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Up-regulation of LOX-1 is implicated in apoptosis in both vascular smooth muscle cells and in endothelial cells. We examined the effects of doxorubicin on LOX-1 expression in H9c2 cardiomyocytes and the role played by LOX-1 up-regulation in doxorubicin-induced apoptosis. Reactive oxygen species (ROS) formation was assessed by DCF flow cytometry. LOX-1 mRNA and protein expression was assessed by RT-PCR and Western blotting. Apoptosis was evaluated by flow cytometry with annexin/PI double staining. Doxorubicin-induced LOX-1 expression in a concentration- and time-dependent fashion. The doxorubicin-induced ROS formation and the LOX-1 expression were significantly attenuated by pre-treatment with antioxidants. By exposing cells that had been pre-treated with doxorubicin to oxidized-LDL, a LOX-1 agonist, in the presence or in the absence of k-carrageenan, a LOX-1 receptor antagonist, we documented that doxorubicin-induced LOX-1 expression plays a role in inducing apoptosis. These findings suggest that LOX-1 up-regulation is redox-sensitive and may contribute to doxorubicin-induced cardiotoxicity.

The clinical use of doxorubicin, a highly active anticancer drug, is limited by its severe cardiotoxic side effects. Increased oxidative stress and apoptosis have been implicated in the cardiotoxicity of doxorubicin. Carvedilol is an adrenergic blocking agent with potent anti-oxidant activity. In this study we investigated whether carvedilol has protective effects against doxorubicin-induced free radical production and apoptosis in cultured cardiac muscle cells, and we compared the effects of carvedilol to atenolol, a beta-blocker with no anti-oxidant activity. Reactive oxygen species (ROS) generation in cultured cardiac muscle cells (H9c2 cells) was evaluated by flow cytometry using dichlorofluorescein (DCF) and hydroethidine (HE). Apoptosis was assessed by measuring annexin V-FITC/propidium iodide double staining, DNA laddering, levels of expression of the pro-apoptotic protein Bax-alpha and the anti-apoptotic protein Bcl-2, and caspase-3 activity. Pre-treatment with carvedilol significantly attenuated the doxorubicin-induced increases in DCF (P < 0.001 compared to cells not pre-treated with carvedilol) and HE (P < 0.01) fluorescence. Doxorubicin increased the fraction of annexin V-FITC-positive fluorescent cells, while pre-treatment with carvedilol reduced the number of positive fluorescent cells (P < 0.01). Doxorubicin-induced DNA fragmentation to a clear ladder pattern, while carvedilol prevented DNA fragmentation. Doxorubicin-induced a fall in mRNA expression of the anti-apoptotic Bcl-2 and an increase in the expression of the pro-apoptotic Bax-alpha. Carvedilol pre-treatment blunted both the decrease of Bcl-2 (P < 0.01) and the increase of Bax-alpha mRNA expression (P < 0.01). Caspase-3 activity significantly increased after the addition of doxorubicin. Concurrently, carvedilol partially inhibited the doxorubicin-induced activation of caspase-3 (P < 0.01). Atenolol did not produce any effect in preventing doxorubicin-induced ROS generation and cardiac apoptosis. Our results suggest that carvedilol is potentially protective against doxorubicin cardiotoxicity by decreasing free radical release and apoptosis in cardiomyocytes.