Cardioprotective effects of the flavonoid quercetin against doxorubicin-Induced cardiotoxicity in H9c2 cardiomyoblasts

Doxorubicin (DOX) is an effective chemotherapy drug to treat diverse cancers such as breast, ovarian, small cell lung, and leukaemia. However, its use is limited by dose-dependent cardiotoxicity, leading to conditions such as cardiac arrhythmias, cardiomyopathy, and congestive heart failure. The precise mechanism of DOX-induced cardiotoxicity is still unclear but oxidative stress is a pivotal mechanism in DOX-induced cardiotoxicity. DOX causes senescence in cardiac tissues, a key driver of aging and damage-associated loss of tissue homeostasis mediated by oxidative stress. Therefore, attenuation of DOX-induced oxidative stress could be a key strategy in alleviating DOX-induced cardiotoxicity. Accumulative evidence suggests antioxidants such as quercetin (a naturally occurring flavonoid abundant in fruits and vegetables) have beneficial effect in the protection of oxidative stress-induced cell death.

The aim of this study is to investigate the putative cardioprotective effects of antioxidants against DOX and hydrogen peroxide-induced H9c2 cell death.

H9C2 cells were seeded (5000 cells/well) onto 96-well plates and pre-treated with either quercetin (50μM), melatonin (1μM) or in combination for 24 hours. Thereafter, some cells were treated with DOX (10-400 μM), DMSO (10-40%), or hydrogen peroxide (0.25-1 mM) for 2 hours. In some assays, cells were pre-treated with either ferrostatin (50uM) or necrostatin (100μM) for 24 hours before treatment with DOX (200μM) or hydrogen peroxide (500μM) for 2 hours. Cell viability was assayed using the MTT and alamarBlue assays. Statistical analyses were conducted using a one-way ANOVA with Tukey’s post hoc test and data plotted as mean ± SD.

Doxorubicin, hydrogen peroxide and DMSO dose-dependently reduced H9c2 cell viability (n=5 separate experiments,10 wells per treatment group) when compared to untreated control cells (p<0.0001). Doxorubicin (200uM), DMSO (20%) and hydrogen peroxide (500uM) treatment (2 hours) resulted in 55+/-7%, 65 +/- 6% and 19+/- 5.6% cell viability, respectively. However, pre-treatment with quercetin (50μM) completely blocked DOX-cardiotoxicity and attenuated hydrogen-peroxide induced death by increasing cell viability from 19% to 58 +/- 6% (p<0.05). Ferrostatin (50 μM) pretreatment also provided a similar profile of inhibition for DOX and hydrogen-peroxide induced cell death. Necrostatin had no effect on drug-induced cell death. Cells pre-treated with quercetin (50μM), ferrostatin (50μM and necrostatin (50μM) had no significant effect on cell viability (p>0.05).

Simulated oxidative stress (DOX and hydrogen peroxide) induced dose-dependent cell death in h9c2 cells which was attenuated by quercetin and ferrostatin. It is conceivable that a key mechanism of quercetin is via the blockade of ferroptosis an provides a platform as novel natural therapeutics against DOX-induced cardiotoxicity.