Cunha-de 2017 Thesis

Cunha-de Padua MM (2017) Mecanismos citotóxicos da galactomanana de sementes de Schizolobium amazonicum e seus complexos com oxovanádio em células de hepatocarcinoma humano (HepG2) e efeito do silenciamento da enzima glucose-6-fosfato isomerase (GPI) na sobrevivência de células de adenocarcinoma de cólon humano (LS174T). Doctoral Thesis p160.

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Cunha-de Padua MM (2017) Doctoral Thesis

Abstract: The aim of this study, was evaluated the toxicity of galactomannan preparations isolated from seeds of S. amazonicum and its complexes with oxovanadium in HepG2 cells, focusing on effects of energy metabolism. Native galactomannan (SAGM) was obtained from endosperm of S. amazonicum seeds by aqueous extraction at 25 ºC. SAGM was subjected to partial acid hydrolysis resulting in the MSAGM fraction. HPSEC/MALLS/RI elution profiles of native and modified galactomannans confirmed partial hydrolysis. Molecular mass of MSAGM obtained by MALLS was 1.56 × 10⁴ g/mol. The Man: Gal ratio of 3.2:1 determined by GC, was not modified in relation to SAGM. SAGM and its partially hydrolyzed form (MSAGM) were complexed with oxovanadium, and was called SAGM:VO and MSAGM:VO, respectively. The complexation of biopolymers with oxovanadium (IV/ V) was characterized by potentiometric titration, FT-IR, NMR ⁵¹V, ¹³C. Atomic absorption technique was used to determine the presence of the metal in the complexes. The values of the complexation constants for the chemical equilibria were similar for both preparations, except for the equilibrium [L₂(OH)₂(VO)₂]/[L]₂.[OH]₂.[VO]₂ that was detected only for MSAGM:VO. For SAGM:VO complex the vanadium content was 3.8 fold greater than MSAGM:VO complex. Among the polymers, SAGM and MSAGM:VO promoted a greater reduction (~ 50%) in the viability of HepG2 cells at concentration of 250 μg/mL during 72h of incubation. Under the same conditions only MSAGM:VO promoted ~ 50% of inhibition in the proliferation of these cells. Both polymers also inhibited all states of respiration (basal: ~ 70%, decoupled: ~ 80%, and leak: ~ 40%). This effect was also repeated in permeabilized cells, in which respiration inhibition was observed in ~ 50%, which was maintained after the addition of ADP, glutamate/malate and succinate (substrates for complexes I and II, respectively). Lactate concentration of HepG2 cells incubated with SAGM (250 μg/mL) for 72h increased by ~ 25%, but for SAGM:VO, MSAGM and MSAGM:VO, there was a ~ 20% reduction. Under the same conditions, the presence of the biopolymers did not significantly alter the concentration of pyruvate. ATP levels were reduced by ~ 30% after treatment of 72 h at 250 μg/ml with all preparations. MSAGM:VO increased ROS levels by ~ 149%, while mitochondrial transmembrane potential (ΔΨm) was reduced by ~ 47%. The analysis of cell death in normoxic condition, after 72h, showed that MSAGM:VO (250 μg / mL) promoted an increase of caspase activity (3 and 7) and, to a lesser extent, protein BAX. However, under hypoxia conditions, the biopolymer increased expression of the anti-apoptotic proteins MCL- 1 and BCL-XL. Also in this condition, treatment with MSAGM:VO promoted increase of LC3-II proteins and reduction of p62, suggesting the autophagy process. Expression levels of HIF-1α under hypoxia conditions were reduced after treatment with MSAGM:VO (250 μg/mL and 72 h). These results demonstrate that the preparations of galactomannans from S. amazonicum, especially SAGM and MSAGM:VO, render HepG2 cells susceptible to death and suggest that these polymers have a potential antitumor activity, motivating future studies that allow their therapeutic application.

In addition to the study carried out with galactomannans from S. amazonicum, the other aim of this study was to evaluate the impact of disruption glucose - 6 - phosphate isomerase (GPI) on LS174T cells. GPI is a cytosolic enzyme that performs glucose - 6 - phosphate interconversion to fructose - 6 - phosphate. Genetic disruption of this enzyme in LS174T cells (GPIKO) was performed in a way to divert the metabolism of these cells to the phosphate pentose pathway. After GPI silencing under conditions of normoxia, a reduction of glucose and lactate was observed, reprogramming GPIKO cells for oxidative phosphorylation in order to raise mitochondrial ATP levels, thus maintaining viability. Incubation with phenformin in GPIKO cells led the cells to death, indicating sensitivity to the inhibitor of the complex I of the electron transport chain. Under hypoxia conditions, GPIKO had a reduced cell growth, characterized by decreased proliferation and cellular respiration. Despite in vitro growth restriction under hypoxia, tumor growth in vivo moderately reduced compared to wild type, which did not show the mutation. These results indicate that the exclusive use of oxidative metabolism has the ability to provide metabolic precursors for tumor growth, pointing out that the plasticity of tumor cell metabolism has a strong limitation for antitumor strategies. • Keywords: Galactomannan, Oxovanadium (IV/V), HepG2 cells, LS174T cells, Metabolism, GPI, Cytotoxic effects, Antitumor, Hypoxia. • Bioblast editor: Kandolf G • O2k-Network Lab: BR Parana Iacomini M

Labels: MiParea: Respiration, Pharmacology;toxicology  Pathology: Cancer  Stress:Hypoxia  Organism: Human  Tissue;cell: Liver, Other cell lines  Preparation: Intact cells, Permeabilized cells 

Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: N, NS, ROX  HRR: Oxygraph-2k 

Labels, 2018-02