Prazosin displays anticancer activity against human prostate cancers: targeting DNA and cell cycle

Abstract

Quinazoline-based alpha1-adrenoceptor antagonists, in particular doxazosin and terazosin, are suggested to display antineoplastic activity against prostate cancers. However, there are few studies elucidating the effect of prazosin. In this study, prazosin displayed antiproliferative activity superior to that of other alpha1-blockers, including doxazosin, terazosin, tamsulosin, and phentolamine. Prazosin induced G2 checkpoint arrest and subsequent apoptosis in prostate cancer PC-3, DU-145, and LNCaP cells. In p53-null PC-3 cells, prazosin induced an increase in DNA strand breaks and ATM/ATR checkpoint pathways, leading to the activation of downstream signaling cascades, including Cdc25c phosphorylation at Ser216, nuclear export of Cdc25c, and cyclin-dependent kinase (Cdk) 1 phosphorylation at Tyr15. The data, together with sustained elevated cyclin A levels (other than cyclin B1 levels), suggested that Cdk1 activity was inactivated by prazosin. Moreover, prazosin triggered mitochondria-mediated and caspase-executed apoptotic pathways in PC-3 cells. The oral administration of prazosin significantly reduced tumor mass in PC-3-derived cancer xenografts in nude mice. In summary, we suggest that prazosin is a potential antitumor agent that induces cell apoptosis through the induction of DNA damage stress, leading to Cdk1 inactivation and G2 checkpoint arrest. Subsequently, mitochondria-mediated caspase cascades are triggered to induce apoptosis in PC-3 cells.

Keywords: Cdc25c; DNA damage; Prazosin; cell cycle; mitochondria-involved apoptosis.

Figure 1

Antineoplastic effect of α-adrenoceptor antagonists on several human prostate cancer cell lines. The compound, at the indicated concentration, was added to cells for 48 hours. Then the cells were fixed and stained with SRB. After a series of washings, bound SRB was subsequently solubilized and absorbance was read at a wavelength of 515 nm. Data are expressed as the mean ± SEM of four independent determinations (each in triplicate) (A). IC₅₀ values were calculated as described in the Materials and Methods section (B). PC-3 cells were treated with or without prazosin (30 µM) for the indicated times. Cell morphology was detected by microscopic examination (C). PC-3 cells were treated with or without prazosin (30 µM) for 36 hours. Apoptosis was detected by TUNEL and Hoechst 33342 reaction techniques (D). Scale bar, 20 µm. *IC50 > 100 µM.

Figure 2

Effect of α-adrenoceptor antagonists on cell-cycle progression. Cells were treated with the indicated drug at various concentrations for 48 hours. Then the cells were fixed and stained with PI to analyze DNA content by FACScan flow cytometric analysis. Data are representative of three independent experiments (A and B). The inhibition of cell growth by doxazosin and prazosin using SRB assay (solid curves) is demonstrated. The data show that growth inhibition is correlated with the population in G₂/M phase in response to prazosin action, but not with PC-3 cells in response to doxazosin action (B). Prazosin (30 µM)-mediated time-dependent change in G₂/M phase and sub-G₁ population were detected by FACScan flow cytometric analysis in PC-3 cells. Data are expressed as the mean ± SEM of three independent determinations. *P < .05 and **P < .01 compared with the respective controls (zero time) (C).

Figure 3

Effect of prazosin on the expression of several cell-cycle regulators. (A and B) PC-3 cells were incubated in the absence or in the presence of prazosin (30 µM) for the indicated times. Then the cells were harvested and lysed for the detection of protein expression with the antibody by Western blot analysis. For Western blot analysis, the amount of proteins (40 µg) was separated by electrophoresis in a 10% or a 15% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoreacted with the indicated antibody.

Figure 4

Effect of prazosin on DNA damage stress in PC-3 cells. (A) Cells were treated with the indicated agent for 18 hours. Then the cells were fixed and stained with PI to analyze DNA content by FACScan flow cytometric analysis. Data are expressed as the mean ± SEM of three independent determinations. (B) Comet assay was employed to examine the integrity of chromosome DNA on treatment with prazosin (1 hour). Etoposide (50 µM) was included as a positive control. One hundred cells were scored to calculate the overall percentage of Comet tail-positive cells. Data are expressed as the mean ± SEM of three independent experiments. *P < .05 and ***P < .001 compared with controls.

Figure 5

Effect of prazosin on the expression of several proteins in PC-3 cells. (A–D) Cells were incubated in the absence or in the presence of prazosin (30 µM) for the indicated times. Then the cells were harvested and lysed for the detection of protein expression with the antibody by Western blot analysis. For Western blot analysis, the amount of proteins (40 µg) was separated by electrophoresis in a 10% or a 15% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoreacted with the indicated antibody.

Figure 6

In vivo antitumor study against PC-3 cells. The nude mice were subcutaneously injected with PC-3 cells (10⁷ cell/mouse). The tumors were measured every 2 to 3 days. When the tumors had reached a volume of 100 to 140 mm³, the mice were divided into three groups (n = 7), and vehicle (0.5% CMC) or prazosin (3 and 10 mg/kg) was given orally every day. Tumor volume was measured every 2 to 3 days.