The human NSCLC cell line, A549, was purchased from the American Type Culture Collection. RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc.). The GAPDH (cat. no. 32233) and fibronectin (cat. no. 18825) antibodies were purchased from Santa Cruz Biotechnology, Inc. Vascular endothelial (VE)-cadherin (cat. no. 2500), Ephrin type-A receptor 2 (EpHA2; cat. no. 6997), phosphorylated (p-)EpHA2^Ser897 (cat. no. 6347), 3-phosphoinositide-dependent kinase 1 (PDK1; cat. no. 3062), p-PDK1^Ser241 (cat. no. 3061), AKT (cat. no. 9272), p-AKT^Ser473 (cat. no. 4060), mTOR (cat. no. 2972), mTOR^Ser2448 (cat. no. 2971), P70s6k (cat. no. 9202), p-P70s6k^Thr389 (cat. no. 9234), ERK (cat. no. 9102), p-ERK^{Thr202/Tyr204} (cat. no. 9101) and vimentin (cat. no. 5741) antibodies were purchased from Cell Signaling Technologies, Inc. VEGF-A (cat. no. ab1316) antibodies were purchased from Abcam. Matrix metalloproteinase (MMP)-2 (cat. no. AB19167), MMP-9 (cat. no. AB19016) and Laminin 5γ2 (cat. no. MAB19562) antibodies were purchased from Millipore. Matrigel was purchased from BD Biosciences. Anti-mouse (cat. no. 115-035-062) and anti-rabbit (cat. no. 111-035-045) IgGs were purchased from Jackson ImmunoResearch Laboratories, Inc. Doxazosin (cat. no. D9815), thiazolyl blue tetrazolium blue (MTT; cat. no. M2128) and sulforhodamine B (SRB; cat. no. S9012) were purchased from Sigma-Aldrich.

A549 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin-amphotericin B solution. Cell cultures were maintained in a 37°C incubator with 5% CO₂. Adherent cell cultures were passaged using 0.05% trypsin-EDTA after reaching ~80% confluence.

After a 24-h treatment with the indicated concentrations of doxazosin, the cells were incubated with MTT (final concentration 0.5 mg/ml) for 2 h. Then, the medium was removed and replaced with 100 µl DMSO to dissolve the formed purple formazan. An ELISA reader (570 nm) was used to assess the absorbance values (25).

Firstly, cells were seeded into 96-well plates. After overnight incubation, cells in partial wells were fixed with 10% trichloroacetic acid (TCA) for 10 min at room temperature and washed with ddH₂O. Cells at this stage represented the cell population at the time of drug addition (T0). The other cells were treated with (Tx) or without [control (C) in 0.1% DMSO] the indicated concentrations of doxazosin for an additional 48 h. Then, the cells (T0 and Tx) were fixed with 10% TCA for 10 min at room temperature and washed with ddH₂O. All cells were stained with 0.4% (w/v) SRB in 1% acetic acid for 10 min at room temperature, and then washed with 1% acetic acid to remove unbound dye. SRB bound cells were solubilized with 10 mM trizma base. Using the absorbance (515 nm) measurements for T0, C and Tx, the percentage of doxazosin effect was calculated as follows: [1-(Tx-T0)/(C-T0)] ×100% (26,27).

Cells were harvested by trypsinization, fixed with 70% (v/v) ethanol for 30 min at 4°C and washed with PBS. The cells were then centrifuged at 500 × g for 10 min at room temperature and re-suspended with 0.3 ml PI solution containing Triton X-100 (0.1%, v/v), RNase (100 µg/ml) and PI (80 µg/ml). The cellular DNA content was analyzed using an FACScan flow cytometer and CellQuest software (V6.0.4; Becton, Dickinson and Company) (28).

VM formation assays were performed in 48-well culture plates coated with 100 µl Matrigel (9.5 mg/ml). Following Matrigel polymerization at 37°C for 30 min, the cells were seeded at 3.2×10⁵ cells/ml in serum-free RPMI medium onto the Matrigel. After cell adhesion to the Matrigel for 4 h (basal condition, representing non-VM forming condition), 0 or 25 µM doxazosin was added to the serum-free medium and a medium change was performed every 2 days at 37°C for the indicated time, before western blotting and microscopic examination.

