Determinação de efeito de drogas antitumorais in vitro em diferentes estratégias de cultura celular

• Autor(a) principal: Fontoura, Julia Crispim da
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da PUC_RS
• Texto Completo: http://tede2.pucrs.br/tede2/handle/tede/8968
• topic: Câncer; HspBP1; Cultura 3D; Eletrofiação; SCPL; CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Cancer has caused millions of deaths every year across the world, evidencing the importance of drug discovery and improving in vitro drug testing. With that in mind, those two approaches have been considered for this work. First, was studied a protein which can inhibit Hsp70, thus potentially acting as a new anticancer therapy. The chaperone Hsp70 has been shown to be upregulated in various tumors, where it can inhibit cell death and promote tumor growth. In order for Hsp70 to work properly, it associates with different cochaperones, one of which is the Hsp70 Binding Protein 1 (HspBP1). Preliminary experiments from our group showed that tumors overexpressing HspBP1 grew considerably less than regular ones. As such, the potential for HspBP1 as an anticancer therapy in combination or not with chemotherapy was analyzed. Cells were cultured in vitro and treated for 48 h before analysis of: viability (MTT), cell density (Optical microscopy, static and over 48 h), cell death (LDH) and apoptosis (Annexin/PI). Although there were slight antitumor effects, they were not significant, showing that in vitro HspBP1 was unable to promote neither cytotoxic, nor cytostatic effects. For the second part, the usage of three-dimensional (3D) models for in vitro culture were studied, since it has been shown that growing cells on scaffolds may approximate in vitro to in vivo cell growth. Thus, cell growth, morphology, response to chemotherapy and RNA expression were analyzed across different culture models. These models encompassed classical 2D culture; culture on a gel made from a tumor extract (EHS gel, such as Matrigel®); an electrospun; a scaffold produced by Solvent Casting Particle Leaching (SCPL); and cells grown in vivo. Through Scanning Electron Microscopy (SEM) 3D scaffolds were analyzed regarding pore size. It could be seen that the gel had the smallest pore diameters, followed by the electrospun and then the SCPL membrane. Melanoma B16F10 GFP cells were cultured in vitro for 7 and in vivo for 10 days, after which morphology was also analyzed through SEM. As expected, cells grown on a glass slide appeared flat and elongated, while cells on 3D had different morphologies, including the formation of cell aggregates and spheroids. Cells on 3D had morphologies more similar to those grown in vivo. When analyzing resistance to chemotherapy, after culture cells were treated for 48h with cisplatin, followed by viability analysis through MTT. Though 3D cultures in general had a tendency for cisplatin resistance, only those grown on electrospun membranes had significant resistance. At last, RNA analysis showed similarities across 3D culture models, confirming morphological results. As it is, cell growth on any of the 3D culture models shown was able to better reproduce in vivo culture than the 2D model, showing the potential of synthetic scaffolds to reduce the usage of animal derived models for 3D growth.