The Prophylactic Effect of Pinocembrin Against Doxorubicin-Induced Cardiotoxicity in an In Vitro H9c2 Cell Model

Figure 1. The effect of pinocembrin (Pin) and the co-administrative effect of doxorubicin (Dox) plus Pin on cell viability. (A) Cytotoxicity effect of Pin on H9c2 cardiomyoblasts and MCF-7 breast cancer cells. (B) Co-administrative effect of Dox plus Pin on Dox-induced cardiotoxicity. Briefly, H9c2 and MCF-7 cells were exposed to log concentration (0.01–1,000 µM), of Pin for 6 days to attain the most therapeutic dose. Thereafter, chronic cardiotoxicity was induced by subjecting the H9c2 cells to Dox (2 µM) for 6 days. Cells were also co-treated with Dox (2 µM) plus Pin (1 µM) and Dox (2uM) plus Dexrazoxane (Dex) (20 µM) or treated with Pin (1 µM) for the same treatment duration. Untreated cells served as the control. The results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are presented as the mean ± SEM of 5 biological experiments with 3 technical repeats (n=15). Significance is indicated as ap ≤ 0.05, aap ≤ 0.01, versus the H9c2 cells, ###p ≤ 0.001 versus the control and ***p ≤ 0.001 versus Dox.

Figure 2. The effect of pinocembrin (Pin) on doxorubicin (Dox)-induced oxidative stress. (A) ROS production was quantified using a fluorogenic dye (2,7-dichlorofluorescin diacetate (DCFH-DA) which measures hydroxyl and peroxyl radicals. (B) Lipid peroxidation was quantified by measuring the malondialdehyde (MDA) which reacts with thiobarbituric acid (TBA) to generate an MDA-TBA adduct. H9c2 cells were treated with Dox (2 μM), Pin (1 μM), Dox (2 μM) plus Pin (1 μM) and Dox plus Dex (20 μM) for 6 days. The results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are shown as the mean ± SEM of 3 biological experiments with 3 technical repeats (n=9). Significance is indicated as ##p ≤ 0.01, ###p ≤ 0.001 versus the control;**p ≤ 0.01, ***p ≤ 0.001 versus Dox. Relative fluorescence units (RFU). Arbitrary units (AU).

Figure 3. The co-administrative effect of doxorubicin (Dox) plus pinocembrin (Pin) on the antioxidant capacity of H9c2 cells. (A) Superoxide dismutase activity, (B) reduced glutathione (GSH), (C) oxidized glutathione (GSSG) and (D) total GSH content (ratio of GSH/GSSG). H9c2 cells were treated with Dox (2 μM), Pin (1 μM), Dox (2 μM) plus Pin (1 μM) and Dox plus Dex (20 μM) for 6 days The results were analyzed by One-way Anova, student t-tests, or non-parametric tests where applicable. Data are shown as the mean ± SEM of 3 biological experiments with 3 technical repeats (n=9). Significance is indicated as ##p ≤ 0.01, ###p ≤ 0.001 versus the control; *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001 versus Dox.

Figure 4. (A) Reduced glutathione (GSH), (B) Oxidized gutathione (GSSG), (C) Total GSH content (ratio of GSH/GSSG). The co-administrative effect of doxorubicin (Dox) plus pinocembrin (Pin) on total glutathione content. To induce cytotoxicity, MCF-7 breast cancer cells were treated with Dox (2 µM), Pin (1 µM) or co-treated with Dox (2 µM) plus Pin (1 µM) and Dox (2 µM) plus Dex (20 µM) for 6 days. Untreated cells served as the control. The results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are presented as the mean ± SEM of 3 biological experiments with 3 technical repeats (n=9). Significance is indicated as #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 versus the control; **p ≤ 0.01, ***p ≤ 0.001 versus Dox.

Figure 5. Co-treatment with doxorubicin (Dox) plus pinocembrin (Pin) improves mitochondrial respiration. (A) Mitochondrial oxygen consumption rate (OCR) (B) extracellular acidification rate (ECAR), (C) basal respiration, (D) ATP-linked respiration, (E) ATP turnover, (F) maximal respiration and (G) spare respiratory capacity Basal respiration was acquired after subtraction of non-mitochondrial respiration. ATP turnover measured by ATP-linked respiration subtracted from the basal OCR. Maximum respiration calculated by non-mitochondrial respiration subtracted from FCCP-stimulated OCR. Coupling efficiency was calculated as the fraction of basal mitochondrial OCR used for ATP synthesis (ATP-linked OCR/basal OCR). Results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are presented as the mean ± SEM of 3 biological experiments with 6 technical repeats (n=9). Significance is indicated as ##p ≤ 0.01, ###p ≤ 0.001 versus the control; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus Dox.

Figure 6. The co-administrative effect of doxorubicin (Dox) plus pinocembrin (Pin) on the mitochondrial integrity of the H9c2 cardiomyoblasts and MCF-7 breast cancer cells. (A) JC-1 staining and mitochondrial membrane potential (MMP) of H9c2 cells, (B) metabolic activity of H9c2 cells, (C) JC-1 staining and MMP of MCF-7 cells and (D) metabolic capacity of MCF-7. Results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are shown as the mean ± SEM of 3 biological experiments with 4 technical repeats (n=12). Significance is indicated as #p ≤ 0.05, ###p ≤ 0.001 versus the control; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus Dox.

Figure 7. The effect of pinocembrin (Pin) on Doxorubicin-induced apoptosis. (A) Viable, (B) early apoptotic, (C) late apoptotic, and (D) necrotic H9c2 cells. (E) Viable MCF-7 breast cancer cells, (F) early apoptotic, (G) late apoptotic, and (H) necrotic MCF-7 cells. Annexin V and propidium iodide (PI) staining was evaluated using a BD Accuri C6. Upper right quadrant: Demonstrates cells showing Annexin V and PI positive and late apoptotic. Lower right quadrant: Cells that are Annexin V positive but are PI negative and are early apoptotic. Lower left quadrant: Consists of viable cells that are both Annexin V and PI negative. Upper left quadrant: Cells stained PI positive and are considered necrotic. The results were analyzed using One-way Anova, student t-tests, or non-parametric tests where applicable. Data are shown as the mean ± SEM of 3 biological experiments with 3 technical repeats (n=9). Significance is indicated as ### p ≤ 0.001 versus the control;**p ≤ 0.01, ***p ≤ 0.001 versus Dox.

Figure 8. Pinocembrin attenuates Doxorubicin-induced cardiotoxicity. Pinocembrin as adjunct to the chemotherapeutic drug Doxorubicin protects H9c2 cardiomyoblasts from Doxorubicin-mediated oxidative stress and lipid peroxidation by boosting the activity of superoxide dismutase and the ratio of reduced glutathione to oxidized glutathione content. This increase in antioxidant capacity thereby ameliorates the mitochondrial membrane potential of the heart cells and protects against resultant apoptosis.