Cells were cultured on 18×18 mm glass coverslips coated with Matrigel at 37°C for 30 min. After 72 h treatment with different concentration of doxazosin, 3D culture cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, then quickly washed with PBS. Vascular channels were stained using a PAS kit (cat. no. SI-395B; Sigma) for 10 min at room temperature, washed with PBS for 10 min and treated with Schiff reagent (cat. no. SI-395B; Sigma-Aldrich) for 20 min at room temperature. The stained cells were washed with PBS for 15 min and vasculogenic morphogenesis was visualized using fluorescence microscopy (Zeiss Axio Imager, M1). Vascular channels were quantified using MetaMorph software (V7.8.0; Molecular Devices, LLC) (29). For 3D reconstruction, the 3D culture cells were stained with PAS and observed using a ZEISS Cell Observer SD Confocal Microscope (Zeiss GmbH) and ZEN software (V2.3; Zeiss GmbH) (30). The results were determined as follows: Total tube length=total length of tube (excluding nodes); mean tube length=(total tube length)/(number of segments); total tube area=total tube area (excluding nodes); mean tube area=(total tube area)/(number of segments); segments=total number of tube segments connecting branch points and/or ends. IC₅₀ is the half maximal inhibitory concentration.

RT-qPCR were performed to assess mRNA expression levels. Corning Cell Recovery Solution (Corning, Inc.) was used to recover cells from 3D Matrigel cultures according to the manufacturer's instructions. Total RNA was extracted from the cells using an RNAspin Mini Kit (Cytivia), then cDNA was reverse transcribed from the total RNA (0.7 µg) using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc.). The reverse transcription was performed at 37°C for 60 min, and then at 85°C for 5 min, according to the manufacturer's protocol. qPCR was performed using iTag Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc.) and primer sequences as follows: Human VEGF-A forward (F), 5′-CTACCTCCACCATGCCAAGT-3′ and reverse (R), 5′-GCAGTAGCTGCGCTGATAGA-3′; human EPHA2 F, 5′-CCTCTAGTGCCTTCTTTAG-3′ and R, 5′-GAATGTTTGACACCCTCT-3′; human VE-cadherin F, 5′-CGTGTTCGCCATTGAGAG-3′ and R, 5′-TTCGCCAGTGTCCTTGTC-3′; human N-cadherin F, 5′-AGTACAGAAGCACTGGGATT-3′ and R, 5′-AAGCGTGTTGAAGCATATCAT-3′; human vimentin F, 5′-AGTCCACTGAGTACCGGAGAC-3′ and R, 5′-CATTTCACGCATCTGGCGTTC-3′; human FAK F, 5′-GTAGCGTGGCGTAAGTTA-3′ and R, 5′-TTCCTTGACAAGTGAATTATGC-3′; and human GAPDH F, 5′-CAGGGCTGCTTTTAACTCTGGT-3′ and R, 5′-GATTTTGGAGGGATCTCGCT-3′. DNA was amplified with an initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 5 sec, and 60°C for 30 sec. GAPDH was chosen as the internal reference. The data were expressed as relative mRNA levels by Cq values and then subsequently converted to fold change (31).

Corning Cell Recovery Solution (Corning, Inc.) was used to recover cells from 3D Matrigel cultures according to the manufacturer's instructions. The cells were harvested, centrifuged at 500 × g for 10 min at 4°C and lysed in 80 µl ice-cold lysis buffer (150 mM NaCl, 1% Triton X-100, 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 µg/ml leupeptin, 1 mM Na₃VO₄, 1 mM NaF and 1 mM dithiothreitol) for 30 min. The concentration of the total protein was quantified using the Bradford method. Total protein was mixed with sample buffer and heated at 95°C for 10 min. An equal amount of protein (30 µg) per lane was separated by 8 or 12% SDS-PAGE, transferred to PVDF membranes. The membrane was then blocked with 5% skimmed milk for 1 h at room temperature. Protein expression levels were detected with specific antibodies (1:1,000 dilution for the primary antibodies at 4°C overnight and 1:7,000 dilution for the secondary antibodies at room temperature for 2 h). The membranes were washed three times with PBS-T (0.1% Tween 20) for 10 min after incubations with the primary and secondary antibodies. The immunoreactive proteins were detected with an enhanced chemiluminescence detection kit (LumiFlash™ Prime Chemiluminescent Substrate; cat. no. LF01-500; Visual Protein; Energenesis Biomedical Co., Ltd.) and the images were captured using a ChemiDoc™ MP System (Bio-Rad Laboratories, Inc.). Lab^TM Software (V6.0; Bio-Rad Laboratories, Inc.) was used to semi-quantify the data from western blotting.

