Apoptosis induction by doxazosin and other quinazoline α1-adrenoceptor antagonists: a new mechanism for cancer treatment?

Natasha Kyprianou 0 1 Taylor B. Vaughan 0 1 Martin C. Michel 0 1 0 M. C. Michel Department of Pharmacology and Pharmacotherapy, Academic Medical Center , Amsterdam, The Netherlands 1 N. Kyprianou Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center and Markey Cancer Center , Combs Building, Room 306, 800 Rose Street, Lexington, KY 40536, USA Doxazosin and related, quinazoline-based 1adrenoceptor antagonists can induce apoptosis in prostate and various other normal, benign, smooth muscle, endothelial and malignant cells. Such apoptosis-inducing effects occur independently of 1-adrenoceptor antagonism and typically require much high concentrations than those required for receptor occupancy. Several studies have invested efforts towards the elucidation of the molecular mechanisms underlying doxazosin-induced apoptosis. These include various tumor cells, cardiomyocytes, endothelial cells and bladder smooth muscle cells. While the high concentrations of doxazosin required to induce apoptosis challenge the use of this and related drugs for clinical optimization of apoptosis induction, such quinazoline structure may represent chemical starting points to develop more potent apoptosis-inducing agents free of 1-adrenoceptor antagonistic action and suitable for cancer treatment with minimal and well-tolerated side effects. - 1-Adrenoceptor antagonists have originally been used for the treatment of arterial hypertension. However, the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the 1antagonist doxazosin provided less protection from cardiovascular events than the diuretic chlorthalidone (ALLHAT Research Group 2000). Additionally, in the Vasodilator Heart Failure Trial 1, prazosin was shown to be associated with greater mortality among heart failure patients vs those treated with other vasodilators (Cohn et al. 1986). Therefore, 1-antagonists are no longer considered to be a firstline treatment in cardiovascular medicine. On the other hand, this class of drugs has later been introduced to treat voiding symptoms attributed to benign prostatic hyperplasia (BPH) where they have become the most widely used option of medical therapy. Initially, the therapeutic success of this drug class was assumed only to be a direct result of 1-adrenoceptor blockade resulting in smooth muscle relaxation within the prostate. In this model, smooth muscle relaxation, in turn, provides relief from obstructive lower urinary tract symptoms caused by the enlarged prostate (Kirby 1995). Since the late 1990s, rapidly growing evidence has mounted to support the paradigm shift concept that certain 1-antagonists not only have action on smooth muscle but also have 1-adrenoceptor-independent effects including apoptosis induction and suppression of tissue vascularity. Early studies in mice showed that doxazosin, an 1adrenoceptor antagonist with a quinazoline-based structure, induced apoptosis in murine prostatic stromal and epithelial cells (Yang et al. 1997). Small retrospective cohort studies in humans provided the initial evidence that the therapeutic benefit of doxazosin in BPH patients may involve an apoptotic action against both prostate stromal and epithelial cells (Kyprianou et al. 1998). A series of subsequent studies have confirmed the proapoptotic activity of quinazoline 1antagonists including doxazosin, terazosin (Chon et al. 1999), and more recently, prazosin (Lin et al. 2007) in normal and malignant prostate cells. While the induction of apoptosis by doxazosin and chemically related compounds was initially shown in prostate cells and most of the subsequent work has also been conducted in such cells, later studies demonstrated doxazosin-induced apoptosis to also occur in a range of other normal and tumor cells, indicating that this may be a global effect that is not cell type-dependent. Such other cell types include cardiac myocytes (Rodriguez-Feo et al. 2000; Eiras et al. 2006) and the H9C2 cell line derived thereof (Yang et al. 2009), vascular endothelial cells (Keledjian et al. 2005), bladder smooth muscle cells (Austin et al. 2004), urothelial cancer cells (Siddiqui et al. 2005), pituitary adenoma cells (Fernando and Heaney 2005), breast cancer cells (Hui et al. 2008), colon cancer cells, and HeLa cells (Gan et al. 2008). One of the most interesting aspects of this effect is the fact that apoptosis induction occurs independently of the 1antagonistic properties of these drugs (Kyprianou and Benning 2000; Anglin et al. 2002). Support for this concept emerges from evidence suggesting a significantly increased apoptotic index in prostatic cells exposed to doxazosin and terazosin (quinazoline-based 1-antagonists) but not following tamsulosin treatment (a sulfonamide-based 1antagonist). Furthermore, the irreversible 1-antagonist phenoxybenzamine had no effect on the antigrowth action of doxazosin or terazosin (Kyprianou and Benning 2000). Finally, it should be noted that doxazosin and related quinazoline 1-adrenoceptor antagonists block all subtypes of those receptors in the nanomolar range (Michel et al. 1995) whereas micromolar concentrations of these drugs are required to induced apoptosis in vitro (Kyprianou and Benning 2000; Benning and Kyprianou 2002). In some animal studies, the induction of apoptosis in response to doxazosin treatment in vivo was achieved at relatively high concentrations and supratherapeutic doses (Yang et al. 1997; Benning and Kyprianou 2002). Despite the initial reservations regarding this issue, one must recognize that the retrospective human studies have reported quinazolineinduced apoptosis in the prostate upon exposure to therapeutically relevant doses (Kyprianou et al. 1998; Chon et al. 1999). While the reason for this discrepancy is not fully clear, it should be noted that many of those human in vivo studies have largely relied on the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique as an apoptosis marker, which may yield false-positive results in some cases (see below). Moreover, multiple large placebo-controlled studies have failed to detect reduction of prostate size upon long-term treatment with, e.g., doxazosin (McConnell et al. 2003) or alfuzosin (Roehrborn 2006), which would argue against a clinically relevant degree of apoptosis induction upon standard therapeutic doses of these drugs. Several studies have attempted to elucidate the cellular and molecular mechanisms underlying the apoptosis-inducing effects of doxazosin and related quinazolines. While in vitro approaches with cultured cell lines initially demonstrated that at least part of these effects occur directly by an interaction of the quinazolines with their target cells (Anglin et al. 2002; Partin et al. 2003), subsequent studies in more complex models indicate additional indirect effects. For example, some experimental studies have identified anoikis (programme (...truncated)