Efeito citoprotetor do canabidiol contra o insulto citotóxico induzido pela cisplatina em modelo in vitro de mioblastos e miotubos C2C12 e in vivo utilizando o Caenorhabditis elegans
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Data
2024-05-16
Autores
Santos, Michele Rosana Maia [UNIFESP]
Orientadores
Trindade, Claudia Bincoletto [UNIFESP]
Tipo
Dissertação de mestrado
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A caquexia, e consequente sarcopenia secundária, são efeitos adversos graves da quimioterapia e impacta na qualidade de vida e sobrevida do paciente. Compostos platinados, especialmente a cisplatina (CDDP), são muito utilizados no tratamento de tumores sólidos e estão diretamente relacionados à sarcopenia, uma vez que levam à atrofia muscular esquelética, principalmente pela via ubiquitina-proteossoma, impedindo a regeneração muscular por interferir na miogênese das células satélites. Diversos estudos com o canabidiol (CBD), canabinóide derivado da Cannabis sativa, tem demonstrado a capacidade citoprotetora da molécula em modelos celulares in vitro e in vivo, enquanto também apresenta atividade antitumoral. Objetivo: Verificar os efeitos do CBD em mioblastos e miotubos C2C12 e Caenorhabditis elegans diante da toxicidade da CDDP. Métodos: Foram realizados ensaio de redução MTT à formazan e citometria de fluxo com marcação por 7-AAD e anexina V-FITC para avaliação da viabilidade celular e detecção de morte celular em mioblastos C2C12 e adenocarcinoma mamário humano MCF-7 tratados com CBD (1-50 μM), CDDP (12,5-200 μM) e em cotratamento por 24 horas (h); Imunofluorescência (IF) para identificação dos receptores CB1, CB2 e TRPV1 em miotubos C2C12 e quantificação de myosin heavy chain (MHC)/núcleo nos miotubos tratados com CBD (10 μM), CDDP (50 μM) e cotratamento, e agonista inverso ou antagonistas dos receptores CB1 ou TRPV1 (AM-251 ou capsazepina 10 μM, respectivamente) por 48h; Ensaio de locomoção (movimentos/20 segundos) na cepa selvagem N2 (Bristol-WT) e na knockout para o receptor do sistema endocanabinóide RB1668 (npr- 19(ok2068)) de C. elegans tratados com CBD (15 μM) por 48h e CDDP (25 μM) por 24h e cotratamento. Resultados: Nenhuma das concentrações de CBD foi citotóxicas aos mioblastos C2C12 enquanto que, a partir de 10 μM, foi tóxica à MCF- 7, reduzindo a viabilidade celular e aumentando morte celular por apoptose e apoptose tardia; o CBD 10 e 15 μM protegeu os mioblastos frente à toxicidade da CDDP, mas não alterou as respostas da linhagem tumoral MCF-7 no cotratamento; imagens de IF identificaram a presença dos receptores CB1 e TRPV1, mas não de CB2; o CBD a 10 μM protegeu parcialmente os miotubos C2C12 aumentando a relação MHC/núcleo; os receptores CB1 e TRPV1 parecem estar envolvidos nos efeitos de citoproteção do CBD; O CBD 15 μM protegeu a capacidade locomotora em C. elegans expostos à CDDP via npr-19 Conclusões: O CBD induziu citoproteção à linhagem C2C12 agredida com o quimioterápico CDDP via receptores
CB1 e TRPV1 enquanto que não afetou a ação antitumoral desse fármaco, abrindo perspectiva para a utilização futura do CBD como tratamento citoprotetor antitumoral.
