title: Unraveling the mechanisms behind the enhanced MTT conversion by irradiated breast cancer cells.
abstract: Previously, we described the radiation-induced (RI) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) effect as the increased MTT metabolization at the intermediate dose region after the irradiation of an MCF-7/6 cell monolayer with an X-ray dose gradient. We wondered if the cell monolayer at the intermediate dose region was characterized by an increased metabolic activity. In this study, we unraveled the mechanisms behind the RI MTT effect. Comparison of the MTT, sulforhodamine B (SRB), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium (WST-8), and nitroblue tetrazolium (NBT) assays indicated that the RI MTT effect is not due to an increased cell density, but to an exclusively intracellular MTT conversion. Our results for the MTT and NBT assays after digitonin pretreatment of the irradiated cell monolayer indicated a role of the plasma membrane permeability in the RI MTT effect. Assessment of the radiation impact on the oxidative phosphorylation system by Western blot analysis, spectrophotometric measurement and Blue Native gel electrophoresis showed a dose-dependent downregulation of the oxidative phosphorylation system complexes, whereby the radiosensitivity of each complex was proportional to the number of mitochondrial DNA-encoded subunits. Further, only treatment of the irradiated cell monolayer with a cocktail and not with the individual inhibitors of complexes I, II and IV during the MTT assay prevented the RI MTT effect. In general, our results demonstrate that the RI MTT effect is not due to an increased metabolic activity, but rather to an enhanced cellular MTT entry and mitochondrial MTT conversion.
authors: Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Belgium. stephanie.blockhuys@ugent.be
authors: Stéphanie
authors: S
authors: Blockhuys
authors: Barbara
authors: B
authors: Vanhoecke
authors: Joél
authors: J
authors: Smet
authors: Boel
authors: B
authors: De Paepe
authors: Rudy
authors: R
authors: Van Coster
authors: Marc
authors: M
authors: Bracke
authors: Carlos
authors: C
authors: De Wagter
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D015820:Cadherins / Q000502:physiology; D002450:Cell Communication / Q000528:radiation effects; D002452:Cell Count; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010539:Permeability; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D015820:Cadherins; D013778:Tetrazolium Salts; D013844:Thiazoles; D009580:Nitroblue Tetrazolium; C022616:thiazolyl blue