Melissa officinalis Protects against Doxorubicin-Induced Cardiotoxicity in Rats and Potentiates Its Anticancer Activity on MCF-7 Cells

Figures

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  Fig 1. (A) Cardio protective effect of MO against DOX-induced cardiac damage, (A) serum cardiac injury markers: AST, CK and CK-MB activities in serum. Data are represented mean ± S.E.M. of seven independent rats of each group. a P<0.05 vs. control; b P<0.05 vs. DOX. (B) Histopathological changes. (B) Histopathological changes. Light micrographs of heart sections from control rats showing normal heart architecture, heart sections from DOX groups (DOX1-DOX4) showing focal necrosis with eosinophilic cytoplasm and pyknotic nuclei (arrows) and degeneration of myocardial fibers (d), hemorrhage (h) with inflammatory cell infiltration () and edema (e), In the DOX + LD of MO group, less focal necrosis of muscle fiber and inflammatory cell infiltration (*) with mild edema (e) were noted on cardiac tissue sections. DOX and MD of MO groups showed less degree of degeneration (d), edema (e) and inflammation (*). Animals treated with DOX and HD of MO showed better-preserved appearance of myocardial fibers with mild degree of (e). X 200.

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  Fig 2. Effect of MO on oxidative stress biomarkers, (A) serum TOC and (B) cardiac MDA and P.Carbonyl levels and (C) antioxidant enzyme activities in DOX-treated rats. Data are represented as mean ± S.E.M. of seven independent rats of each group. a P<0.05 vs. control; b P<0.05 vs. DOX.

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  Fig 3. Effect of MO on inflammation related parameters. (A) Gel photograph depicting representative analysis of NF-κB, COX-2 and TNF-α mRNA expression. Data are expressed as fold change (relative to control group) and as mean ± S.E.M. of three independent rats of each group. (E) Cardiac MPO activity. Data represent as mean ± S.E.M. of seven independent rats of each group. a P<0.05 vs. control; b P<0.05 vs. DOX.

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  Fig 4. Effect of MO on cardiac apoptotic effect of DOX-treated rats. (A) Gel photograph depicting western blot analysis of bax and caspase-3 protein expression in heart of experimental animals. (B) Graphs present the relative expression of bax and caspase-3. Data are expressed as fold change (relative to control group) and as mean ± S.E.M. of seven independent rats of each group. a P<0.05 vs. control; b P<0.05 vs. DOX.

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  Fig 5. Effect of MO, DOX and their combination on viability and apoptosis of MCF-7 cells. (A and B) the effect of MO on the dose-response curve of DOX in MCF-7 cells. Cells were exposed to serial dilution of MO, DOX or combination of MO with DOX for 72h. Cell viability was after 72 h was measured using MTT assay. (C) Quantification of apoptosis induced by MO, DOX and their combination. The cells were treated with IC50 for 48 hrs. a P<0.05 vs. MO; b P<0.05 vs. DOX. Apoptosis was quantified by ELISA as single-stranded DNA. The data are expressed as percentages of control cells. Each point represents the mean ± S.E.M. of three independent experiments. a P<0.05 vs. control; b P<0.05 vs. MO; c P<0.05 vs. DOX; d P<0.05 vs. MO+DOX.

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  Fig 6. Effects of MO and DOX and their combination on pro-apoptotic proteins in MCF-7 cells. The protein expression of (A) Bax, (B) cytochrome c and (C) p53 levels were examined by ELISA in supernatant from MCF7 cells treated with IC50 of MO and DOX for 48 h. The data are expressed as percentages of control cells. Each point represents the mean ± S.E.M. of three independent experiments. a P<0.05 vs. control; b P<0.05 vs. MO; c P<0.05 vs. DOX; d P<0.05 vs. MO+DOX.

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  Fig 7. Effects of MO and DOX and their combination on intracellular redox state in MCF-7 cells. (A) MDA, (B) NO and (C) GSH levels were examined in supernatant from MCF7 cells treated with IC50 of MO and DOX for 48 h. The data are expressed as percentages of control cells. Each point represents the mean ± S.E.M. of three independent experiments. a P<0.05 vs. control; b P<0.05 vs. MO; c P<0.05 vs. DOX; d P<0.05 vs. MO+DOX.

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  Fig 8. (A) ABTS, and (B) DPPH radical scavenging activities of MO ethanol extract and Trolox as reference antioxidant at various concentrations. (C) Representative HPLC phenolic acid profile of MO ethanol extract at 254 nm. Gallic acid (peak 1), caffeic acid (peak2), syringic acid (peak3), ferulic acid (peak4) and rosmarinic acid (peak 5). Values are means ± S.E.M. of three experiments.

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  Fig 9. MO blocks DOX-induced apoptosis, -ROS formation and –necrosis and down regulates the induced inflammation in vivo. In human breast cancer cells (MCF-7), MO improves the anticancer efficacy of DOX and potentiates oxidative damage and apoptosis.