morte por cancro em todo o
mundo. Deste grupo de tumores faz parte o glioblastoma (GBM), astrocitoma de grau
IV e a forma mais maligna de gliomas, com elevada heterogeneidade celular, grande
capacidade infiltrativa, baixa metastização, reincidência local, resistência às
terapêuticas e baixa taxa de sobrevivência. Os tratamentos disponíveis não têm
apresentado resultados satisfatórios, por isso é necessário recorrer a metodologias
inovadoras e direcionadas para as singularidades das células de GBM.
O uso de derivados de Cannabis sativa L. tem suscitado um interesse particular devido
aos seus efeitos anti-proliferativos, pró-apoptóticos e anti-tumorais, particularmente
em células de GBM. Nessas células, os canabinóides atuam através dos recetores do
tipo 1 e 2 (CB1 e CB2, respetivamente), acoplados a proteína G. Os genes dos recetores
dos canabinóides são regulados pela metilação das suas regiões promotoras. A
adenosina (Ado), a qual se acumula nas regiões de hipoxia encontradas no GBM, pode
regular a metilação de diversos genes. O presente trabalho teve como objetivo,
investigar a possibilidade de concentrações elevadas de Ado puderem modular o efeito
anti-tumoral dos canabinóides e a expressão de recetores destes em GBM. Para tal, o
efeito de um agonista sintético não seletivo dos recetores CB1 e CB2, o Win 55 212-2
(Win), na viabilidade/proliferação celular e na citotoxicidade em linhas celulares GBM
foi avaliado usando os ensaios de MTT e de libertação da desidrogenase do lactato
(LDH), respetivamente. A expressão do recetor CB1, mais comum no sistema nervoso,
foi avaliada por PCR em tempo real (RT-PCR).
Começou-se por testar qual a concentração de Win com melhor efeito anti-proliferativo
em três linhas de GBM (U373, U87 e SNB19) e em células de astrócitos humanos
normais (HA). As células foram incubadas por 72 h na presença de Win. Inicialmente,
foi estudada uma gama de concentrações de Win entre 0,1 µM e 30 µM; a concentração
de 5 µM foi selecionada para estudos posteriores, pois produziu 60%-90% de inibição
da proliferação/viabilidade celular em todas as células estudadas. Noutras experiências
as células foram incubadas durante 72h com Win (5 µM), na presença ou na ausência
de Ado (30 µM) e na presença ou na ausência de um cocktail de antagonistas dos
recetores da adenosina (C.A.). Em algumas experiências, a concentração endógena de
Ado foi aumentada usando um inibidor da cinase da Ado, o ABT-702 (30 µM) (ABT).
Na presença de C.A., a Ado foi capaz de reduzir a inibição da proliferação/viabilidade
celular induzida pelo Win, em células U373, enquanto que o ABT na presença de C.A.
reduziu a inibição da proliferação/viabilidade celular induzida por Win nas linhas U373, U87, SNB19 e em HA. O facto da atenuação do efeito anti-proliferativo do Win
pela Ado exógena, nas células U373, e pelo aumento da Ado endógena pelo ABT, em
todos os tipos de células estudadas, não ser prevenida pelo bloqueio dos recetores da
Ado pelo CA, indica que esta atenuação não é mediada por ativação dos recetores da
Ado.
No caso dos ensaios de citotoxicidade, o Win (5 µM) produziu um aumento da
libertação de LDH em todas as células estudadas, quer na ausência quer na presença do
C.A. O ABT (30 µM) na presença de C.A. reduziu a inibição da libertação de LDH
induzida por Win apenas na linha U87 e em HA.
A expressão do gene do recetor CB1 dos canabinóides foi avaliada nas três linhas
celulares de GBM e em HA, tratadas com Ado ou ABT, e observou-se que na linha U87
houve uma redução na expressão do recetor CB1 na presença de ABT.
Em conclusão, Win causou uma diminuição acentuada na proliferação/viabilidade
celular e um aumento na morte celular avaliada pela liberação de LDH em todas as três
linhas celulares de GBM e em HA. O efeito anti-proliferativo e citotóxico de Win foi
significativamente atenuado pelo aumento da concentração de Ado através de um
mecanismo independente de recetor em todas as três linhas celulares GBM estudadas.
