Resveratrol reverses doxorubicin-resistance and alleviates doxorubicin-induced myocardial injury in hepatocellular carcinoma

Figures

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  FIGURE 1. RES and DOX exert an efficacy-enhancing combined effect in eliminating drug-resistant liver cancer cells. (A–D) HepG2-R (A,B) and SMMC-7721-R (C,D) cells were treated with increasing concentrations of RES, DOX, or their combination (Com) for 24 h (A,C) or 48 h (B,D). Cell viability was evaluated via CCK-8 assay. All in vitro experiments were performed in biological triplicates. Statistical analysis was conducted using one-way ANOVA followed by Tukey´s multiple comparisons test. (E) Microscopic observation of morphological changes in HepG2-R and SMMC-7721-R cells after 24 h treatment with 40 μM RES and/or 2 μM DOX. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 100 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05 vs. Com group.

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  FIGURE 2. RES and DOX inhibit the migratory ability of drug-resistant liver cancer cells. (A,B) Representative scratch wound healing assay images of HepG2-R (A) and SMMC-7721-R (B) cells at 0 h, 24 h, and 48 h after treatment with 40 μM RES, 2 μM DOX, or their combination. (C–F) Quantitative analysis of wound healing rate. All experiments were repeated three biologically independent times. One-way ANOVA with Tukey´s post hoc test was used for statistical analysis. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 100 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05 vs. Com group.

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  FIGURE 3. RES and DOX inhibit the migratory ability of drug-resistant liver cancer cells. (A,B) Representative Transwell migration assay images of HepG2-R (A) and SMMC-7721-R (B) cells after 24 h and 48 h treatment. (C–F) Quantitative statistics of migrated cell numbers. Data were obtained from three biological replicates. Statistical differences were analyzed by one-way ANOVA followed by Tukey´s test. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 50 μm*p < 0.05, **p < 0.01 vs. NC group; #p < 0.05 vs. Com group.

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  FIGURE 4. RES and DOX regulate EMT-related protein expression in drug-resistant liver cancer cells. (A) Representative Western blot images of N-cadherin, E-cadherin, Vimentin, Slug, and Snail. Tubulin was used as a loading control. (B–F) Quantitative analysis of relative protein expression. All Western blot experiments were performed in triplicate biological repeats. One-way ANOVA and Tukey´s multiple comparison test were applied for statistical analysis. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05 vs. Com group.

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  FIGURE 5. RES and DOX jointly enhance apoptosis in drug-resistant liver cancer cells. (A) Representative TUNEL fluorescence staining images of HepG2-R and SMMC-7721-R cells. (B,C) Statistical analysis of cell apoptotic rate. (D) Representative Western blot images of apoptosis-related proteins. (E–H) Quantitative analysis of relative protein levels. All experiments were conducted with three biological replicates. Statistical analysis was performed using one-way ANOVA with Tukey´s post hoc test. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 50 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. Com group.

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  FIGURE 6. RES regulates DOX-induced oxidative stress injury in cardiomyocytes. (A) Representative ROS fluorescence staining images. (B) Cardiomyocyte viability after 24 h treatment. (C) Quantitative analysis of ROS fluorescence intensity. (D,E) Detection of SOD activity and MDA content. All assays were repeated three times biologically. Statistical analysis was performed via one-way ANOVA and Tukey´s multiple comparisons test. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 50 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. Com group.

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  FIGURE 7. RES alleviates DOX-induced cardiomyocyte apoptosis. (A) TUNEL staining images of cardiomyocytes. (B) Statistical analysis of cardiomyocyte apoptotic rate. (C–G) Western blot detection and quantitative analysis of apoptosis-related proteins. All experiments were performed in three independent biological replicates. One-way ANOVA with Tukey´s post hoc test was used for statistical analysis. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 50 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. Com group.

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  FIGURE 8. In vivo anti-tumor and myocardial protective effects of RES combined with DOX in H22 tumor-bearing mice. (A) Dynamic tumor volume curves. (B) Mouse body weight changes. (C–G) Serum CK-MB, LDH, GOT levels; MDA and SOD levels from myocardial tissue homogenates. (H,I) Serum cTnI and BNP levels. (J,K) Myocardial injury score and representative H&E staining images. (L,M) Myocardial CVF and representative Masson’s trichrome staining images. In vivo experiments included six mice per group (biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. NC: Negative control; RES: Resveratrol; DOX: Doxorubicin; Com: RES + DOX. Scale bar: 20 μm *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05 vs. Com group.