Western blot analysis revealed the maspin expression in DU-145 transfectants. As shown in , the DU-145-maspin cells, clone M7 (which was used for the subsequent experiments in this study), exhibited high expression of maspin protein in contrast to DU-145-neo control transfectants that totally lacked maspin. Bax expression was slightly increased in M7 cells as compared to the neo control transfectants. In response to treatment with tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) (50 ng/ml; 12h), DU-145-maspin cells exhibit increased sensitivity to apoptosis, as determined by poly(ADP-ribose) polymerase (PARP) cleavage and caspase-3 activation ().
Figure 1 Characterization of apoptotic response of maspin-expressing DU-145 transfectants to TRAIL. (a) Western blot analysis revealed that DU-145-maspin cells exhibit strong maspin expression (M7). (b) DU-145-maspin expressing cells undergo apoptosis in response (more ...)
A significant decrease in cell viability was observed for the DU-145-neo transfectants cells at a concentration of 15 μm doxazosin (82% viability, P<0.005) with a continuous decrease at higher doxazosin concentrations (P<0.0001). The DU-145-maspin cells exhibited a significant reduction of cell viability at a concentration of 25 μm (39.8%).
On the basis of the methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell viability assay, there was a higher sensitivity for the DU-145-maspin cells to increasing doses of doxazosin compared to the DU-145-neo control cells (). At both 25 and 50 μm of doxazosin, significant differences were detected between the two cell lines (67.2±8.6 versus 103.2±3.7% and 3.8±0.5 versus 51.5±11.4%, P<0.005).
Figure 2 (a) Effect of doxazosin on cell viability of prostate cancer cells (MTT assay). DU-145-maspin cells were treated with increasing concentrations of doxazosin as shown. Error bars represent average±s.e. (b) Doxazosin-mediated apoptosis in maspin-expressing (more ...)
As shown in , a dose-dependent increase in the number of apoptotic cells was observed after treatment with doxazosin as determined by Hoechst staining. At the concentration of 25 μm, a significantly higher number of DU-145-maspin cells underwent apoptosis compared to neo controls (34.5±9 versus 13.8±6.6%, respectively, P<0.0001).
We subsequently investigated the effect of doxazosin on cell proliferation. indicates a decrease in [3H]thymidine uptake in both cell lines after doxazosin treatment that was statistically significant at 25 μm and there was a further significant decrease at 50 μm (46.1±7.7 and 43.1±5.4% for the DU-145-maspin and -neo cells, respectively). DU-145-maspin cells exhibited a higher sensitivity at 25 μm of doxazosin, showing a significant reduction in thymidine uptake compared to neo controls (52.3±5.9 versus 82.1±3.6%, P<0.005).
Figure 3 Effect of doxazosin on DNA synthesis: [3H]thymidine uptake assay. Prostate cancer DU-145-maspin cells treated with increasing concentrations of the drug as shown are more sensitive at the concentration of 25 μm doxazosin (P<0.005). Error (more ...)
Considering the ability of maspin to increase adhesion to ECM components, we subsequently examined the attachment potential of prostate cancer cells after doxazosin treatment. Overall, untreated prostate cancer maspin transfectants exhibited an enhanced ability to attach to coated plates, which was more pronounced on fibronectin-coated plates (maspin: 89.9±7.3 versus neo: 63.1±4.1 cells/100 × power field, P<0.005, ), whereas the difference on collagen was not that prominent. There was progressive loss of cell attachment to collagen for both cell lines treated with doxazosin (); however, the DU-145-maspin cells exhibited a relatively higher ability to remain attached under treatment with doxazosin compared to DU-145-neo controls (at 50 μm, P<0.01). A similar significant decrease in cell attachment to fibronectin was observed after doxazosin treatment at both concentrations ().
Figure 4 Effect of doxazosin on cell attachment potential of prostate cancer cells overexpressing maspin. Doxazosin-treated DU-145-maspin cells exhibit increased adhesion to collagen compared to neo controls (P<0.01). (Panel a) Untreated maspin transfectants, (more ...)
