A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A

Abstract

Fanconi Anemia (FA) is a rare recessive genetic disorder characterized by aplastic anemia due to a defective DNA repair system. In addition, dysfunctional energy metabolism, lipid droplets accumulation, and unbalanced oxidative stress are involved in FA pathogenesis. Thus, to modulate the altered metabolism, Fanc-A lymphoblast cell lines were treated with quercetin, a flavonoid compound, C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid), a fatty acid synthesis inhibitor, and rapamycin, an mTOR inhibitor, alone or in combination. As a control, isogenic FA cell lines corrected with the functional Fanc-A gene were used. Results showed that: (i) quercetin recovered the energy metabolism efficiency, reducing oxidative stress; (ii) C75 caused the lipid accumulation decrement and a slight oxidative stress reduction, without improving the energy metabolism; (iii) rapamycin reduced the aerobic metabolism and the oxidative stress, without increasing the energy status. In addition, all molecules reduce the accumulation of DNA double-strand breaks. Two-by-two combinations of the three drugs showed an additive effect compared with the action of the single molecule. Specifically, the quercetin/C75 combination appeared the most efficient in the mitochondrial and lipid metabolism improvement and in oxidative stress production reduction, while the quercetin/rapamycin combination seemed the most efficient in the DNA breaks decrement. Thus, data reported herein suggest that FA is a complex and multifactorial disease, and a multidrug strategy is necessary to correct the metabolic alterations.

Divergent Oxidative Stress in Normal Tissues and Inflammatory Cells in Hodgkin and Non-Hodgkin Lymphoma

“…Data were acquired on a FACSCan (Becton Dickinson, Milan, Italy) and data analysis was performed with FlowJo software (Version 9.96). The analysis of PBMC was restricted to viable cells, cellular viability was assessed by propidium iodide (PI) exclusion assays and cells positive for PI fluorescence were considered dead cells [ 19 ]. After gating procedures based on forward- and side-scatter features the recovery of lymphocytes and monocytes in the expected gate was confirmed by staining with CD3, CD19, and CD14 (Miltenyi Biotec, Bergisch Gladbach, Germany).…”

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“…Control cells seeded in parallel received only fresh medium. Cells were then collected and prepared for WB analysis as described in Section 2.12 [ 35 ].…”

Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging

“…The reaction medium contained: 100 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 0.2 mM ATP, 10 mM phosphoenolpyruvate, 0.15 mM NADH, 10 IU adenylate kinase, 25 IU pyruvate kinase, and 15 IU of lactate dehydrogenase. ATP/AMP ratio was calculated as the ratio between the intracellular concentration of ATP and AMP, expressed in mM/mg of total protein [ 31 ].…”