Previous evidence demonstrated that doxazosin exerts an apoptotic effect against prostate cancer cells via inhibition of protein kinase B (PKB)/Akt activation and the activation of latent apoptotic machinery involving transforming growth factor-beta 1 (TGF-β1) signaling and IκBα. In this study the molecular events targeted by this apoptotic action were further dissected in vitro using the human androgen-independent prostate cancer cell line, PC-3. Pharmacological exploitation and structural function analysis of doxazosin-based quinazoline analogues was undertaken towards the development of a novel class of anti-tumor agents. Quantitative microarray assays were performed on malignant prostate cells after treatment with doxazosin (25μM, 6 and 24hrs) and identified transient changes in the expression of several apoptosis regulators. Specifically, we observed upregulation of Bax, FADD, and Fas/CD95, and downregulation of Bcl-xL and TRAMP/Apo3 in response to doxazosin. Moreover, there was activation of caspase-8 and -3 within the first 6-12 hours of treatment with doxazosin. Doxazosin-induced apoptosis was blocked upon pretreatment of cells with specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of FADD and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex (DISC) formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of doxazosin’s effect in prostate epithelial cells. In addition, structural modification of doxazosin’s chemical nucleus allowed for the development of a novel class of apoptosis-inducing agents as well as a class of angiostatic agents, the most effective being DZ-50. Compound DZ-50 was effective at reducing endothelial cell viability (10μM, 48hrs) via a non-apoptotic mechanism. Moreover, treatment with DZ-50 effectively prevented in vitro tube formation and in vivo chorioallantoic membrane (CAM) vessel development. Confocal microscopy examination revealed a significant reduction in the efficiency of tumor cells to migrate through monolayers of endothelial cells by DZ-50 (10μM, 3, 6, 9, and 12hrs). DZ-50 treatment resulted in reduced xenograph tumor volume and blood vessel formation in vivo. These results demonstrate that doxazosin exerts its apoptotic effects against malignant prostate cells via a death-receptor mediated mechanism with significant clinical implications for therapeutic targeting. Development of quinazoline-based compounds has considerable promise as novel anti-angiogenic drugs for the treatment of advanced prostate cancer.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]