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Efeito anti tumoral dos canabinóides em glioblastoma: modulação pela adenosina
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Abstract(s)
Dentro do grupo dos gliomas, o glioblastoma (GB) é considerado o mais maligno. Este tumor é classificado como um astrocitoma de grau 4 e é caracterizado pela elevada proliferação celular e intensa angiogénese. Devido à sua significativa heterogeneidade, bem como à barreira hematoencefálica exclusiva do sistema nervoso central e ao microambiente imunossupressor, as terapias convencionais não têm demonstrado eficácia satisfatória no tratamento do GB. Diante da escassez de alternativas terapêuticas eficazes, surge uma necessidade urgente de desenvolver abordagens inovadoras que considerem as características peculiares do GB. Os derivados da Cannabis sativa L. despertaram crescente interesse como potenciais agentes terapêuticos para o GB, atribuídos às suas variadas propriedades: psicotrópicas, analgésicas, anticancerígenas, anti-inflamatórias, anticonvulsivas, antibacterianas e antifúngicas. Os canabinóides atuam nas células de GB via recetores dos canabinóides (recetor tipo 1 e recetor tipo 2), através de três mecanismos principais: indução da morte celular, inibição da divisão celular e ações antiangiogénicas. Em condições de hipoxia associadas ao GB, há um aumento da concentração de nucleótidos e nucleósidos extracelulares, como a adenosina (Ado). Este aumento de Ado está associado à superexpressão do fator de crescimento endotelial vascular (VEGF), que favorece a angiogénese e o crescimento tumoral. No entanto, de acordo com o trabalho prévio do nosso grupo, a inibição da adenosina cinase (ADK) pelo ABT-702 diminui a proliferação celular do GB, sugerindo que a regulação das concentrações de Ado pode ter um papel crucial na dinâmica tumoral. Embora ainda não se conheça a interação entre os inibidores da ADK e canabinóides, a possibilidade de que os canabinóides possam potencializar os efeitos da inibição da ADK representa uma área promissora para futuras investigações. Assim, os canabinóides apresentam-se como potenciais agentes terapêuticos para o GB, uma vez que interferem em processos celulares fundamentais, reduzindo a proliferação e divisão celular, bem como a angiogénese, essenciais para o suprimento de nutrientes às células tumorais. O objetivo desta dissertação foi investigar de que forma os canabinóides afetaram a proliferação e a apoptose em linhas celulares de GB, com especial atenção para a modulação mediada pela Ado e para a interação com fármacos que inibem o seu metabolismo. Foram avaliados os efeitos na presença como na ausência de inibidores dos recetores de Ado. Para tal, foram realizados ensaios de MTT a fim de avaliar a proliferação/viabilidade celular, utilizando linhas celulares de GB (U87 e U373) que foram incubadas por 72 horas com o canabinóide sintético WIN (5µM). Este tratamento foi realizado na presença ou ausência do inibidor da ADK, o ABT-702 (30µM) e também na presença ou ausência de um cocktail de antagonistas dos recetores de Ado, para distinguir entre efeitos mediados e não mediados pelos recetores da Ado. Em ambas as linhas celulares de GB, a combinação de WIN com ABT-702 resultou numa redução significativa da viabilidade celular, embora a presença de altas concentrações de Ado intracelular tenha atenuado ligeiramente o efeito do WIN. Quanto à combinação de cocktail de antagonistas dos recetores de Ado e WIN, o bloqueio dos recetores de Ado pelo cocktail de antagonistas permitiu que o WIN apresentasse um efeito antitumoral mais acentuado, implicando efeito protetor da Ado extracelular não é mediado através destes recetores, contrastando com o efeito anti proliferativo da Ado intracelular. Nos ensaios de migração celular, o WIN (5µM) reduziu a migração celular nas linhas celulares U87 e U373, bem como nos astrócitos primários humanos. A combinação de WIN com ABT-702, resultou numa ligeira atenuação do efeito do WIN. Na linha celular SNB19, os resultados sugerem uma ligeira redução na migração celular com o WIN, embora sem significado estatístico. Relativamente aos ensaios de imunocitoquímica, não foi possível demonstrar que o WIN (5µM) induza apoptose na linha celular U373, sugerindo um efeito essencialmente citoestático do WIN. Em síntese, o WIN provocou uma diminuição significativa na viabilidade celular nas linhas U87 e U373, reduzindo também a migração celular nas linhas U87 e U373, bem como nos astrócitos humanos, corroborando o seu efeito antitumoral. Contudo, os resultados indicaram que a inibição da ADK pelo ABT-702 atenuou o efeito do WIN, sugerindo que a interação entre o ABT-702 e o WIN é menos que aditiva, devido ao aumento da Ado intracelular.
