Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia

Abstract

Fanconi anemia (FA) is a rare genetic disease characterized by a dysfunctional DNA repair and an oxidative stress accumulation due to defective mitochondrial energy metabolism, not counteracted by endogenous antioxidant defenses, which appear down-expressed compared to the control. Since the antioxidant response lack could depend on the hypoacetylation of genes coding for detoxifying enzymes, we treated lymphoblasts and fibroblasts mutated for the FANC-A gene with some histone deacetylase inhibitors (HDACi), namely, valproic acid (VPA), beta-hydroxybutyrate (OHB), and EX527 (a Sirt1 inhibitor), under basal conditions and after hydrogen peroxide addition. The results show that VPA increased catalase and glutathione reductase expression and activity, corrected the metabolic defect, lowered lipid peroxidation, restored the mitochondrial fusion and fission balance, and improved mitomycin survival. In contrast, OHB, despite a slight increase in antioxidant enzyme expressions, exacerbated the metabolic defect, increasing oxidative stress production, probably because it also acts as an oxidative phosphorylation metabolite, while EX527 showed no effect. In conclusion, the data suggest that VPA could be a promising drug to modulate the gene expression in FA cells, confirming that the antioxidant response modulation plays a pivotal in FA pathogenesis as it acts on both oxidative stress levels and the mitochondrial metabolism and dynamics quality.

Keywords: aldehyde dehydrogenase; antioxidant defenses; catalase; energy metabolism; glutathione reductase; histone deacetylase inhibitors; lipid peroxidation; oxidative phosphorylation.

Figure 1

Modulation of catalase and glutathione reductase expression and activity in FA lymphoblasts treated with VPA, OHB, or EX527. (A) Catalase (CAT) gene expression; (B) glutathione reductase (GR) gene expression; (C) CAT activity; (D) GR activity. In each panel, the effects were evaluated in lymphoblasts after 3 and 24 h from VPA, OHB, or EX527 addition. Data are reported as mean ± SD, and each graph is representative of 6 independent experiments. Statistical significance was tested with a one-way ANOVA. **, ***, and **** represent a significant difference for p < 0.01, 0.001, or 0.0001, respectively, between FA and FA-corr cells used as control. ### and #### represent a significant difference for p < 0.001 or 0.0001, respectively, between untreated and treated FA cells. °, °°°, and °°°° represent a significant difference for p < 0.05, 0.001, or 0.0001, respectively, between the same treatment at 3 and 24 h.

Figure 2

VPA, OHB, and EX527 effects on energy metabolism parameters and lipid peroxidation in FA lymphoblasts. (A) Oxygen Consumption Rate (OCR); (B) ATP synthesis through F_oF₁ ATP synthase; (C) P/O ratio as an OxPhos efficiency marker. For Panels A-C, pyruvate/malate (P/M) and succinate (Succ) were employed as respiratory substrates. (D) Electron transfer between complexes I and III; (E) ATP/AMP ratio as a cellular energy status marker; (F) cellular lipid concentration; (G) aldehyde dehydrogenase (ALDH) activity; (H) malondialdehyde (MDA) intracellular concentration as a lipid peroxidation marker. In each panel, the effects were evaluated in lymphoblasts after 24 h from the VPA, OHB, or EX527 addition. Data are reported as mean ± SD, and each graph is representative of 6 independent experiments. Statistical significance was tested opportunely with a one-way ANOVA. *, **, ***, and **** represent a significant difference for p < 0.05, 0.01, 0.001, or 0.0001, respectively, between FA and FA-corr cells used as control. #, ##, ###, and #### represent a significant difference for p < 0.05, 0.01, 0.001, or 0.0001, respectively, between untreated and treated FA cells.

Figure 3

VPA, OHB, or EX527 treatment effect on mitomycin survival. The graph shows the mitomycin (MMC) survival rate of FA lymphoblasts treated with VPA, OHB, or EX527. Data are reported as mean ± SD, and each graph is representative of 6 independent experiments. Statistical significance was tested with a one-way ANOVA. * and ** represent a significant difference for p < 0.05 or 0.01, respectively, between FA and FA-corr cells used as control.

