Omentin protects H9c2 cells against docetaxel cardiotoxicity

Fig 1. Effect of OMT in viability, apoptosis and Caspase 3 expression in H9c2 cells exposed to DTX. (A) Histograms showing cell viability measured by MTT assay in H9c2 cell treated with DTX after 24 h. (B) MTT cell viability assay in H9c2 cells treated with DTX and/or OMT after 24 hours. (C) Annexin V flow cytometry assay on H9c2 cell treated with DTX and/or OMT. (D) Representative density-plots images of Annexin V-FITC/propidium iodide (PI) double staining flow cytometry. (E) Caspase-3 levels in H9c2 cells treated with DTX and/or OMT after 24 hours. (F) Representative western blot autoradiographic images. Data are expressed as mean ± SEM of three or four independent experiments. Statistical significance *, ** and ***p < 0.05, 0.01, and 0.001 vs. control; #, ##, ### p< 0.05, 0.01 and 0.001 DTX-OMT vs. DTX.

Fig 2. Effect of OMT in ROS production and NADPH oxidase (NOX4) expression in H9c2 treated with DTX. (A) ROS levels determined by flow cytometry using DHE staining in H9c2 cells treated with DTX and/or OMT after 24 hours and/or pre-treated with NAC for 1h. (B) Representative histogram images of DHE staining. (C) Histograms showing NOX4 mRNA expression measured by real-time polymerase chain reaction in H9c2 cell treated with DTX after 24 h. Data represent the means ± SEM from at least three independent experiments. Statistical significance *, ** and ***p < 0.05, 0.01, and 0.001 vs. control; #, ##, ### p< 0.05, 0.01 and 0.001 DTX-OMT vs. DTX.

Fig 3. Effect of OMT in CAT, GPx and SOD1 antioxidant enzymes gene and protein expression in H9c2 treated with DTX. mRNA levels of (A) CAT, (B) GPx and (C) SOD1 determined by real-time RT-PCR and protein levels of (D) CAT, (E) GPx and (F) SOD1 determined by Western blot in H9c2 cells treated with DTX and/or OMT after 24 hours. Data represent the means ± SEM from at least three independent experiments. Statistical significance *, ** and ***p < 0.05, 0.01, and 0.001 vs. control; #, ##, ### p< 0.05, 0.01 and 0.001 DTX-OMT vs. DTX.

Fig 4. Effect of OMT in ERS gene and protein expression in H9c2 treated with DTX. mRNA levels of (A) BIP, (B) CHOP, (C) ATF6, (D) XBP1, (E) GADD34 and (F) MCPIP determined by real-time RT-PCR. Protein levels of (G) ATF6α, (H) peIF2α/eIF2α ratio and (I) BIP determined by western blot. All experiments were performed in H9c2 cells treated with DTX and/or OMT after 24 hours. Data represent the means ± SEM from at least three independent experiments. Statistical significance *, ** and ***p < 0.05, 0.01, and 0.001 vs. control; #, ##, ### p< 0.05, 0.01 and 0.001 DTX-OMT vs. DTX.

Fig 5. Proposed mechanism of DTX induced cardiotoxicity: Involvement of ER stress. DTX induces BIP, CHOP, ATF6 expression and reduces GADD34 expression increasing phosphorylated eIF2a levels. Unresolved ER stress will eventually lead to cell death (caspase-mediated apoptosis). Rounded ends indicate inhibitory pathways; arrows indicate stimulatory pathways.