The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression

Abstract

Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.

Figure 1. Treatment with doxazosin decreases endothelial cell migration, proliferation, and capillary-like tubule formation

(A) The effect of doxazosin treatment on VEGF-stimulated endothelial cell migration was assessed using Boyden Transwell chambers. Cells were treated with increasing concentrations of doxazosin, fixed, and then stained with H&E. The numbers of migrated cells were calculated under a light microscope. Three independent experiments were assayed in triplicate. Data are presented as the means ± SDs. *P<0.05 vs. the control group. (B) The inhibitory effects of doxazosin on endothelial cell proliferation. Cells were incubated for 3 days with or without VEGF. The c.p.m. of [³H] thymidine was evaluated using a liquid scintillation counter. The data are presented as the means ± SDs of three independent experiments. *P<0.05 vs. the control group. (C) Cells were treated with doxazosin and then grown on growth factor-reduced Matrigel in the presence or absence of 10 ng/ml of VEGF. Tubular-like structure formation was monitored using inverted microscopy. Tube length was quantified and expressed as means ± SD. Experiments were repeated three times, and representative data are shown. *P<0.05 vs. control.

Figure 2. Doxazosin downregulates PI3K activity and Akt/mTOR/p70S6K phosphorylation in vitro

(A) SKOV-3 cells were treated with increasing concentrations of doxazosin, harvested, lysed, and analyzed using an in vitro PI3K assay (lower panel) and immunoblotting with anti-p85 polyclonal antibodies; PI3K was used to verify equal sample loading (upper panel). Data are representative of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. *, P<0.05; **, P<0.01 compared with the controls. (B) SKOV-3 cells were treated with various concentrations of doxazosin, and Akt phosphorylation was evaluated using Western blotting. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. The data are presented as the means ± SDs of three independent experiments. *P<0.05; **, P<0.01 vs. the control group. (C) SKOV-3 cells were treated with doxazosin, the mTOR inhibitor rapamycin, rapamycin plus doxazosin, or the PI3K inhibitor wortmannin. After 24 h, the cells were collected and then analyzed using immunoblotting; total Akt and 4E-BP1 were used as loading controls. The data are presented as the means ± SDs of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. *P<0.05; **, P<0.01 vs. the control group. (D) After treatment with doxazosin, cells were harvested and analyzed by Western blotting with antibodies against phosphorylated and total PDK1, mTOR, and VEGF and its downstream targets including p70S6K and HIF-1α. β-Actin was used as a loading control. Data are representative of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. **, P<0.01 compared with the controls.

Figure 3. Doxazosin suppresses VEGF-dependent Akt, PDK1, and mTOR phosphorylation

SKOV-3 cells were treated with doxazosin and then harvested for immunoblotting with antibodies against phosphorylated or total Akt, PDK1, mTOR, and ERK1/2. The data are presented as the means ± SDs of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. *P<0.05; **, P<0.01; ***, P<0.005; ns, no significant vs. the control group.

Figure 4. Doxazosin inhibits VEGF-induced HIF-1α and VEGF

Cells were incubated with 10 ng/ml VEGF and then control-treated or treated with doxazosin in HUVECs (left panel) or SKOV-3 ovarian carcinoma cells (right panel). HIF-1α and VEGF protein expression levels were then analyzed using Western blotting. Data are representative of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. *, P<0.05; **, P<0.01 compared with the controls.

Figure 5. Doxazosin inhibits VEGF-induced VEGFR-2 phosphorylation and VEGFR-2-dependent transcription

(A) SKOV-3 ovarian carcinoma cells were incubated with 10 ng/ml VEGF followed by control or doxazosin. Total cell lysates were prepared, and VEGFR-2 phosphorylation was assessed using immunoblotting. Total VEGFR-2 and β-actin were used as loading controls. Data are representative of three independent experiments. *, P<0.05; **, P<0.01 compared with the controls. (B) The suppression of VEGFR-2-dependent transcription by doxazosin. Cells were co-transfected with 500 ng of VEGFR-2-Luc, 500 ng of VEGFR-2 expression plasmid (pcDNA3.1/VEGFR-2), and increasing concentrations of doxazosin (0, 5, 15, and 25 μM/ml). Data are presented as means ± SDs.*P<0.05 vs. the control.

Figure 6. Doxazosin suppresses tumor growth by inhibiting angiogenesis in vivo

(A) SKOV-3 ovarian cancer cells were injected subcutaneously into nude mice. (A) Mean tumor weight at the end of the experiments. *P<0.05 vs. the control group (left panel). Sections of doxazosin-treated and control tumors were stained with H&E (right panel). Bar = 50 μm. (B) Tumor tissues were evaluated by immunoblotting with primary antibodies against PCNA, cyclin D1, VEGF, survivin, p-VEGFR-2, and PARP. Data are representative of three independent experiments. Protein levels were calculated by densitometric analysis and normalized to levels of the loading control. *, P<0.05; **, P<0.01 compared with the controls. (C) CD31 staining was performed to visualize the blood vessels (arrows) in tumor tissues. Endothelial cells within frozen tumor sections were stained using anti-CD31 (PECAM-1) antibodies. Bar = 50 μm. *P<0.05 vs. the control.