Vanillin Prevents Doxorubicin-Induced Apoptosis and Oxidative Stress in Rat H9c2 Cardiomyocytes

Figure 1. Effect of doxo and vanillin on cell viability. Cell viability was evaluated by MTT assay in H9c2 cells exposed for 24 h to (A) increasing concentration of doxo (from 0.1 to 20 μM) and to (B) different concentrations of vanillin (20, 50, 100 and 150 μM) UT—untreated cells. Data are expressed as average percentage of MTT reduction ± SD relative to untreated cells from triplicate wells from 5 separate experiments. *: p < 0.05; ***: p < 0.001 by unpaired t-test. Other experimental details are described in Materials and Methods section.

Figure 2. Effect of vanillin on doxo-induced cytotoxicity. Cell viability was evaluated by MTT assay in H9c2 cells exposed to doxo for 24 h, (A) pretreated and (B) co-treated with vanillin at the indicated concentrations. Data are expressed as average percentage of MTT reduction ± SD relative to untreated cells from triplicate wells from 5 separate experiments. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Pretr—vanillin pretreated cells for 18 h; Cotr—vanillin cotreated cells; Van: cells exposed to 150-μM Van (C) Phase contrast microscopy images taken at the end of treatment time with a higher vanillin concentration. **: p < 0.01; ***: p <0.001 by one-way ANOVA. Other experimental details are described in Materials and Methods section.

Figure 3. Effect of vanillin on doxo-induced cell death and nuclear alteration. (A) Dead cells number were evaluated by Trypan Blue assay in H9c2 cells exposed to 20 μM doxo for 24 h, pretreated (Pretr) and co-treated (Cotr) with 100-μM vanillin. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Van—cells exposed to 100-μM Van. Scale bar represents 50 μm. Data represent the average of three independent experiments ± SD; (B) representative fluorescence images of cells stained by DAPI nuclear staining. The fluorescence intensity was estimated by Image J software. *: p < 0.05; **: p < 0.01; ***: p < 0.001 by two-way ANOVA. Other experimental details are described in Materials and Methods section.

Figure 4. Effect of vanillin on doxo-induced sub G1-phase alteration. (A) Representative experiments of PI-stained H9c2 cells exposed to 20-μM doxo for 24 h, pretreated (Pretr) and co-treated (Cotr) with 100-μM vanillin. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Van—cells exposed to 100-μM Van; (B) sub-G1 phase quantification of not less than two. Data represent the average of three independent experiments ± SD. *: p < 0.05; **: p < 0.01 by one-way ANOVA. Other experimental details are described in Materials and Methods section.

Figure 5. Effect of vanillin on caspase 3 and PARP1 activation. (A) Western blot analysis of cleaved caspase-3 (CC3) (A), PARP1 and cleaved PARP1 (c-PARP) (B) in H9c2 cells exposed to 20-μM doxo for 24 h, pretreated (Pretr) and cotreated (Cotr) with 100-μM vanillin. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Van—cells exposed to 100-μM Van. Images are representative of western blotting from two different cellular extract with similar results. Data represent the average of three independent experiments ± SD. **: p < 0.01; ***: p < 0.001 by one-way ANOVA. Other experimental details are described in Materials and Methods section.

Figure 6. Effect of vanillin on doxo-induced ROS production and MAPK signaling. (A) ROS production was evaluated by DCFHDA assay in H9c2 cells exposed to 20-μM doxo for 24 h, pretreated (Pretr) and co-treated (Cotr) with 100-μM vanillin. The data are presented as mean ± SD of three replicates in five independent experiments; (B) western blot analysis of ERK activation. The image is representative of western blotting from two different cellular extract with similar results. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Van—cells exposed to 100-μM Van; H2O2—cells treated with 1-mM H2O2; ** p < 0.01; *** p ˂ 0.001, by one-way ANOVA. Other experimental details are described in Materials and Methods section.

Figure 7. Effect of vanillin on doxo-induced antineoplastic properties in U2OS osteosarcoma cells. Number of living (A) and dead (B) U2OS cells exposed to 20-μM doxo for 24 h, pretreated (Pretr) and cotreated (Cotr) with 100-μM vanillin. UT—untreated cells; doxo—cells exposed to 20-μM doxo; Van—cells exposed to 100-μM Van; * p < 0.05; *** p ˂ 0.001 by one-way ANOVA.