Doxazosin induces apoptosis in cardiomyocytes cultured in vitro by a mechanism that is independent of α1-adrenergic blockade

Cardiology Research Unit, University Clinical Hospital, Santiago de Compostela, Spain.
Circulation (Impact Factor: 14.43). 02/2003; 107(1):127-31. DOI: 10.1161/01.CIR.0000043803.20822.D1
Source: PubMed


The alpha1-adrenoceptor-blocking antihypertensive doxazosin has been associated with increased risk of heart failure and is known to induce prostate cell apoptosis. We hypothesized that it might also induce apoptosis in cardiomyocytes.
Hoechst dye vital staining and flow cytometry provided evidence that doxazosin induced apoptosis time- and dose-dependently in cardiomyocytes of the HL-1 cell line. TUNEL assays and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability test confirmed that doxazosin induced DNA damage and cell death in these cells. MTT tests showed that doxazosin treatment decreased cell viability in primary cultures of neonatal rat cardiomyocytes, and Hoechst dye vital staining demonstrated doxazosin-induced apoptosis in primary cultures of human adult cardiomyocytes. The proapoptotic effect of doxazosin on cardiomyocytes seems not to depend on alpha1 blockade, because it was not modified by cotreatment with alpha- or beta-adrenergic agonists or with the irreversible alpha1-blocker phenoxybenzamine and because doxazosin also decreased the viability of NIH 3T3 cells, which lack alpha1-adrenoceptors. It also does not involve calcineurin, being unaffected by the presence of the calcineurin inhibitors cyclosporin A and FK506. Three other alpha1-blockers were also investigated; prazosin was proapoptotic, like doxazosin, but 5-methylurapidil and terazosin were not.
The alpha1-blockers doxazosin and prazosin induce the apoptosis of cardiomyocytes cultured in vitro by a mechanism that is independent of alpha1 blockade and calcineurin.

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Available from: Roberto Piñeiro, Jun 21, 2014
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    • "Moreover, hERG channels control cell proliferation and apoptosis [12]. Targeting of hERG channels by the small molecule α1-adrenoceptor antagonist doxazosin induces apoptosis in vitro independent of its anti-adrenergic function [13]–[15]. This pro-apoptotic mechanism of action was extended to structurally unrelated compounds, suggesting broader significance [11], [16]. "
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    ABSTRACT: Glioblastoma (GB) is associated with poor patient survival owing to uncontrolled tumor proliferation and resistance to apoptosis. Human ether-a-go-go-related gene K(+) channels (hERG; Kv11.1, KCNH2) are expressed in multiple cancer cells including GB and control cell proliferation and death. We hypothesized that pharmacological targeting of hERG protein would inhibit tumor growth by inducing apoptosis of GB cells. The small molecule hERG ligand doxazosin induced concentration-dependent apoptosis of human LNT-229 (EC50 = 35 µM) and U87MG (EC50 = 29 µM) GB cells, accompanied by cell cycle arrest in the G0/G1 phase. Apoptosis was associated with 64% reduction of hERG protein. HERG suppression via siRNA-mediated knock down mimicked pro-apoptotic effects of doxazosin. Antagonism of doxazosin binding by the non-apoptotic hERG ligand terazosin resulted in rescue of protein expression and in increased survival of GB cells. At the molecular level doxazosin-dependent apoptosis was characterized by activation of pro-apoptotic factors (phospho-erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2, phospho-p38 mitogen-activated protein kinase, growth arrest and DNA damage inducible gene 153, cleaved caspases 9, 7, and 3), and by inactivation of anti-apoptotic poly-ADP-ribose-polymerase, respectively. In summary, this work identifies doxazosin as small molecule compound that promotes apoptosis and exerts anti-proliferative effects in human GB cells. Suppression of hERG protein is a crucial molecular event in GB cell apoptosis. Doxazosin and future derivatives are proposed as novel options for more effective GB treatment.
    PLoS ONE 02/2014; 9(2):e88164. DOI:10.1371/journal.pone.0088164 · 3.23 Impact Factor
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    • "Because of these attributes, even though HL-1 cells were originally derived from atrial myocytes, they have proven to be useful as a general model for studying contracting (working) cardiomyocytes because of their organized structure and ability to contract in culture [31]. They have been used as a cell culture model in numerous studies including apoptosis [58], [59], [60], [61], cell-cycle [15], electrophysiology [62], [63], [64], oxidative stress [65], [66], signal transduction [67], [68], [69], transcriptional regulation [65], [66], [70] and cellular transplantation [71]. However, it should be noted, that the results obtained are in adult cardiomyocytes and may differ for pre-natal or post natal myocytes. "
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    ABSTRACT: Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice. Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy. Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.
    PLoS ONE 06/2013; 8(6):e66789. DOI:10.1371/journal.pone.0066789 · 3.23 Impact Factor
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    • "Apoptosis was studied in an established model of mouse atrial cardiac tumour cells (HL-1 cells) exposed to doxazosin (González-Juanatey et al., 2003; Eiras et al., 2006). The antihypertensive a 1-adrenoceptor antagonist doxazosin is implicated in HF and triggers apoptosis in cardiomyocytes and in experimental cells, independent of its antagonism of adrenoceptors (ALLHAT Trial Investigators, 2000; González-Juanatey et al., 2003; Thomas et al., 2008; Kyprianou et al., 2009; Staudacher et al., 2011). "
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    ABSTRACT: Background and purpose: Heart failure and atrial fibrillation are associated with apoptosis of cardiomyocytes, suggesting common abnormalities in pro-apoptotic cardiac molecules. Activation of the receptor tyrosine kinase EphA2 causes apoptosis in vitro, and dysregulation of EphA2-dependent signalling is implicated in LEOPARD and Noonan syndromes associated with cardiomyopathy. Molecular pathways and regulation of EphA2 signalling in the heart are poorly understood. Here we elucidated the pathways of EphA2-dependent apoptosis and evaluated a therapeutic strategy to prevent EphA2 activation and cardiac cell death. Experimental approach: EphA2 signalling was studied in an established model of doxazosin-induced apoptosis in HL-1 cells. Apoptosis was measured with TUNEL assays and as cell viability using a formazan method. Western blotting and siRNA for EphA2 were also used. Key results: Apoptosis induced by doxazosin (EC(50) = 17.3 μM) was associated with EphA2 activation through enhanced phosphorylation (2.2-fold). Activation of pro-apoptotic downstream factors, phospho-SHP-2 (3.9-fold), phospho-p38 MAPK (2.3-fold) and GADD153 (1.6-fold) resulted in cleavage of caspase 3. Furthermore, two anti-apoptotic enzymes were suppressed (focal adhesion kinase, by 41%; phospho-Akt, by 78%). Inactivation of EphA2 with appropriate siRNA mimicked pro-apoptotic effects of doxazosin. Finally, administration of lithocholic acid (LCA) protected against apoptosis by increasing EphA2 protein levels and decreasing EphA2 phosphorylation. Conclusions and implications: EphA2 phosphorylation and activation of SHP-2 are critical steps in apoptosis. Reduction of EphA2 phosphorylation by LCA may represent a novel approach for future anti-apoptotic treatment of heart failure and atrial fibrillation.
    British Journal of Pharmacology 07/2012; 167(7). DOI:10.1111/j.1476-5381.2012.02117.x · 4.84 Impact Factor
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