Protective Effect of Secretory APE1/Ref-1 on Doxorubicin-Induced Cardiotoxicity via Suppression of ROS and p53 Pathway

Figures

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  Figure 1. Visual representation of the experimental strategies to develop the doxorubicin‐induced cardiotoxicity models. (A) Establishment of DOX‐induced cardiotoxicity model in vitro. H9C2 cells were treated with conditioned media of HEK293T cells that were transfected by 500 multiplicity of infection of Ad‐PPTLS‐APE1/Ref‐1 for 48 h. After 24 h of treatment with the conditioned media, the cells were treated with 2 μM DOX. (B) Establishment of DOX‐induced cardiotoxicity model in vivo. Eight‐week‐old C57BL/6 mice (n = 16) were transfected with Ad‐APE1/Ref‐1 (5 × 109 virus particles), or Adβ‐Gal. After 24 h, a single dose of DOX (15 mg/kg) was administered intraperitoneally. Normal saline was injected into the control group. The mice were sacrificed after 48 h of DOX treatment for analysis.

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  Figure 2. Expression of secretory APE1/Ref‐1 and redox activity in the conditioned media containing HEK293T cells transfected with Adβ‐Gal or Ad‐PPTLS‐APE1/Ref‐1. (A) Western blot analysis of the cell lysate (first row) and supernatant (second row) of HEK293T cells after transfection. (B) Quantification of secretory APE1/Ref‐1 in the conditioned media using ELISA showing increased secretory APE1/Ref‐1 concentration. (C) The redox activity of secretory APE1/Ref‐1 in the conditioned media. The fluorescence level of cells treated with Ad‐PPTLS‐APE1/Ref‐1 or β‐gal was measured. A higher fluorescence level was detected in the Ad‐PPTLS‐APE1/Ref‐1‐transfected cells. (D) Thiol activity of secretory APE1/Ref‐1 and β‐gal determined using the 5,5′‐dithio‐bis(2‐nitrobenzoic acid) assay. Higher thiol activity was observed in the Ad‐PPTLS‐APE1/Ref‐1 group. Data show the means ± SEM of three replications/independent experiments. **P < 0.001; *P < 0.05 vs. control (Adβ‐gal).

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  Figure 3. Effects of doxorubicin on the in vitro model evaluated based on the drug concentration and time. (A) Representative images of the microscopic examination of the suspended H9C2 cells treated with different concentrations of DOX (0.2, 0.5, 1.0, 2.0, and 5.0 μM) compared with the control at a magnification of 40× (first row) and 200× (second row). The dose‐dependent effects of DOX on cell viability were evaluated; cell death and morphological changes were observed when the DOX concentration exceeded 2 μM. (B) Western blot analysis of cellular APE1/Ref‐1 and p53 expression depending on the DOX concentration. (C) Quantification of the cellular APE1/Ref‐1 expression shows that the expression level remained unchanged. (D) Quantification of the cellular p53 expression shows a significantly increased p53 level with DOX concentrations greater than 2 μM. (E–G) The time‐dependent effects of DOX on cell viability were evaluated after 12, 24, and 48 h of DOX treatment at concentrations of 1.0, 2.0, or 5.0 μM; cell death was initially induced by DOX concentrations greater than 1 μM within 24 h and consistently induced after 48 h. Data show the means ± SEM of three replications/independent…

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  Figure 4. Secretory APE1/Ref‐1 alleviates doxorubicin‐induced cardiomyocyte damage in vitro and in vivo. (A) Representative images of the microscopic examinations of the suspended H9C2 cells. The disarray of cells with morphological changes and cell death was observed in H9C2 cells treated with DOX (2 μM) while cells pretreated with secretory APE1/Ref‐1 partially recovered cell arrays. (B) Quantification of cell viability using the CCK‐8 assay showed an increased number of viable H9C2 cells with secretory APE1/Ref‐1 treatment, showing an inhibitory effect of DOX‐induced cell death. (C) The level of NT‐proBNP in H9C2 cells decreased with secretory APE1/Ref‐1 treatment in vitro compared with the DOX‐treated group. (D) The serum level of NT‐proBNP decreased with APE1/Ref‐1 overexpression through Ad‐APE1/Ref‐1 transfection in vivo compared with mice treated with a dose of 15 mg/kg DOX (n = 12). Data show the means ± SEM of three replications/independent experiments. **P < 0.001, *P < 0.05 compared with the control.

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  Figure 5. Inhibitory effect of secretory APE1/Ref‐1 on doxorubicin‐induced cell death in vitro and in vivo. (A) Western blot analysis of H9C2 cells show decreased expression of p53, Bax/Bcl‐2, and caspase‐3 after 48 h of secretory APE1/Ref‐1 treatment, indicating reduced apoptotic signalling. (B–D) Quantification of the expression of p53, Bax/Bcl‐2, and caspase‐3. (E–H) Ad‐APE1/Ref‐1 transfection has no effect on the expression of p53, Bax/Bcl‐2, and caspase‐3 in H9C2 cells after 48 h of DOX administration. (I–L) Secretory APE1/Ref‐1 decreased the expression of p53, Bax/Bcl‐2, and caspase‐3 in the homogenized heart tissue of DOX‐treated mice, indicating that secretory APE1/Ref‐1 has an inhibitory effect on DOX‐induced apoptosis in vivo. (M, N) Cellular expression of APE1/Ref‐1 does not change with Ad‐APE1/Ref‐1 transfection in DOX‐treated mice. β‐Actin was used as a loading control in all western blot analyses. Data show the means ± SEM of three replications/independent experiments. **P < 0.001, *P < 0.05 compared with the control.

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  Figure 6. Effect of APE1/Ref‐1 on doxorubicin‐induced ROS generation in H9C2 cells. (A) The 2′,7′‐dichlorofluorescein diacetate assay revealed significantly decreased ROS generation, shown as a reduced area of fluorescence in H9C2 cells pre‐treated with secretory APE1/Ref‐1 (third image). (B) Quantitative measurement of fluorescence intensity further confirmed this finding. The data are expressed as the means ± SEM. **P < 0.001, *P < 0.05 compared to the control.