The effects of Mitoquinone on simulated ischemia/reperfusion injuries in H9c2 cells

Introduction: Reperfusion to an ischemic myocardium could result in damage termed myocardial ischemia/reperfusion (I/R) injury. Mitochondrial dysfunction is a major factor in I/R injury, producing less ATP and generating more reactive oxygen species (ROS). Mitoquinone (MitoQ) is an antioxidant that highly accumulates in the mitochondria. However, the dose-response effects and underlying mechanisms of MitoQ on simulated I/R injury have not been well established. Objectives: We hypothesized that H9c2 myoblast cells would be damaged by simulated I/R. Moreover, MitoQ would attenuate myocardial injury, characterized by increased cell viability, compared to non-treated control. Methods: The H9c2 myoblast cells (less than 20 passages) were treated with or without various concentrations of MitoQ (0.005, 0.05, 0.1, 0.5, 1, 2, 5 μM) under 3 different mediums: normal (containing 4.5 g glucose and pyruvate), low glucose (containing 1 g glucose and pyruvate), and no glucose/pyruvate medium. Three different experiments were conducted on the cells. The first experiment aimed to determine if MitoQ alone exerts different effects under different medium conditions by treating the cells with MitoQ for 24 hrs in a normal incubator. The second experiment aimed to determine if MitoQ increased cell viability under simulated ischemia conditions after MitoQ pretreatment. The third experiment aimed to determine if MitoQ increased cell viability under simulated I/R conditions after MitoQ pretreatment. Cell viability was measured by absorbance at 450 nm after adding a cell counting agent. The change in cell viability was expressed as ratios relative to the untreated controls. Results: Low concentrations of MitoQ alone slightly increased cell viability in all three mediums. The maximum increased cell viability was 1.25 ± 0.07 (n=9) at 0.005 μM MitoQ in the normal medium, 1.35 ± 0.23 (n=5, p MitoQ pretreatment exerts protection to cells in simulated ischemia conditions at certain MitoQ concentrations. The maximum increased cell viability was 1.37 ± 0.3 (n=4) at 0.01 μM MitoQ in normal medium, 1.20 ± 0.13 (n=4) at 1.0 μM MitoQ in low glucose medium, and 1.45 ± 0.24 (n=3) at 0.1 μM MitoQ in no glucose medium compared to the untreated control. MitoQ effects on simulated I/R injury will be reported in the future. Discussion: Preliminary data shows the effects of MitoQ alone and MitoQ pretreatment in ischemic conditions on cell viability is influenced by different mediums and concentrations of MitoQ

Comparison of the inhibition of an OCT3 transporter inhibitor, Nilotinib, on Doxorubicin’s effects on cardiac and cancer cell lines

Introduction Doxorubicin (DOX)-induced cardiotoxicity remains a significant barrier limiting its clinical application due to a lack of effective resolution. Targeting how DOX enters cardiac and cancer cells is a promising new strategy. Research suggests that an OCT3 transporter significantly contributes to DOX entry into the heart tissue. By contrast, it expresses much lower on breast cancer cell lines. Moreover, Nilotinib (NIB) can suppress OCT3 transporter function by 80%. Therefore, exploring the impact of NIB on the DOX’s effects on cardiac and cancer cell lines by altering DOX intracellular accumulation is intriguing. Objective First, we would establish a dose-response curve of DOX and NIB alone to assess their individual effects on cell viability. Secondly, we would record the impact of NIB on DOX entry within cardiac myoblasts (H9C2) and breast cancer cells (MCF7) through OCT3 transporter antagonism to assess if NIB can exert cardioprotective effects while maintaining DOX’s anticancer effect. Methods H9C2 myoblast and MCF7 breast cancer cells were seeded in 96-well black plates. Cells were treated with only DOX or NIB to establish a dose-response curve. Moreover, NIB was combined with DOX as a cotreatment or pretreatment regimen to evaluate the impacts of NIB on DOX’s effect. Titrated combinations of NIB (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 2 µM, 5 µM) and DOX (10 µM and 40µM) were used. Bioassays were conducted after cells were treated for 24 hours. Intracellular DOX fluorescence intensity was measured at 488/590 nm by fluoroskan. Subsequently, cell viability was detected by measuring absorbance at 450 nm after adding a cell counting reagent. The data were expressed as a ratio relative to untreated or the DOX control. Results DOX dose-dependently reduced viability of H9c2 and MCF7 cells. H9c2 cell showed significantly lower cell viability at 1 µM (0.86±0.04, n=10, p\u3c0.05) and 40 µM (0.40±0.02, n=10, p\u3c0.05) when compared to those of MCF7 cells (1.07±0.05 and 0.68±0.08 for 1 µM and 40 µM, respectively, n=7). By contrast, NIB (10 nM-2 µM) only slightly increased cell viability to 1.13±0.05 (n=11) in H9c2 cells and to 1.16±0.13 (n=7) in MCF7 cells, respectively, when compared to untreated control. The highest tested dose of NIB (5 µM) showed a similar reduction of cell viability to 0.83±0.07 in H9c2 cells and to 0.81±0.10 in MCF7 cells. Furthermore, NIB cotreatment mitigated DOX-induced damages in H9c2 by increasing cell viability to 1.28±0.07 (n=5) and 1.26±0.11 (n=7) when compared to the DOX controls (10 µM and 40µM), respectively. Interestingly, NIB cotreatment enhanced DOX’s anti-cancer effects in by decreasing MCF7 cell viability to 0.66±0.10 (n=7) and 0.70±0.09 (n=6) when compared to the DOX controls (10 µM and 40µM), respectively. The intracellular DOX fluorescence data and NIB pretreatment results are still being gathered. Conclusion DOX, not NIB, dose-dependently induced H9c2 and MCF7 cell death. Moreover, DOX-induced damage was more potent in H9c2 cells than in MCF7 cells. NIB cotreatment mildly protected H9c2 cells against DOX, whereas it increased DOX’s anti-cancer effects in MCF7 cells