title: 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 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.
authors.affiliation: Laboratory of Cardiovascular Biology, Division of Cardiology, Department of Internal Medicine, University of Genova, Viale Benedetto XV, 6 Genova 16132, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Spallarossa
authors.forename: Silvano
authors.initials: S
authors.lastname: Garibaldi
authors.forename: Paola
authors.initials: P
authors.lastname: Altieri
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fabbi
authors.forename: Valeria
authors.initials: V
authors.lastname: Manca
authors.forename: Sabina
authors.initials: S
authors.lastname: Nasti
authors.forename: Pierfranco
authors.initials: P
authors.lastname: Rossettin
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ghigliotti
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
authors.forename: Franco
authors.initials: F
authors.lastname: Patrone
authors.forename: Antonio
authors.initials: A
authors.lastname: Barsotti
authors.forename: Claudio
authors.initials: C
authors.lastname: Brunelli
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000037:antagonists & inhibitors* / Q000633:toxicity; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D001262:Atenolol / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology*; D000077261:Carvedilol; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004317:Doxorubicin / Q000037:antagonists & inhibitors* / Q000633:toxicity; D005609:Free Radicals / Q000378:metabolism*; D015870:Gene Expression; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D011412:Propanolamines / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D051028:bcl-2-Associated X Protein
chemical_list: D000903:Antibiotics, Antineoplastic; D000975:Antioxidants; C494828:Bax protein, rat; D002227:Carbazoles; D005609:Free Radicals; D011412:Propanolamines; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D000077261:Carvedilol; D001262:Atenolol; D004317:Doxorubicin; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases