Cinnamic Acid Derivatives as Cardioprotective Agents against Oxidative and Structural Damage Induced by Doxorubicin

Figures

• 

  Figure 1. Protective effect of compounds 5, 10, and 15 against doxorubicin-induced cytotoxicity (A), oxidative stress (B), and mitochondrial damage (C,D). The study was performed in two cellular models: H9c2 and hiPSC-CMs. Cells were cultured with tested compounds—5, 10, 15 (25 µM), and quercetin (20 µM)—as a reference standard for 5 h. Then, the medium was removed, cells were incubated with doxorubicin (DOX, 5 or 10 µM) for 90 min, and the MTT (A) or ROS-Glo™ H2O2 (B) assays were performed. Values represent the viability of cells in the percentage of control (untreated cells) ± SEM of six replicates of three independent experiments (A) or average RLU (relative light unit) ± SEM of six replicates of three independent experiments (B). The RLU value increases in direct proportion to the level of oxidative stress generated by the cells. Statistical significance (*) was calculated relative to DOX (p < 0.05). The effect of cinnamic acid derivatives and doxorubicin (CA–DOX) co-treatment on mitochondrial morphology was analyzed via fluorescent photomicrographs representing MitoTracker fluorescent staining of H9c2 cells (C). Bar graph representing JC-1 fluorescent mitochondrial depolarization of…

• 

  Figure 2. The protective effects of compounds 5, 10, and 15 against doxorubicin-induced apoptosis. (A) represents the results for H9c2 and (B) for hiPSC-CMs. Cells were seeded into 96-well plates; after 24 h, cells were pre-incubated in the presence of the compounds (5, 10, 15, and QUE) for 24 h, and then DOX was added for the next 24 h. Luminescent detection of caspase-3 and -7 was performed according to the manufacturer’s specifications. Caspase-3/7 activation results are expressed as a percentage (mean value for condition × 100/control) ± SEM, n = 9. Statistical significance (*) was calculated relative to control (p < 0.05) separately for DOX (–) and DOX (+) conditions. Cells were incubated with or without the wide-ranging caspase inhibitor (INH) Z-VAD-FMK at a concentration of 20 µM, in order to indicate the caspase-dependent mechanism of apoptosis.

• 

  Figure 3. Effect of DOX in combination with compounds (5, 10, and 15) on the migration of rat cardiomyocytes in the wound-healing model. Cells were seeded at 1 × 105 per well in 12-well plates and cultivated overnight in order to achieve 100% confluence, in the presence of the examined compound (25 µM). Artificial wounds were made manually with a pipette tip, after which the culture medium was replaced with a fresh one containing additional DOX (0.5 µM). Representative images show the most significant results of the wound-healing assay in vitro at indicated time steps. The area of the wound is outlined in order to be measured. Scale bar is 300 µm (A). Graph presenting the dynamics of wound closure. The average cell-covered area from multiple fields of view is presented with 30-min time steps. Only the most relevant conditions are presented, in order to increase the transparency of the results (B). Complete summary of analyzed conditions. Each bar shows the average cell-covered area within the entire experiment (after 16 h of recording) ± SEM; n > 10; * p < 0.05; statistical significance was determined using the Brown–Forsythe and Welch’s ANOVA tests, along with a post-hoc unpaired t-test…

• 

  Figure 4. Compound 15 protected rat cardiomyocytes’ (H9c2) cytoskeletons from DOX-induced damage. Pictures show the changes in the actin and microtubule cytoskeletons of rat cardiomyocytes (H9c2) preincubated with compound 15 at a concentration of 25 μM for 8 h and then exposed to DOX at a concentration of 0.5 μM for next 16 h. Controls were: cells exposed only to the culture medium (ctrl), cells exposed only to DOX (DOX), and cells exposed only to compound 15. Bar = 50 μm.

• 

  Figure 5. Safety assessment of CA derivatives. Hepatotoxicity analysis was conducted on the HepG2 cell line (A). Cells were incubated in the presence of analyzed compounds for 24 h, then the CytoTox-Glo assay was performed. Graph represents the % of cytotoxicity ± SEM of six replicates of three independent experiments. DOX was used as a reference standard. Statistical significance (*) was calculated relative to control (p < 0.05). ADME-Tox cardiotoxicity was investigated using the hERG-CHO cell line in an automated patch clamp assay performed by Eurofines. The degree of inhibition (%) was obtained by measuring the tail current amplitude, which was induced by a 1-s test pulse to –40 mV after a 2-spulse to +20 mV, before and after incubation with the compounds (5, 15) (the difference in current was normalized to control and multiplied by 100 in orderto obtain the percent of inhibition). Compound E4031 was used as a reference standard. The experiment was performed in triplicate (B). Mutagenicity of compounds 5 and 15 in the absence (–S9) or presence (+S9) of the S9 fraction using the Ames test. Graphs represent the average number of revertants/plate of TA98 and TA100 bacteria strains growing…