In the group of gliomas, glioblastoma (GB) is regarded as the most malignant. This type of glioma is classified as a grade 4 astrocytoma and is characterised by high cell proliferation and intense angiogenesis. Due to the significant genetic heterogeneity, as well as the unique blood-brain barrier of the central nervous system and the immunosuppressive microenvironment, conventional therapies have not demonstrated satisfactory efficacy in the treatment of GB. Considering the scarcity of effective therapeutic alternatives, there is an urgent need to develop innovative approaches that take into account the peculiar characteristics of GB. Derivatives of Cannabis sativa L. have recently aroused growing interest as potential therapeutic agents for GB, due to their varied properties: psychotropic, analgesic, anticancer, anti-inflammatory, anticonvulsant, antibacterial and antifungal. Cannabinoids exert their effects on GB cells through interaction with cannabinoid receptors (type 1 and type 2), via three principal mechanisms: induction of cell death, inhibition of cell division and anti-angiogenic actions. In hypoxic conditions associated with GB, there is an increase in the concentration of extracellular nucleotides and nucleosides, such as adenosine (Ado). This increase in Ado is associated with overexpression of vascular endothelial growth factor (VEGF), which favours angiogenesis and tumour growth. However, according to previous research conducted by our group, the inhibition of adenosine kinase (ADK) by ABT-702 leads to a decrease in GB proliferation, suggesting that the regulation of Ado concentrations may play a crucial role in tumour dynamics. Although the interaction between ADK inhibitors and cannabinoids is not yet known, the possibility that cannabinoids may potentiate the effects of ADK inhibition represents a promising area for future research. Thus, cannabinoids present themselves as potential therapeutic agents for GB, since they interfere in fundamental cellular processes, reducing cell proliferation and division, as well as angiogenesis, which are essential for the supply of nutrients to tumour cells. The present dissertation aimed to investigate the impact of cannabinoids on the proliferation and apoptosis of GB cell lines, with particular attention to the role of Ado mediated modulation and its interaction with drugs that inhibit their metabolism. The effects were evaluated in the presence and absence of Ado receptor inhibitors. In order to assess cell proliferation/viability, MTT assays were carried out using GB cell lines (U87 and U373). These cell lines were incubated for 72 hours with the synthetic cannabinoid WIN (5 µM). This treatment was conducted in the presence or absence of the ADK inhibitor ABT-702 (30 µM) and also in the presence or absence of a cocktail of adenosine receptor antagonists. This was done in order to distinguish between effects mediated and not mediated by Ado receptors. In both GB cell lines, the combination of WIN with ABT-702 resulted in a significant reduction in cell viability, although the presence of high concentrations of intracellular Ado slightly attenuated the effect of WIN. The combination of the Ado receptor antagonist cocktail and WIN demonstrated a more pronounced anti-tumour effect, suggesting that the protective effect of extracellular Ado is not mediated through these receptors, in contrast to the anti-proliferative effect of intracellular Ado. In cell migration assays, WIN (5µM) reduced cell migration in the U87 and U373 cell lines, as well as in primary human astrocytes. Combining WIN with ABT-702 resulted in a slight attenuation of WIN's effect. In the SNB19 cell line, the results suggest a slight reduction in cell migration with WIN, although without statistical significance. In the context of the immunocytochemistry tests, it was not possible to demonstrate that WIN (5 µM) induces apoptosis in the U373 cell line, suggesting an essentially cytostatic effect of WIN. In summary, WIN caused a significant decrease in cell viability in the U87 and U373 lines and also reduced cell migration in the U87 and U373 lines, as well as in human astrocytes, corroborating its antitumour effect. However, the results indicated that the inhibition of ADK by ABT-702 attenuated the effect of WIN, suggesting that the interaction between ABT-702 and WIN is less than additive, due to the increase in intracellular Ado.
