BGP-15 Protects against Doxorubicin-Induced Cell Toxicity via Enhanced Mitochondrial Function

Figures

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  Figure 1. Effect of BGP-15 pretreatment on cell viability and LDH release in DOX-induced cardiotoxicity model. (A) Cell viability effects of BGP-15 (with or without 50 μM, 24 h pretreatment) on cardiomyocytes after 12 h (n = 6) and (B) 24 h (n = 8) incubation in the concentration range of 0.1–3 μM DOX. (C) LDH release % of BGP-15 (with or without 50 μM, 24 h pretreatment) on cardiomyocytes with 24 h incubation in the concentration range of 0.1–3 μM DOX, n = 5. Data are presented as mean ± SEM, *, **, ***, and **** represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001.

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  Figure 2. Effect of BGP-15 pretreatment on mitochondrial ROS generation and activation of caspase-3 apoptosis marker in DOX-induced cardiotoxicity. (A) ROS production was measured after MitoSOX Red staining by flow cytometry and expressed as mean ± SEM of MitoSOX Red fluorescence intensity (with or without BGP-15 (50 μM, 24 h pretreatment) on DOX-exposed H9c2 cells in the concentration range of 0.1–3 μM). n = 4. The images were captured using a 63× oil immersion objective lens. Data are presented as mean ± SEM, *, **, and **** represent p < 0.05, p < 0.01, and p < 0.0001, respectively. The significance of differences among groups was evaluated with a one-way analysis of variance (ANOVA) followed by Tukey’s posttest. (B) Representative images of MitoSOX Red staining; the nuclear fluorescence in DOX-treated H9c2 derives from DOX. (C) Analysis of the protein level of cleaved-caspase-3/pro-caspase-3 ratio after the cardiomyocyte cells were exposed to 1 μM DOX for 24 h in the absence or presence of the pretreatment of 50 μM BGP-15 for 24 h by Western blot. Red arrows indicate the bands for cleaced-caspase-3 (17; 19 kDa). Values were normalized to the total protein level and expressed as the…

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  Figure 3. BGP-15 preserves the mitochondrial membrane potential in DOX-induced cardiotoxicity. (A) Effect of BGP-15 on DOX-induced mitochondrial membrane depolarization in H9c2 cells. Cells were exposed to 0.1, 1, and 3 μM DOX for 24 h in the absence or presence of 50 μM BGP-15 pretreatment for 24 h, then stained with JC-1, a membrane potential-sensitive fluorescent dye. Data are presented as mean ± SEM of % of control and ratio of red/green fluorescence intensity. n = 5. Data are presented as mean ± SEM, *, ***, and **** represent p < 0.05, p < 0.001, and p < 0.0001, respectively. (B) Representative images of JC-1 staining. Green channel: JC-1 monomeric form; Red channel: JC-1 aggregated form; Blue channel: DAPI as nucleus staining (the nuclear fluorescence in DOX-treated H9c2 derives from DOX); Merged images. The images were captured using a 63× oil immersion objective lens.

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  Figure 4. Effect of BGP-15 pretreatment and DOX-induced cardiotoxicity on autophagy flux. (A) Analysis of protein level of LC3B-II (B) and p62 after the cardiomyocyte cells were exposed to 1 μM DOX for 24 h in the absence or presence of the pretreatment of 50 μM BGP-15 for 24 h by Western blot. Values were normalized to the total protein level and expressed as the mean ± SEM, n = 14 and 14. The significance of differences among groups was evaluated with a one-way analysis of variance (ANOVA) followed by Tukey’s posttest. ** p < 0.01; *** p < 0.001; **** p < 0.0001, respectively. Red arrows indicate the bands for LC3B-I and LC3B-II. (C) Autophagy flux determined by fluorescent microscopy of LC3B or (D) p62 immunostaining. Cells were exposed to 1 μM DOX for 24 h in the absence or presence of 50 μM BGP-15 pretreatment for 24 h, and 10 μM chloroquine for 18 h, then stained. Representative images show the following: Blue channel: DAPI as nucleus staining (the nuclear fluorescence in DOX-treated H9c2 derives from DOX); Red channel: Lysotracker Red; Green channel: LC3B or p62 immunostaining; Merged images. The images were captured using a 63× oil immersion objective lens.