Gliomas, central nervous system tumors, include most malignant brain tumors and constitute a major cause of cancer related death around the world. This group of tumors includes glioblastoma (GBM), a grade IV astrocytoma and the most malignant form of gliomas, with high cellular heterogeneity, great infiltrative capacity, low metastazation rate, high local recurrence, resistance to therapies and low survival rate. The available treatments have not shown satisfactory results, thus it is necessary to seek innovative and targeted methods to deal with GBM cell’s singularities. Cannabis sativa L. derivatives’ use has raised an interest due to its anti-proliferative, pro-apoptotic and anti-tumoral effects, especially in GBM cells. In these cells, cannabinoids act through coupled protein G type 1 and 2 receptors (CB1 and CB2, respectively). Cannabinoid receptor genes are regulated by methylation of their promoter regions. Adenosine (Ado) accumulates in hypoxic regions found in GBM and can regulate the methylation of multiple genes. The present study aimed to investigate the possibility that high concentrations of Ado could modulate the anti-tumoral effect of cannabinoids and the expression of their receptor in GBM. For this purpose, the effect of a non-selective synthetic agonist of CB1 and CB2 receptors, Win 55212-2 (Win), on cellular viability/proliferation and cytotoxicity in GBM cell lines was evaluated using MTT assays and lactate dehydrogenase assays, respectively. CB1 receptor expression, the most common in the nervous system, was evaluated by real time PCR (RT-PCR). It was started by testing which Win concentration had the best anti-proliferative effect on three GBM cell lines (U373, U87 and SNB19) and normal human astrocyte cells (HA). The cells were incubated for 72h in the presence of Win. Initially, it was studied a Win concentration range between 0,1 µM and 30 µM; 5 µM was the selected concentration to future studies, since it produced a 60%-90% inhibition of proliferation/viability of all the studied cells. In the following experiments, the cells were incubated for 72h with Win (5 µM), in the presence or absence of Ado (30 µM) and the presence or absence of an antagonist cocktail of adenosine receptors (CA). In some experiments, the endogenous Ado concentration was increased using an adenosine kinase inhibitor, ABT-702 (30 µM) (ABT). In the presence of CA, Ado could reduce the cellular proliferation/viability inhibition induced by Win, in U373 cells, while ABT in the presence of CA reduced the cellular proliferation/viability inhibition induced by Win in U373, U87, SNB19 cell lines and HA. The mitigation of Win’s anti-proliferative effect by exogenous Ado, in U373 cells, and the increase of endogenous Ado by ABT, in all studied cells, is not prevented by the blockage of Ado’s receptors by CA, which indicates that this effect is not mediated by Ado receptors activation. In cytotoxicity assays, Win (5 µM) produced an increase of LDH release in all studied cells, in thes presence and absence of CA. ABT (30 µM) in the presence of CA reduced the inhibition of the LDH’s release induced by Win, in U87 and HA. Cannabinoid CB1 receptor gene expression was evaluated in the three GBM cell lines and HA, treated with Ado or ABT and it was found that there was a reduction in the expression of CB1 receptor in the U87 cell line, in the presence of ABT. In conclusion, Win caused an accentuated decrease in cell proliferation/viability and an increase in cell death evaluated by LDH assay in all three GBM cell lines and HA. The anti-proliferative and cytotoxic effects of Win was significantly mitigated by the increase of Ado’s concentration by a receptor independent mechanism in all studied GBM cell lines.
Gliomas, central nervous system tumors, include most malignant brain tumors and constitute a major cause of cancer related death around the world. This group of tumors includes glioblastoma (GBM), a grade IV astrocytoma and the most malignant form of gliomas, with high cellular heterogeneity, great infiltrative capacity, low metastazation rate, high local recurrence, resistance to therapies and low survival rate. The available treatments have not shown satisfactory results, thus it is necessary to seek innovative and targeted methods to deal with GBM cell’s singularities. Cannabis sativa L. derivatives’ use has raised an interest due to its anti-proliferative, pro-apoptotic and anti-tumoral effects, especially in GBM cells. In these cells, cannabinoids act through coupled protein G type 1 and 2 receptors (CB1 and CB2, respectively). Cannabinoid receptor genes are regulated by methylation of their promoter regions. Adenosine (Ado) accumulates in hypoxic regions found in GBM and can regulate the methylation of multiple genes. The present study aimed to investigate the possibility that high concentrations of Ado could modulate the anti-tumoral effect of cannabinoids and the expression of their receptor in GBM. For this purpose, the effect of a non-selective synthetic agonist of CB1 and CB2 receptors, Win 55212-2 (Win), on cellular viability/proliferation and cytotoxicity in GBM cell lines was evaluated using MTT assays and lactate dehydrogenase assays, respectively. CB1 receptor expression, the most common in the nervous system, was evaluated by real time PCR (RT-PCR). It was started by testing which Win concentration had the best anti-proliferative effect on three GBM cell lines (U373, U87 and SNB19) and normal human astrocyte cells (HA). The cells were incubated for 72h in the presence of Win. Initially, it was studied a Win concentration range between 0,1 µM and 30 µM; 5 µM was the selected concentration to future studies, since it produced a 60%-90% inhibition of proliferation/viability of all the studied cells. In the following experiments, the cells were incubated for 72h with Win (5 µM), in the presence or absence of Ado (30 µM) and the presence or absence of an antagonist cocktail of adenosine receptors (CA). In some experiments, the endogenous Ado concentration was increased using an adenosine kinase inhibitor, ABT-702 (30 µM) (ABT). In the presence of CA, Ado could reduce the cellular proliferation/viability inhibition induced by Win, in U373 cells, while ABT in the presence of CA reduced the cellular proliferation/viability inhibition induced by Win in U373, U87, SNB19 cell lines and HA. The mitigation of Win’s anti-proliferative effect by exogenous Ado, in U373 cells, and the increase of endogenous Ado by ABT, in all studied cells, is not prevented by the blockage of Ado’s receptors by CA, which indicates that this effect is not mediated by Ado receptors activation. In cytotoxicity assays, Win (5 µM) produced an increase of LDH release in all studied cells, in thes presence and absence of CA. ABT (30 µM) in the presence of CA reduced the inhibition of the LDH’s release induced by Win, in U87 and HA. Cannabinoid CB1 receptor gene expression was evaluated in the three GBM cell lines and HA, treated with Ado or ABT and it was found that there was a reduction in the expression of CB1 receptor in the U87 cell line, in the presence of ABT. In conclusion, Win caused an accentuated decrease in cell proliferation/viability and an increase in cell death evaluated by LDH assay in all three GBM cell lines and HA. The anti-proliferative and cytotoxic effects of Win was significantly mitigated by the increase of Ado’s concentration by a receptor independent mechanism in all studied GBM cell lines.
Description
Keywords
Adenosina Astrócitos e Recetores Cb1 Canabinóides Glioblastoma