IN VITRO STUDIES ON CYANIDIN PROTECTION AGAINST DOXORUBICIN CARDIOMYOCYTE CYTOTOXICITY AND ANTICANCER ACTIVITY

Abstract

Mitochondrial reactive oxygen species (ROS) are recognized for their role in several health related problems when produced at excessively high concentrations. Due to their potent antioxidant activity and potential mitochondriotropic behavior, the anthocyanidins may have the potential to lower mitochondrial ROS levels. Nevertheless, the effect of anthocyanidins remains overlooked due to their presumed low stability and bioavailability. In addition, this instability has lead to a general belief that the phenolic degradation products, protocatechuic acid (PCA) and phloroglucinaldehye (PGA), exert the bioactivity rather than the parent compound. In this work, doxorubicin-induced cytotoxicity in differentiated H9c2 cardiomyocytes was initially established as a model in which the mitochondrial antioxidant activity of the selected flavonoids would be examined. First, we delineated the mechanisms by which doxorubicin affected H9c2 cell survival and mitochondrial function. The results showed that the early effects of doxorubicin on mitochondrial superoxide generation led to a delayed effect on cell survival. Using this model, we then revealed the protective ability of cyanidin against doxorubicin-induced cytological damage, showing protection to mitochondria. While cyanidin co-incubation with doxorubicin did not show protection when cell survival was assessed after 24 h, it gave delayed protection after a further 24 h drug-free period. Using the delayed protection model, we also showed that cyanidin had greater bioactivity over other flavonoids tested (quercetin, catechin and cyanidin-3-glucoside (C3G)). The protection by cyanidin also exceeded that of its degradation products (PCA and PGA), suggesting that the parent compound has additional bioactivity. The cytoprotective ability of the flavonoids was related to their ability to lower mitochondrial superoxide at early time points, with cyanidin being the most effective. Experiments on doxorubicin cytotoxicity to HepG2 (liver cancer) and K562 (erythroleukemia) cells showed no protective effect with cyanidin. These results suggest cyanidin protects cardiomyocytes but does not interfere with the cytotoxic activity of doxorubicin in the cancer cell lines. Investigations on the degradation of cyanidin in physiological media, UV-vis, HPLC and MS analytical techniques provided evidence that cyanidin does not degrade immediately to PCA and PGA. Instead, intermediate compounds (hemiketal and chalcone) survived for sufficient periods to exert putative bioactivity. Studies on the influence of different media on the degradation of cyanidin showed that the stability in human serum was significantly higher (t½ 43.2 min at room temperature, 22 ¬± 1°C) compared to phosphate buffered saline and Dulbecco’s Modified Eagle’s Medium with and without 10% fetal bovine serum (t½ 10.2-32.6 min). In conclusion, using differentiated H9c2 cells, our results show an ability of cyanidin to survive long enough in cell culture media, and presumably intracellularly, to exert cytoprotection against doxorubicin which exceeded that of other flavonoids (quercetin, catechin, C3G) and its degradation products (PCA and PGA). The results present cyanidin as a possible antioxidant choice to use in clinical practice to protect the heart from the mitochondrial toxicity of doxorubicin and warrants investigation into this possible therapeutic application.