Figure 4

VPA modulation of mitochondrial dynamic and uncoupling protein 2 expression in FA primary fibroblasts. (A) WB signals and (B) relative densitometry of DRP1 (mitochondrial fission), MFN2 (mitochondrial fusion), uncoupling protein 2 (UCP2), and Actin (housekeeping protein used for signal normalization) in FA-corr and FA primary fibroblasts treated or not with VPA for 24 h. Each signal was normalized on the actin signal. (C) Confocal imaging of FA-corr and FA fibroblasts stained with antibody against TOM20 (red) and DAPI (blue) to show the mitochondrial reticulum and nuclei, respectively. White scale bars correspond to 10 μm. The higher magnification inserts, corresponding to the area enclosed by the white square, represent an example of mitochondrial network distribution. Data reported in the histogram on the right are expressed as mean ± SD. Each panel is representative of at least 6 independent experiments. Statistical significance was tested with a one-way ANOVA. **** represents a significant difference for p < 0.0001 between FA cells and FA-corr. ### and #### represent a significant difference for p < 0.001 or 0.0001, respectively, between untreated and VPA-treated FA fibroblasts.

Figure 5

Modulation of catalase and glutathione reductase expression and activity in FA-corr and FA lymphoblasts treated with VPA, OHB, or EX527 after the hydrogen peroxide addition. In each graph, 0.5 mM hydrogen peroxide was added to induce oxidative insult. (A,B) Catalase (CAT) expression after 3 h and 24 h from VPA, OHB, or EX527 addition. (C,D) Glutathione reductase (GR) expression after 3 h and 24 h from VPA, OHB, or EX527 addition. (E,F) Catalase (CAT) activity after 3 h and 24 h from VPA, OHB, or EX527 addition. (G,H) Glutathione reductase (GR) activity after 3 h and 24 h from VPA, OHB, or EX527 addition. Data are reported as mean ± SD, and each graph is representative of 6 independent experiments. Statistical significance was tested with a one-way ANOVA. **** represents a significant difference for p < 0.0001 between FA and FA-corr cells in the same treatment condition. ## and #### represent a significant difference for p < 0.01 or 0.0001, respectively, between untreated and VPA-, OHB-, or EX537-treated samples. §, §§, and §§§ represent a significant difference for p < 0.05, 0.01, or 0.001, respectively, between H₂O₂-treated and untreated samples.

Figure 6

VPA, OHB, and EX527 effects on energy metabolism parameters and lipid peroxidation in FA-corr and FA lymphoblasts after hydrogen peroxide addition. In each graph, 0.5 mM hydrogen peroxide was added to induce oxidative damage. (A) Oxygen Consumption Rate (OCR) in the presence of pyruvate/malate (P/M). (B) ATP synthesis through F_oF₁ ATP synthase in the presence of P/M. (C) P/O ratio in the presence of P/M as an OxPhos efficiency marker. (D) Oxygen Consumption Rate (OCR) in the presence of succinate (Succ). (E) ATP synthesis through F_oF₁ ATP synthase in the presence of Succ. (F) P/O ratio in the presence of Succ as an OxPhos efficiency marker. (G) Electron transfer between complexes I and III. (H) ATP/AMP ratio as a cellular energy status marker. (I) Cellular lipid concentration. (J) Aldehyde dehydrogenase (ALDH) activity. (K) Malondialdehyde (MDA) intracellular concentration as a lipid peroxidation marker. In each panel, the effects were evaluated in lymphoblasts after 24 h from VPA, OHB, or EX527 addition. Data are reported as mean ± SD, and each graph is representative of 6 independent experiments. Statistical significance was tested opportunely with a one-way ANOVA or two-way ANOVA. *, **, and **** represent a significant difference for p < 0.05, 0.01, or 0.0001, respectively, between FA and FA-corr cells in the same treatment condition. #, ##, ###, and #### represent a significant difference for p < 0.05, 0.01, 0.001, or 0.0001, respectively, between untreated and VPA-, OHB-, or EX537-treated samples. §, §§, and §§§§ represent a significant difference for p < 0.05, 0.01, or 0.0001, respectively, between H₂O₂-treated and untreated samples.