Hydroxytyrosol Prevents Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes

Figures

• 

  Figure 1. Chemical structure of hydroxytyrosol (HT).

• 

  Figure 2. Evaluation of Dox cytotoxicity in cardiomyoblasts. Cell viability was evaluated by MTT assay in H9c2 cells exposed for 24 and 48 h to an increasing concentration of Dox (from 0.1 to 20 μM). CTR: untreated cells. Data are expressed as average percentage of MTT reduction ± SD relative to untreated cells from triplicate wells from 5 separate experiments. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR.

• 

  Figure 3. Effect of HT on Dox-induced cytotoxicity. (A) Cell viability was evaluated by MTT assay in H9c2 cells exposed to 0.1 µM Dox (Dox), and cotreated with 20 (Dox-HT 20), 50 (Dox-HT 50) and 70 (Dox-HT 70) µM HT for 24 and 48 h. Data are expressed as average percentage of MTT reduction ± SD relative to untreated cells (CTR) from triplicate wells from 5 separate experiments. *** p ˂ 0.001 versus CTR, ### p ˂ 0.001 versus Dox. (B) Phase contrast microscopy images (magnification 10×) of H9c2 cells after 48-h incubation. CTR: untreated cells; Dox: cells exposed to 0.1 µM Dox; Dox–HT 50/70: cells cotreated with 50 and 70 µM of HT. Other experimental details are described in the Materials and Methods section.

• 

  Figure 4. Role of HT in Dox-induced oxidative stress. H9c2 cells were exposed to 0.1 µM Dox (Dox) and co-incubated with 50 µM HT (Dox–HT), and both ROS production by DCFH-DA assay (A) and SOD2 expression by western-blot analysis (B) were evaluated. CTR: untreated cells; HT: cells treated with HT only; CTR+: cells treated with 1.0 mM H2O2. Data are expressed as average ± S.D. from five independent experiments carried out in triplicate. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR, ### p ˂ 0.001 versus Dox.

• 

  Figure 5. Role of HT in Dox-induced apoptosis. Western blot analysis of Bcl2 and Bax in H9c2 cells exposed to 0.1 μM Dox (Dox) and co-treated with 50 μM HT (Dox-HT). CTR: untreated cells. The images are representative of Western blotting from three different cellular extracts with similar results. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR, ### p ˂ 0.001 versus Dox.

• 

  Figure 6. Role of HT in Dox-induced apoptosis. Western blot analysis of cleaved caspase-3 (C-C3) and phosphorylated p38 (p-p38) in H9c2 cells exposed to 0.1 μM Dox (Dox) and co-treated with 50 μM HT (Dox-HT). CTR: untreated cells. The p38 phosphorylation was quantified through the evaluation of the p-p38/p38 ratio. The images are representative of Western blotting from three different cellular extract with similar results. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR, ### p ˂ 0.001 versus Dox.

• 

  Figure 7. Role of HT in Dox-induced DNA damage. Western blot analysis of phospho-histone H2AX (γ-H2AX) in H9c2 cells exposed to 0.1 μM Dox (Dox) and co-treated with 50 μM HT (Dox-HT). CTR: untreated cells. The images are representative of Western blotting from three different cellular extracts with similar results. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR, ### p ˂ 0.001 versus Dox.

• 

  Figure 8. Effect of HT on Dox-induced antineoplastic properties in U2OS osteosarcoma cells. Number of living (A) and dead (B) cells for U2OS cells exposed to 0.1 μM Dox for 48 h, pretreated (Dox-HT pre) and co-treated (Dox-HT) with 50 μM HT. CTR: untreated cells; Dox: cells exposed to 0.1 μM Dox; Dox–HT pre: HT pretreated cells for 18 h; Dox–HT: HT co-treated cells. HT: cells exposed to 50 μM HT. Other experimental details are described in the Materials and Methods section. *** p ˂ 0.001 versus CTR.

• 

  Figure 9. Working hypothesis on the role of HT in Dox-induced cardiotoxicity.