Original ArticleCarvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro
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.
Section snippets
Materials
Fetal bovine serum (FBS), phosphate buffered saline (PBS), and cell culture medium were purchased from Euroclone (UK). Bicinchoninic acid (BCA) protein assay was bought from Pierce (Rockford, IL, USA). Hydroethidine (HE) was purchased from Polysciences Inc. (Warrington, PA, USA). Carvedilol was kindly provided by Roche. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), or ICN (Irvine, CA, USA).
Cell culture
Rat heart cell line H9c2 (2-1) was obtained from American Type Culture
Viability test
To study the effects of various concentrations of doxorubicin and carvedilol we first determined the number of viable cells with trypan blue exclusion test. Myocytes exposed to 0.1, 0.5, and 5 μM doxorubicin for 12 h excluded trypan blue similarly to untreated myocytes, while myocytes exposed to 10 μM doxorubicin had an increased uptake of trypan blue, suggesting an early fall in membrane integrity and rapid myocyte degeneration. However, when myocytes were exposed to doxorubicin for a longer
Discussion
The salient results of the study are that carvedilol in doxorubicin-treated myocytes significantly inhibits free radical production, increases the number of viable cells, inhibits apoptotic pathways, prevents DNA fragmentation, and decreases the number of apoptotic cells.
The measurement of oxidative free radical production was carried out by means of two probes with different specificity. DCFH detects the intracellular production of hydrogen peroxide, [27] other hydroperoxides, hydroxyl
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