Carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro

Abstract

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 β-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-α 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-α. Carvedilol pre-treatment blunted both the decrease of Bcl-2 (P < 0.01) and the increase of Bax-α 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.

Introduction

Doxorubicin is the mainstay of treatment of a variety of hematological malignancies and solid tumors. Unfortunately, the clinical use of this drug is limited by cumulative dose-related cardiotoxicity which may ultimately lead to a severe and irreversible form of cardiomyopathy [1], [2]. In an effort to improve the therapeutic index of doxorubicin several preventive measures are recommended. Firstly, to limit the lifetime cumulative dose. Secondly, to modify the methods of administration in order to prevent high peak serum concentrations [3]. Thirdly, to adjust the doxorubicin total dose, or to avoid it altogether, in relation to risk factors for cardiotoxicity (co-existing heart disease, irradiation of mediastinum, hypertension, inappropriate nutrition, diabetes, younger or older age, and female gender) [4]. Finally, to periodically perform cardiac examinations and discontinue therapy when significant anatomical or functional changes are detected [5]. It goes without saying that due to these limitations several patients are precluded from receiving a highly effective treatment.

An alternative approach is the use of cardioprotective drugs. Agents which prevent cardiotoxic effects would allow us to exploit the full therapeutic potential of doxorubicin, with a tremendous impact on cancer therapy. The supposed mechanism of doxorubicin-induced cardiotoxicity is the redox activation to a semi-quinone intermediate and the formation of reactive oxygen species (ROS) which ultimately results in myocyte apoptosis (or programmed cell death) [6]. Scavengers of free radicals can inhibit doxorubicin-induced cardiomyocyte apoptosis, suggesting that ROS are involved in apoptotic cell death [7]. However, several attempts to prevent doxorubicin-induced cardiotoxicity by using anti-oxidants have produced conflicting results [8], [9], [10], [11], [12].

Carvedilol is a non-selective β-blocker initially used in the treatment of hypertension and angina, and which has more recently become the backbone for the treatment of patients with congestive heart failure. By means of a comprehensive adrenergic blockade (β₁, β₂, and α), carvedilol may provide greater protection of the heart against the deleterious consequences of sympathetic activation than selective, conventional adrenergic blockade does [13]. Carvedilol also acts as a potent anti-oxidant (it is approximately 10 times more potent than α-tocopherol) and is unique among β-blockers in this respect [14]. In physicochemical, biochemical, and cellular assays carvedilol and several of its metabolites inhibit the formation of reactive oxygen radicals and lipid peroxidation, scavenge oxygen free radicals, and prevent the depletion of endogenous anti-oxidants, such as vitamin E and glutathione [15], [16]. Carvedilol also prevented cardiomyocyte apoptosis in an experimental model of ischemia/reperfusion [17].

Carvedilol is already indicated in the treatment of clinically documented doxorubicin-induced cardiomyopathy to halt the further decline in left ventricular function, to ameliorate symptoms, and to improve prognosis [18]. Cardiotoxicity is known to occur cumulatively, starting with the first dose of treatment [19], while symptoms of congestive heart failure develop late in the course of the natural history of doxorubicin cardiomyopathy and are often preceded by a progressive, asymptomatic, dose-related impairment of ventricular function. We think it is of interest to examine whether carvedilol has therapeutic potential not only in advanced doxorubicin cardiomyopathy, but also at the beginning of chemotherapy, in an attempt to prevent myocardial damage. With this aim in mind we investigated whether carvedilol has protective effects against doxorubicin-induced free radical production and apoptosis in cultured cardiac muscle cells.