Doxazosin inhibits vasculogenic mimicry in human non‑small cell lung cancer through inhibition of the VEGF‑A/VE‑cadherin/mTOR/MMP pathway

Abstract

Lung cancer is the leading cause of cancer-related death worldwide, and ~85% of lung cancers are non-small cell lung cancer (NSCLC), which has a low 5-year overall survival rate and high mortality. Several therapeutic strategies have been developed, such as targeted therapy, immuno-oncotherapy and combination therapy. However, the low survival rate indicates the urgent need for new NSCLC treatments. Vasculogenic mimicry (VM) is an endothelial cell-free tumor blood supply system of aggressive and metastatic tumor cells present during tumor neovascularization. VM is clinically responsible for tumor metastasis and resistance, and is correlated with poor prognosis in NSCLC, making it a potential therapeutic target. In the present study, A549 cells formed glycoprotein-rich lined tubular structures, and transcript levels of VM-related genes were markedly upregulated in VM-forming cells. Based on a drug repurposing strategy, it was demonstrated that doxazosin (an antihypertensive drug) displayed inhibitory activity on VM formation at non-cytotoxic concentrations. Doxazosin significantly reduced the levels of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase-2 (MMP-2) in the cell media during VM formation. Further experiments revealed that the protein expression levels of VEGF-A and vascular endothelial-cadherin (VE-cadherin), which contribute to tumor aggressiveness and VM formation, were downregulated following doxazosin treatment. Moreover, the downstream signaling Ephrin type-A receptor 2 (EphA2)/AKT/mTOR/MMP/Laminin-5γ2 network was inhibited in response to doxazosin treatment. In conclusion, the present study demonstrated that doxazosin displayed anti-VM activity in an NSCLC cell model through the downregulation of VEGF-A and VE-cadherin levels, and the suppression of signaling pathways related to the receptor tyrosine kinase, EphA2, protein kinases, AKT and mTOR, and proteases, MMP-2 and MMP-9. These results support the add-on anti-VM effect of doxazosin as a potential agent against NSCLC.

Keywords: doxazosin; drug repurposing; non-small cell lung cancer; vascular endothelial growth factor A/vascular endothelial-cadherin pathway; vasculogenic mimicry.

Figure 1.

Characterization of a VM in vitro model of human non-small cell lung cancer. A549 cells were seeded in serum-free RPMI medium onto Matrigel and cultured for 96 h. Medium changes were performed every 2 days, then Corning Cell Recovery Solution was used to recover cells from the 3D Matrigel cultures. (A) Following Periodic Acid Schiff staining to identify the glycoprotein-rich inner area of VM vessels, 3D-reconstruction of a 6-day-old 3D culture of A549 cells was performed, with the resulting color map distinguishing between the planes and lumen-containing tubular structures present in the culture, using the confocal microscopy ZEN program. A view from the top and cross sections are shown. The cross section identified upper (red), middle (green) and lower sections (blue) of a tubular structure. (B) VM-related gene expression after a 96-h cell adhesion to Matrigel was determined by reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± SEM of five experiments. *P<0.05, **P<0.01, ***P<0.001 compared with basal condition (cell adhesion to the Matrigel for 4 h). FAK, focal adhesion kinase; MMP, matrix metalloproteinase; VE-cadherin, vascular endothelial-cadherin; VEGF-A, vascular endothelial growth factor A; VM, vasculogenic mimicry.

Figure 2.

Effect of doxazosin on VM formation using PAS staining. A549 cells were seeded in serum-free RPMI medium onto Matrigel in the absence or presence of doxazosin for 72 h. Medium changes were performed every 2 days. (A) Following PAS staining to identify the glycoprotein-rich inner area of VM vessels, vasculogenic morphogenesis was visualized with using fluorescence microscopy (Zeiss AxioImager, M1) and (B) the results were quantified using MetaMorph software. Data are presented as the mean ± SEM of three experiments. *P<0.05, **P<0.01, ***P<0.001 compared with the control (0 µM doxazosin), determined using one-way ANOVA followed by the Bonferroni post hoc test. C, control; PAS, Periodic Acid Schiff; VM, vasculogenic mimicry.

Figure 3.

Effect of doxazosin on inducing cytotoxicity of A549 cells. A549 cells were incubated in the absence or presence of doxazosin. (A) After a 48-h treatment, the cells were fixed and then stained using Sulforhodamine B for the determination of the cytotoxicity. (B) After a 24-h treatment, the cells were stained using MTT for the determination of cell toxicity. (C) After the indicated treatment times, the cells were harvested for the detection of cell populations at different phases by propidium iodide staining and flow cytometry analysis. Data are presented as the mean ± SEM of three experiments. **P<0.01, ***P<0.001 compared with the control (0 µM doxazosin), determined using one-way ANOVA followed by the Bonferroni post hoc test.

Figure 4.

Effect of doxazosin on the secretion levels of VEGF-A and MMP-2 into the media during vasculogenic mimicry formation. A549 cells were seeded in serum-free RPMI medium onto Matrigel. After cell adhesion to the Matrigel for 4 h (basal condition), the cells were incubated in the absence or presence of 25 µM doxazosin for 96 h. The secretion levels of (A) VEGF-A (B) and MMP-2 were determined using commercial ELISA kits. Data are presented as the mean ± SEM of three experiments. One-way ANOVA by Bonferroni post hoc test is used to perform **P<0.01, ***P<0.001, compared with the control (0 µM doxazosin), determined using one-way ANOVA followed by the Bonferroni post hoc test. MMP-2, matrix metalloproteinase; VEGF-A, vascular endothelial growth factor A.

Figure 5.

Effect of doxazosin on the expression of VM-related proteins. A549 cells were seeded in serum-free RPMI medium onto Matrigel in the absence or presence of 25 µM doxazosin for 96 h. Then, Corning Cell Recovery Solution was used to recover cells from the 3D Matrigel cultures. The protein expression levels of (A) VEGFA, VE-cadherin, p-EphA2, p-PDK1, p-AKT, p-mTOR, p-p70S6k, p-ERK, (B) MMP2, MMP9, laminin 5γ2, and (C) vimentin and fibronectin were determined by western blotting analysis. Data are presented as the mean ± SEM of three to five experiments. *P<0.05, **P<0.01, ***P<0.001, compared with the VM control (0 µM doxazosin). EpHA2, Ephrin type-A receptor 2; MMP, matrix metalloproteinase; p-, phosphorylated; PDK1, 3-phosphoinositide-dependent kinase 1; VE-cadherin, vascular endothelial-cadherin; VEGF-A, vascular endothelial growth factor A; VM, vasculogenic mimicry.