In the group of gliomas, glioblastoma (GB) is regarded as the most malignant. This type of glioma is classified as a grade 4 astrocytoma and is characterised by high cell proliferation and intense angiogenesis. Due to the significant genetic heterogeneity, as well as the unique blood-brain barrier of the central nervous system and the immunosuppressive microenvironment, conventional therapies have not demonstrated satisfactory efficacy in the treatment of GB. Considering the scarcity of effective therapeutic alternatives, there is an urgent need to develop innovative approaches that take into account the peculiar characteristics of GB. Derivatives of Cannabis sativa L. have recently aroused growing interest as potential therapeutic agents for GB, due to their varied properties: psychotropic, analgesic, anticancer, anti-inflammatory, anticonvulsant, antibacterial and antifungal. Cannabinoids exert their effects on GB cells through interaction with cannabinoid receptors (type 1 and type 2), via three principal mechanisms: induction of cell death, inhibition of cell division and anti-angiogenic actions. In hypoxic conditions associated with GB, there is an increase in the concentration of extracellular nucleotides and nucleosides, such as adenosine (Ado). This increase in Ado is associated with overexpression of vascular endothelial growth factor (VEGF), which favours angiogenesis and tumour growth. However, according to previous research conducted by our group, the inhibition of adenosine kinase (ADK) by ABT-702 leads to a decrease in GB proliferation, suggesting that the regulation of Ado concentrations may play a crucial role in tumour dynamics. Although the interaction between ADK inhibitors and cannabinoids is not yet known, the possibility that cannabinoids may potentiate the effects of ADK inhibition represents a promising area for future research. Thus, cannabinoids present themselves as potential therapeutic agents for GB, since they interfere in fundamental cellular processes, reducing cell proliferation and division, as well as angiogenesis, which are essential for the supply of nutrients to tumour cells. The present dissertation aimed to investigate the impact of cannabinoids on the proliferation and apoptosis of GB cell lines, with particular attention to the role of Ado mediated modulation and its interaction with drugs that inhibit their metabolism. The effects were evaluated in the presence and absence of Ado receptor inhibitors. In order to assess cell proliferation/viability, MTT assays were carried out using GB cell lines (U87 and U373). These cell lines were incubated for 72 hours with the synthetic cannabinoid WIN (5 µM). This treatment was conducted in the presence or absence of the ADK inhibitor ABT-702 (30 µM) and also in the presence or absence of a cocktail of adenosine receptor antagonists. This was done in order to distinguish between effects mediated and not mediated by Ado receptors. In both GB cell lines, the combination of WIN with ABT-702 resulted in a significant reduction in cell viability, although the presence of high concentrations of intracellular Ado slightly attenuated the effect of WIN. The combination of the Ado receptor antagonist cocktail and WIN demonstrated a more pronounced anti-tumour effect, suggesting that the protective effect of extracellular Ado is not mediated through these receptors, in contrast to the anti-proliferative effect of intracellular Ado. In cell migration assays, WIN (5µM) reduced cell migration in the U87 and U373 cell lines, as well as in primary human astrocytes. Combining WIN with ABT-702 resulted in a slight attenuation of WIN's effect. In the SNB19 cell line, the results suggest a slight reduction in cell migration with WIN, although without statistical significance. In the context of the immunocytochemistry tests, it was not possible to demonstrate that WIN (5 µM) induces apoptosis in the U373 cell line, suggesting an essentially cytostatic effect of WIN. In summary, WIN caused a significant decrease in cell viability in the U87 and U373 lines and also reduced cell migration in the U87 and U373 lines, as well as in human astrocytes, corroborating its antitumour effect. However, the results indicated that the inhibition of ADK by ABT-702 attenuated the effect of WIN, suggesting that the interaction between ABT-702 and WIN is less than additive, due to the increase in intracellular Ado.
Description
Keywords
Glioblastoma Astrócitos Canabinóides Adenosina Astrocytes Cannabinoids Adenosine