Doxorubicin-Induced Cardiomyopathy: A Preliminary Study on the Cardioprotective Benefits of 7-Hydroxyflavanone

Figure 1. Cytotoxicity screening of 7-Hydroxyflavanone (7H) on the H9c2 cardiomyoblasts. Concisely, cells were treated with various concentrations of 7H for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 4 biological experiments with 6 technical repeats (n = 4). Statistical significance is depicted as * p ≤ 0.05, *** p ≤ 0.001 versus the control.

Figure 2. Cytotoxicity screening of doxorubicin (Dox). (A) Cell viability in H9c2 cells, (B) ATP activity in H9c2 cells and (C) ATP activity in MCF-7 cells. H9c2 cardiomyoblasts and MCF-7 breast cancer cells were treated with varying Dox concentrations (0.25, 0.5, 1 and 2 µM) for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 4 biological experiments with 6 technical repeats (n = 4). Statistical significance is depicted as * p ≤ 0.05 and *** p ≤ 0.001 versus the control.

Figure 3. 7-Hydroxyflavanone (7H) mitigates doxorubicin (Dox)-induced oxidative stress in H9c2 cells. (A) 2,7-dichlorofluorescin diacetate (DCFH-DA, (B) lipid peroxidation, (C) interleukin-6 (IL-6), (D) ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and (E) superoxide dismutase (SOD). The cells were treated with 7H in the absence or presence of Dox, for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 6 technical repeats (n = 3). Statistical significance is depicted as *** p ≤ 0.001 versus the control, and ### p ≤ 0.001 versus Dox.

Figure 4. The effect of 7-Hydroxyflavanone (7H) on impaired mitochondrial bioenergetics. (A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR). H9c2 cells were treated with 7H in the absence or presence of Dox, for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 8 technical repeats (n = 3).

Figure 5. 7-Hydroxyflavanone (7H) ameliorates Doxorubicin-induced losses in (A) ATP activity and (B) mitochondrial membrane potential in H9c2 cells, after 6 days of treatment exposure. H9c2 cells were stained with the JC-1, cationic carbocyanine dye and imaging was performed on a Nikon microscope at a 20× magnification. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 6 technical repeats (n = 3). Statistical significance is depicted as *** p ≤ 0.001 versus the control, and # p ≤ 0.05 and ### p ≤ 0.001 versus Dox.

Figure 6. 7-Hydroxyflavanone (7H) regulates autophagy. (A) Pparg coactivator 1 alpha (PGC-1 α), (B) phosphorylated 5′ adenosine monophosphate-activated protein kinase (pAMPK), (C) phosphorylated mammalian target of rapamycin (pMTOR), (D) Beclin-1, (E) Phosphoinositide 3-kinases (PI3K) and (F) phosphorylated protein kinase B (pAkt). A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 6 technical repeats (n = 3). Statistical significance is depicted as * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 versus the control, and # p ≤ 0.05, ## p ≤ 0.01 and ### p ≤ 0.001 versus Dox.

Figure 7. 7-Hydroxyflavanone (7H) attenuating doxorubicin (Dox)-induced cell death. (A) Bright-field microscopy at 20× magnification, scale bar: 20 μm (B) caspase 3/7 activity, (C) flow cytometry, and Annexin V and propidium iodide staining, and (D) quantification of flow cytometry data. The H9c2 cells were treated with 7H, in the absence or presence of Dox, for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 8 technical repeats (n = 3). Statistical significance is depicted as ** p ≤ 0.01 and *** p ≤ 0.001 versus the control, and # p ≤ 0.05 and ### p ≤ 0.001 versus Dox.

Figure 8. 7-Hydroxyflavanone (7H) attenuating doxorubicin (Dox)-induced mitochondrial damage. (A) Oxidized glutathione (GSSG), (B) reduced glutathione (GSH), (C) total glutathione (ratio of GSH/GSSG), (D) ATP activity, (E) bright-field microscopy at 20× magnification, scale bar: 20 μm and (F) caspase 3/7 activity. The MCF-7 cells were treated with 7H in the absence or presence of Dox, for 6 days. A one-way analysis of variance (ANOVA) and a Bonferroni post hoc test were used to analyze the data, which are presented as the mean ± SEM of 3 biological experiments with 6 technical repeats (n = 3). Statistical significance is depicted as * p ≤ 0.05 and *** p ≤ 0.001 versus the control, ### p ≤ 0.001 versus Dox.

Figure 9. In vitro experimental set-up. Concisely, H9c2 cardiomyoblasts and MCF-7 breast cancer cells were treated every second day for 6 days. The cells were treated or co-treated with 7-Hydroxyflavanone in the absence or presence of Dox. After 6 days of treatment, biochemical assays were conducted.