RESUMEN
Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the hemedependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IMsensitive K562 chronic myeloid leukemia cells (K562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K562 and IMchemoresistant K562 chronic myeloid leukemia cells (K562R), as well as human colorectal carcinoma cells HCT116 (+/+p53) and (/p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2 µM) and/or DCA (4 mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde3phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor1a, heme oxygenase1, NFκB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K562 and considerable (>45%) in HCT116 (+/+p53) cultures, but less in K562R and HCT116 (/p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flowcytometry, in K562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.
Asunto(s)
Neoplasias Colorrectales , Leucemia Eritroblástica Aguda , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Apoptosis , Línea Celular Tumoral , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Metabolismo Energético , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Biomarcadores/metabolismo , Células K562 , Resistencia a Antineoplásicos/genética , Proliferación CelularRESUMEN
Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. ß-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult ß-globin chain. The resulting imbalance of α-/ß-globin chains of adult hemoglobin (α2ß2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant ß-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature ß-globin chain. Notably, the produced human recombinant ß-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-ß-globin protein interacts with the endogenous native α-like globins to form hemoglobin α2ß2-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-ß-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for ß-thalassemias.