There was a significant loss of cell migration after treatment with 15 and 25μm doxazosin in both cell lines (). At a concentration of 15 μm (), DU-145-maspin cells had a decreased ability to migrate compared to neo controls; however, this difference did not reach significance. The addition of vascular endothelial growth factor (VEGF) () did not result in a significant increase in the migration of either cell line. Doxazosin, in the presence of VEGF (), could still significantly inhibit the migration of DU-145 cells, resulting in a comparable decrease to that achieved when used alone (P<0.01).
Figure 5 Effect of doxazosin on cell migration: impact of maspin. (a, c) Reduction of cell migration in DU-145-maspin (a) and DU- 145-neo cells (c) with 15 and 25 μm doxazosin. (b) Example of a wounded area. (d) Quantitative analysis of the results. ( (more ...)
To characterize these effects at the molecular level, we performed reverse transcriptase–polymerase chain reaction (RT–PCR) analysis for a number of key apoptosis and angiogenesis molecules. indicates a dramatic induction of Smad4 mRNA after 24 h in DU-145-maspin cells (, 47.6% increase versus 7.8% increase for controls). RT–PCR analysis of VEGF expression () indicated a similar progressive decrease after doxazosin treatment for the different isoforms of VEGF in both DU-145-maspin and neo controls. As shown in , isoform 189 of VEGF was dramatically downregulated in M7 (by 84%) within the first 6 h of doxazosin treatment compared with a 34% reduction in VEGF mRNA in neo controls. Doxazosin downregulated all other VEGF isoforms in a similar manner in the two groups. A similar downregulation by doxazosin was detected for transforming growth factor βRII (TGFβRII) mRNA (data not shown).
(a) RT–PCR of Smad4 expression in DU-145 cells after treatment with doxazosin. mRNA analysis was performed as described in the ‘Materials and methods’ section. (b) Quantitative analysis of Smad4 expression in DU-145 cells
Figure 7 (a) RT–PCR of VEGF expression in DU-145 cells after treatment with doxazosin, showing the different isoforms of VEGF. (b) Quantitative analysis of VEGF expression (189 isoform) in DU-145 cells indicates differential downregulation in DU-145-maspin (more ...)
The apoptotic nature of the response to doxazosin treatment was confirmed with caspase-3 activation documented by Western blotting (). The caspase-3 activity assay failed, however, to show any significant caspase-3 activation after doxazosin treatment in any cell line (data not shown). Doxazosin- treated DU-145-maspin cells exhibited cleavage of caspase-3, within the first 12h of exposure to doxazosin, compared to neo controls (after 24 h). No significant differences in caspase-8 cleavage were noted in either cell line after doxazosin treatment (data not shown). A significant induction of bax, however, was noted in both cell lines after treatment for 24 h, and was stronger in the maspin-transfected cells (30.5% versus 175% for neo controls, ). Western blotting revealed downregulation of VEGF for both cell lines in response to doxazosin (), while there was a parallel increase in Smad4 protein levels (). Phosphorylated VEGF receptor 2 expression was not detected in either cell line (data not shown).
Figure 8 Caspase-3 activation by doxazosin in prostate cancer cells. DU-145 cells were treated with 15 μm doxazosin for 6, 12 and 24 h, and cell lysates were subjected to Western blotting for caspase-3. A temporally accelerated apoptosis induction and (more ...)
(a) Bax protein expression after treatment with doxazosin in DU-145 cells. (b) Quantitative analysis of bax expression normalized to actin expression. Maspin transfectants exhibit stronger activation of bax after doxazosin treatment
(a) Western blot analysis of VEGF expression in response doxazosin in DU-145 cells. (b) Quantitative analysis of VEGF expression normalized to actin expression. Downregulation of VEGF after a transient peak in both cell lines
(a) Smad4 protein expression after treatment with doxazosin in DU-145 cells. (b) Quantitative analysis of Smad4 expression normalized to actin expression. Earlier induction of Smad4 at 6 h was observed for the maspin transfectants, M7