Cachexia and its consequent sarcopenia are serious chemotherapy side effects that impact in cancer patient's quality of life and survival. Platinum compounds, especially cisplatin (CDDP), are widely used for the treatment of solid tumors and also directly related to sarcopenia development, once they lead to skeletal muscle atrophy mainly through the ubiquitinproteasome pathway, at the same time that prevents muscle from regenerating by interfering in the myogenesis. Several studies with cannabidiol (CBD), a cannabinoid derived from Cannabis sativa, have been demonstrating in vitro and in vivo this molecule citoprotective capability while it also presents antitumor activity. Objective: To verify the CBD effects on C2C12 myoblasts and myotubes, and in Caenorhabditis elegans in face of the CDDP toxicity. Methods: It were performed MTT reduction assay and flow cytometry assay with 7AAD and annexin VFITC labeling to evaluate cellular viability and cell death detection in C2C12 myoblasts and MCF7 cells treated with CBD (150 μM), CDDP (12.5200 μM) and in cotreatment of these two compounds for 24 hours (h); Immunofluorescence (IF) for CB1, CB2 and TRPV1 receptors identification and also myosin heavy chain (MHC)/nucleus quantification in C2C12 myotubes treated with CBD (10 μM), CDDP (50 μM), cotreatment of these two compounds, and also with the CB1 inverse agonist or TRPV1 antagonists (AM251 or Capsazepine 10 μM, respectively) for 48h; Locomotion assay (body bends/20 seconds) in the wild strain N2 (BristolWT) and in the RB1668 (npr19(ok2068)), a knockout strain for endocannabinoid system receptors in C. elegans treated with CBD (15 μM) for 48h and CDDP (25 μM) for 24h and its cotreatment. Results: CBD was not cytotoxic for C2C12 myoblasts in any of the tested concentrations while it was toxic for MCF7 from 10 μM reducing its viability and increasing its death by apoptosis and late apoptosis; CBD 15 and 10 μM cytoprotected myoblasts against CDDP toxicity, but did not alter the MCF7 tumor line response to CDDP in the cotreatment group; the presence of CB1 and TRPV1 receptors, but not CB2, were identified in IF images; 10 μM of CBD partially protected C2C12 myotubes by increasing the MHC/nucleus ratio; CB1 and TRPV1 receptors seems to be involved in the CBD cytoprotective effects; CBD 15 μM protected locomotor capability in C. elegans exposed to CDDP via npr19 Conclusions: CBD induced cytoprotection to the C2C12 lineage stressed with CDDP via CB1 and TRPV1 receptors, while it did not affect the antitumor action of this drug, it opens up perspectives for the future use of CBD as an antitumor cytoprotective treatment.
Cachexia and its consequent sarcopenia are serious chemotherapy side effects that impact in cancer patient's quality of life and survival. Platinum compounds, especially cisplatin (CDDP), are widely used for the treatment of solid tumors and also directly related to sarcopenia development, once they lead to skeletal muscle atrophy mainly through the ubiquitinproteasome pathway, at the same time that prevents muscle from regenerating by interfering in the myogenesis. Several studies with cannabidiol (CBD), a cannabinoid derived from Cannabis sativa, have been demonstrating in vitro and in vivo this molecule citoprotective capability while it also presents antitumor activity. Objective: To verify the CBD effects on C2C12 myoblasts and myotubes, and in Caenorhabditis elegans in face of the CDDP toxicity. Methods: It were performed MTT reduction assay and flow cytometry assay with 7AAD and annexin VFITC labeling to evaluate cellular viability and cell death detection in C2C12 myoblasts and MCF7 cells treated with CBD (150 μM), CDDP (12.5200 μM) and in cotreatment of these two compounds for 24 hours (h); Immunofluorescence (IF) for CB1, CB2 and TRPV1 receptors identification and also myosin heavy chain (MHC)/nucleus quantification in C2C12 myotubes treated with CBD (10 μM), CDDP (50 μM), cotreatment of these two compounds, and also with the CB1 inverse agonist or TRPV1 antagonists (AM251 or Capsazepine 10 μM, respectively) for 48h; Locomotion assay (body bends/20 seconds) in the wild strain N2 (BristolWT) and in the RB1668 (npr19(ok2068)), a knockout strain for endocannabinoid system receptors in C. elegans treated with CBD (15 μM) for 48h and CDDP (25 μM) for 24h and its cotreatment. Results: CBD was not cytotoxic for C2C12 myoblasts in any of the tested concentrations while it was toxic for MCF7 from 10 μM reducing its viability and increasing its death by apoptosis and late apoptosis; CBD 15 and 10 μM cytoprotected myoblasts against CDDP toxicity, but did not alter the MCF7 tumor line response to CDDP in the cotreatment group; the presence of CB1 and TRPV1 receptors, but not CB2, were identified in IF images; 10 μM of CBD partially protected C2C12 myotubes by increasing the MHC/nucleus ratio; CB1 and TRPV1 receptors seems to be involved in the CBD cytoprotective effects; CBD 15 μM protected locomotor capability in C. elegans exposed to CDDP via npr19 Conclusions: CBD induced cytoprotection to the C2C12 lineage stressed with CDDP via CB1 and TRPV1 receptors, while it did not affect the antitumor action of this drug, it opens up perspectives for the future use of CBD as an antitumor cytoprotective treatment.
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SANTOS, Michele Rosana Maia. Efeito citoprotetor do canabidiol contra o insulto citotóxico induzido pela cisplatina em modelo in vitro de mioblastos e miotubos C2C12 e in vivo utilizando o Caenorhabditis elegans. 2024. 64 f. Dissertação (Mestrado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.