Apoptosis targeting coincides with the goal of successfully treating patients with advanced prostate cancer. A large number of human tumors exhibit down-regulation of, or insensitivity to, proapoptotic molecules such as Fas and FADD or up-regulation of antiapoptotic molecules like FLIP, suggesting that these cells possess regulatory mechanisms blocking the death signal received through the death receptors (19
Induction of apoptosis in response to doxazosin is well documented in benign and malignant prostate epithelial cells and human prostate clinical specimens at intracellular concentrations comparable with the therapeutic doses (13
). The present data indicate that doxazosin also causes a significant loss of cell viability and enhanced apoptosis of benign prostate cells in vitro.
Interestingly enough, benign prostate cells seem to be more sensitive to the apoptotic effect of doxazosin than tumor cells (16
The data from the gene array analysis revealed that a modest change in down-regulation in caspase-8 mRNA in both cells lines after treatment is in agreement with recent evidence indicating no major changes in the mRNA levels of caspase-8 during the period preceding the onset of apoptosis (22
). Doxazosin treatment of benign and malignant prostate cells resulted in cleavage and activation of caspase-8, and caspase activation parallels the decreased cell viability in a time-dependent manner, indicative of a Fas-receptor pathway for apoptosis induction. In a similar temporal pattern to malignant prostate epithelial cells (15
), benign prostate cells exhibited cleavage of caspase-3 after 12 hours of treatment.
Caspase-8 activation is preceded by its association with FADD and their subsequent recruitment by Fas. In addition, all human prostate cancer cell lines express Fas, but most are resistant to apoptosis induced by an agonistic anti-Fas antibody (23
). Some Fas-resistant cell lines will go through apoptosis by concurrent incubation with a Fas antibody and a protein synthesis inhibitor or by sensitizing the cells with chemotherapeutics (24
). This would support the notion that some chemotherapeutic agents can sensitize tumor cells to Fas-mediated apoptosis (25
). A number of anticancer drugs activate the death receptor pathway via enhancement of Fas and Fas ligand expression (27
). We found an increase in both Fas mRNA and protein levels with no change in Fas ligand mRNA expression in either cell line after treatment with doxazosin, consistent with reported evidence that Fas ligand is not required for drug-induced apoptosis because apoptosis is not suppressed by inhibition of Fas/Fas ligand interaction (27
One could argue that FADD expression may contribute to the sensitivity of Fas activation in prostate cancer cells, as shown in tongue carcinoma cells in response to carboplatin (29
). Moreover, an increase in FADD and procaspase-8 expression has been shown in colon carcinoma cells on treatment with cisplatin, doxorubicin, or mitomycin C (30
). In prostate cancer cells, we show an increase in FADD mRNA and protein expression as well as an increased DISC formation in response to doxazosin. In agreement with our data, other groups have shown similar increases in FADD and DISC formation in tumor cells following drug treatment (32
FADD protein has been shown to interact with caspase-8 via its death-effector domain and blocking proteolytic activation of caspase-8 at the DISC prevents apoptosis (33
). Considering this evidence, we examined the functional consequences of WT-FADD overexpression and the expression of DN-FADD (lacking death-effector domain) on doxazosin-induced apoptosis of prostate cells. PC-3 cells overexpressing WT-FADD exhibited a dramatic increase in their apoptotic response to doxazosin whereas cells expressing DN-FADD have a significant decrease in doxazosin-mediated apoptosis. This further confirms our hypothesis that doxazosin-induced apoptosis occurs via increased FADD protein levels and DISC formation.
The role of Akt signaling in regulating death receptor signaling is not understood. We have previously shown that exposure to doxazosin causes a decrease in Akt phosphorylation (18
). Mechanistically, Akt activation promotes the degradation of IκB, which is involved in the apoptotic action of doxazosin in prostate cancer cells (15
). In other cellular systems, such as T lymphocytes, blocking Akt signaling has been shown to increase caspase-8 activity, ultimately resulting in Fas-dependent apoptosis (34
). Moreover, canstatin inhibits Akt activation and induces Fas-dependent apoptosis in endothelial cells (35
). Taken together, this evidence strongly supports Akt signaling as a critical downstream target of the death receptor–mediated apoptotic pathway.
A mutant form of FADD would potentially uncouple caspase signaling and block apoptosis. Indeed, the present data indicate no changes in Akt activation in doxazosin-treated DN-FADD clones correlating with the apoptotic response compared with the untreated controls. A similar pattern has been found in another experimental system of apoptosis in staurosporine treated cells expressing a DN-FADD (36
). Moreover, apoptosis induction and detachment from ECM can be blocked by expression of a DN-FADD mutant with concurrent treatment with various chemotherapeutic agents (31
). Studies by other investigators have also shown that cisplatin and camptothecin induce Fas ligand–independent aggregation of death receptors and recruitment of FADD to death receptors (37
). Further experiments are required to examine the possible crosstalk between Akt and FADD.
In endothelial cells, Fas/Fas ligand interaction, Fas-FADD complex formation, and caspase-8 activation have been implicated as the precursor to anoikis, and inhibition of any of these events blocks anoikis (39
). As shown in the present study, doxazosin-mediated apoptosis may also rely on changes in integrin expression at the cell surface that may cause anoikis. Considering that anoikis-resistant cells are able to detach from the primary tumor without undergoing apoptosis, acquisition of anoikis resistance becomes a critical step in the metastatic process. Overexpression of a number of mediators involved in cell-matrix and cell-cell anchorage can provide resistance to anoikis, as well as the tumor suppressor genes PTEN
, or overexpression of oncogenes such as ras, raf
, and src
). The Fas-receptor signaling pathway and bcl-2 overexpression have also been intimately implicated in anoikis (37
In summary, the present study indicates that doxazosin treatment of benign and malignant prostate epithelial cells leads to a significant increase in DISC formation and subsequent apoptosis via caspase-3 activation. The apoptotic effect of doxazosin provides a molecular basis for therapeutic targeting of prostate cancer, as well as benign disease, potentially via anoikis. Based on the present findings, a model can be proposed featuring cell detachment as the critical event in apoptosis induction by doxazosin, potentially with caspase-3 activation as a consequence of loss of critical cell attachments rather than the contributing executioner of apoptotic cell death. Ongoing studies address the involvement of integrin signaling in apoptosis induction in prostate tumor epithelial and endothelial cells and the development of novel antiangiogenic (quinazoline-based analogs) against androgen-independent prostate tumors.