*3.2. Bladder Cancer*

When evaluating antitumor activity of α1 blockers in terms of cell viability, cell cycle progression, competition, and apoptotic signaling in bladder cancer, Nakagawa et al. (2016) showed that naftopidil was one of the strongest antitumor α1-adrenoceptor antagonists [45]. Significantly enough, oral administration of naftopidil reduced tumor volume in a xenograft model in a concentration (10–100 μmol/L)-dependent manner, suggesting promising outcomes of α1 blockers in bladder cancer treatment [16]. To a lesser extent, prazosin has been shown to reduce survival of human bladder cancer cells at concentrations more than 30 μmol/L [14]. Terazosin, proven to induce apoptosis in prostate cancer cells, reduced tumor vascularity and induced apoptosis in transitional cell carcinoma (TCC) of the bladder in a retrospective case-control study using a pathological examination of specimens from patients undergoing radical cystectomy (Table 1) [49]. An independent retrospective observational study of 27,183 men confirmed these results and found that those treated with the quinazoline based adrenoceptor antagonists terazosin and doxazosin had a 43% lower relative risk of developing bladder cancer than unexposed men [50].

#### *3.3. Colorectal Cancer*

Epidemiological evidence from case-control studies enabled promising insights into the use of doxazosin as therapeutic and a chemopreventive strategy in treating colorectal cancer. An in vitro case-control study found that the α1 adrenoceptor antagonist, doxazosin significantly suppressed the proliferation of RKO colon cancer cell lines within human colorectal cancer cell assays. Recent pre-clinical studies demonstrated in vivo treatment of mice harboring colon cancer xenografts with doxazosin resulted in a significant decrease in tumor numbers and size compared

to control untreated mice [46]. While limited, these results support the ongoing pursuit of the use of α1-adrenoreceptors antagonists in cancer treatment.

#### *3.4. Adrenal Cancer*

While not directly related to the genitourinary system as the other malignancies we have discussed, it is important to address the recent discoveries of the effect of α1-adrenoreceptors antagonists on adrenal cancer, specifically pheochromocytoma. While limited, there are promising preliminary results in the anti-adrenergic effects of α1-blockade in managing unchecked catecholamine production in pheochromocytoma. High circulating catecholamine levels stimulate alpha receptors on blood vessels, thereby causing vasoconstriction and increased total peripheral resistance. Thus, α-adrenergic blockade helps control blood pressure and prevent hypertensive crisis in the preoperative setting of surgical resection for metastatic pheochromocytomas [51]. While randomized controlled trials are lacking, a literature review has shown the effectiveness of doxazosin and phenoxybenzamine in the preoperative treatment of pheochromocytomas; however, further research is needed in better understanding the use of these drugs, especially in combination with β-blockers for preoperative treatment [51,52].

#### **4. Potential Therapeutic Value in Renal Cancer**

Original studies by our group provided initial translational insights into the therapeutic effects of α1-adrenoceptor antagonists in RCC preclinical models [3]. Doxazosin induces apoptosis in cancer cells through similar α1-adrenoreceptor-independent mechanisms as found in human prostate cancer cell models [3]. Molecular assays have demonstrated this quanizoline-based α1-adrenoceptor antagonist induces apoptosis in prostate cancer cells expressing C-Flip, an endogenous inhibitor of FADD-mediated activation, and subsequently cleaving caspase-8 [3]. As illustrated on Figure 1, doxazosin also induces apoptosis in renal cancer cells through activation of TGF-β1 signaling via Smad effector phosphorylation and targeting Akt survival mechanisms [31–34].

Additional cell-based evidence suggests that α1-blockers impair cancer progression to metastasis via anoikis induction at pharmacologically relevant doses, proceeding via an α1-adrenoreceptorindependent mechanism. Structural optimization studies led to the generation of a quinazoline-based derivative, of α1-adrenoreceptors antagonist, DZ-50, that was shown to overcome anoikis resistance in human renal cancer cells by disrupting integrin/FAK-mediated cell survival pathways in vitro and in vivo [3]. Doxazosin and DZ-50 were both found to exert potent antitumor action against human renal cancer cell lines 786-0 (harboring a VHL tumor-suppressor gene mutation and a highly angiogenic phenotype) and Caki cells (without a VHL mutation) [3].

DZ-50 has the chemoprotective potential to suppress angiogenesis and reverse the hypoxic nature of cancer through disrupting the tumor microenvironment [30]. The process of EMT, directed by TGF-β within the tumor microenvironment phenotypic landscape, confers acquisition of an invasive phenotype via resistance to anoikis, promoting angiogenesis, metastatic progression, and treatment failure. We first reported the ability of the novel quinazoline-derivative, DZ-50 to disrupt the ILK-1/integrin β1 complex and reduce phosphorylation of its downstream targets, AKT and GSK-3β [3]. As mentioned, this is an important mechanism in inducing anoikis in cancer cells because ILK regulates several integrin-mediated cellular processes, including cell adhesion, fibronectin-ECM assembly and anchorage-dependent cell growth [35,39]. By inhibiting ILK, DZ-50 is then able to kill tumor cells via blocking AKT and FAK phosphorylation and subsequent cell survival, disrupting integrin adhesion (α5β1 and α2β1), and engaging ECM associated tumor suppressors [3,30]. Through anoikis induction, DZ-50 has been found to significantly impair RCC metastasis in in vitro and in vivo models [3,17]. In vitro metastasis assays found that DZ-50 significantly decreased the adhesion potential of RCC to fibronectin and laminin in a time-dependent manner and subsequently suppressed the cells' migratory and invasive capabilities. Mechanistic analysis of anoikis induction (determined by Annexin V-based flow cytometry) revealed that this novel agent inactivates critical cellular survival pathways through inhibition of FAK phosphorylation, inactivation of AKT and GSK-β in the focal adhesion complex

signaling cascade, and disruption of integrin-mediated focal adhesion complexes, such as FAK, ILK-1 and paxillin [3]. By interfering with this survival signaling, DZ-50 successfully reverses anoikis resistance and induces cancer cell death [3,17]. By sensitizing cells to anoikis through disruption of integrin β1-mediated focal adhesion complexes, the novel quinazoline-derived agent acquires a high therapeutic value by effectively reversing anoikis resistance in metastatic RCC tumors [3]. Temporal analysis of cell death in response to DZ-50, established that anoikis occurred prior to apoptosis [3]. Furthermore, DZ-50 exerted a more potent inhibitor effect than doxazosin on ILK-1, FAK, and paxillin binding to integrin-β1 in vivo in human renal cancer 786-0 and Caki cells [3,17,18]. In both RCC cell lines, DZ-50 led to significantly greater inhibition of tumor cell adhesion, migration and invasion than doxasozin did at pharmacologically relevant doses [3]. These findings support that the structural optimization of this particular quinazoline-based α1-adrenoreceptor antagonist has furthered a promising effect in inducing anoikis and impairing renal tumor vascularity to impair metastasis. Naftopidil has also been investigated in this context, with studies demonstrating in vitro suppression of proliferation in ACHN and Caki-2 RCC cell lines [13,53]. Fluorescence-activated cell sorting (FACS) analysis revealed that renal cancer cells treated with naftopidil underwent G1-cell cycle arrest in vitro; the drug also decreased tumor weight and vascularity in RCC xenograft models in naftopidiol-treated excised human RCC [53]. Therefore, naftopidil provides another putative systemic therapy for the treatment and prevention of RCC that, based on this evidence, warrants further investigation.
