RESUMEN
The idea to use megadoses of ascorbate (vitamin C) for cancer treatment has recently been revived. Despite clear efficacy in animal experimentation, our understanding of the cellular and molecular mechanisms of this treatment is still limited and suggests a combined oxidative and metabolic mechanism behind the selective cytotoxicity of ascorbate towards cancerous cells. To gain more insight into the cellular effects of high doses of ascorbate, we performed a detailed analysis of metabolic changes and cell survival of both luminal and basal-like breast cancer cells treated with ascorbate and revealed a distinctive metabolic shift virtually reversing the Warburg effect and triggering a severe disruption of redox homeostasis. High doses of ascorbate were cytotoxic against MCF7 and MDA-MB231 cells representing luminal and basal-like breast cancer phenotypes. Cell death was dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD+/NADH, associated with a multifaceted metabolic rewiring. This included a sharp disruption of glycolysis at the triose phosphate level, a rapid drop in ATP levels, and redirection of metabolites toward lipid droplet accumulation and increased metabolites and enzymatic activity in the pentose phosphate pathway (PPP). High doses of ascorbate also inhibited the TCA cycle and increased oxygen consumption. Together the severe disruptions of the intracellular metabolic homeostasis on multiple levels "redox crisis and energetic catastrophe" consequently trigger a rapid irreversible cell death.
Asunto(s)
Neoplasias de la Mama , Animales , Ácido Ascórbico/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Supervivencia Celular , Metabolismo Energético , Femenino , Humanos , Oxidación-Reducción , Estrés OxidativoRESUMEN
The human protein kinase R (PKR) recognizes invading RNA viruses and mediates the antiviral immune response by phosphorylating the eukaryotic translation initiation factor 2α (eIF-2α), thus blocking protein translation in infected cells and thus preventing viral replication. The observation that individuals show different degrees of susceptibility to viral infections gives rise to the hypothesis that single nucleotide polymorphisms (SNPs) in the protein kinase R may alter the response to an infection. Using different available servers (e.g. SIFT, PROVEAN, Polyphen2, SNAP2, SNP&GOs, SNP-PhD, I-Mutant Suite), 14 SNPs were identified that were predicted to have deleterious effects on the protein kinase R. Five SNPs, namely D266Y, Y323D, I398â¯K, Y465C and Y472C, were selected for homology modeling and the generated models were investigated with regard to their secondary structure, residue fluctuations and eIF-2αâ¯binding. Analysis with computational tools POLYVIEW-MM, SAAPdap, SRIDE, CMView, elNémo, NMsim and PatchDock revealed structural changes in all mutants yielding a more stable structure at the cost of reduced flexibility (except Y465C) and less conformational freedom compared to the native protein. The conformational changes in the mutant protein structures and the displacement of functional residues from their strategic positions are predicted to affect the functionality of PKR, and consequently will affect the efficiency of the individual's antiviral immune response negatively. This study will aid the physicians in precision medicine field to tailor optimal treatment for the patients.