ELISA was applied to assess VEGF-A secretion (32). 3D culture cells were treated with 25 µM doxazocin for 96 h. Then, the concentration of VEGF-A in the supernatant was determined using a Human VEGF Quantikine ELISA kit (cat. no. DEV00; R&D Systems, Inc.) according to the manufacturer's instructions.

3D culture cells were treated with 25 µM doxazocin for 96 h. Then, the concentration of MMP-2 in the supernatant was determined using a Human MMP-2 Quantikine ELISA kit (cat. no. MMP200; R&D Systems, Inc.) according to the manufacturer's instructions.

Data are presented as the mean ± SEM. Student's unpaired t-test was performed for the statistical analysis of data comparing two groups. One-way ANOVA followed by the Bonferroni's post hoc test was used to perform the statistical analysis of multiple groups. P<0.05 was considered to indicate a statistically significant difference.

A Matrigel-based VM formation assay was performed to examine whether the NSCLC cell line, A549, generated channels in 3D cultures. The results demonstrated that the cells formed vessel-like structures when cultured on Matrigel (Fig. 1A). PAS staining was performed to identify the glycoprotein-rich inner area of VM vessels, and lumen-containing tubular structures were subsequently observed in cultures using confocal microscopy and ZEN software (Fig. 1A and Video S1) (30). VM-related gene expression levels were also determined. The results revealed that VEGF-A and VE-cadherin expression levels were markedly upregulated in VM-forming cells (Fig. 1B). N-cadherin and vimentin (EMT markers) levels were also increased in 3D cultures. In addition, the levels of MMP-9, which is responsible for cell motility and ECM remodeling (33), were raised. MMP-2, which is involved in degrading basement membrane components for cancer metastasis along with MMP-9 (33), expression levels showed an increased trend, albeit this was not significant. FAK expression was also increased, although not significantly These data demonstrated that glycoprotein-rich lined tubular structures and VM-related molecules were present in the constructed in vitro VM model. Therefore, this model was utilized to evaluate the anti-VM effect of doxazosin.

Quinazoline-based α₁-adrenergic receptor blockers are widely used therapeutic drugs for treating hypertension (34), one of which was examined in the present study using the constructed VM cell model. Vasculogenic morphogenesis on a Matrigel surface was visualized in the control group using fluorescence microscopy following PAS staining (Figs. 1A and 2A). Doxazosin inhibited capillary-like tube formation and caused a dispersed morphology in A549 cells (Fig. 2A). The total tube length, mean tube length, total tube area and mean tube area of mimetic vessels (quantified using MetaMorph software) were also reduced by doxazosin, with IC₅₀ values of 36.07±1.38, 28.38±2.10, 31.27±0.32 and 24.17±2.35 µM, respectively (Fig. 2B). The results therefore indicated that doxazosin treatment of the 3D Matrigel cell culture model significantly reduced VM channel formation.

The results of the SRB and MTT assays demonstrated that doxazosin also induced cytotoxic effects in A549 cells (Fig. 3). Doxazosin was more effective in inducing toxicities (6.3 µM, 13.1±4.6%; 12.5 µM, 20.8±5.6%; 25.0 µM, 45.1±3.5%; 37.5 µM, 98.2±9.1%) when using the SRB assay than when using MTT assay (cell survival: 37.5 µM, 72.6±4.4%; 50.0 µM, 47.8±1.8%; 100.0 µM, 14.5±0.1%) (Fig. 3A and B).

The effect of doxazosin on cell cycle progression was also examined, which revealed an increase in the apoptotic sub-G1 phase population with an initial apoptotic effect at 37.5 µM, compared with the 0 µM doxazosin control group (Figs. 3C and S1), which was similar to the aforementioned cytotoxicity assessment (Fig. 3B). The results therefore indicated that doxazosin was more effective in inducing anti-VM activities than in inducing cytotoxic effects.

To further examine the doxazosin-mediated VM-blocking effect on underlying signaling pathways, the levels of two key mediators were determined in the medium during VM formation. The results revealed that the levels of both VEGF-A and MMP-2 in the control treatment group (0 µM doxazosin) were significantly increased compared with basal condition group, supporting VM formation (Fig. 4). Treatment with doxazosin significantly decreased these secretion levels.

VE-cadherin is required for proper vascular development and is typically examined as an indicator of VM formation (35–39). VE-cadherin expression was significantly increased following a 96-h incubation of the 3D Matrigel cultures (Fig. S2). The VM-related protein expression levels were then examined following treatment with doxazosin. For this, 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 of several signaling pathways, including VEGF-A/VE-cadherin/p-EphA2/p-PDK1/p-AKT/p-mTOR/p-p70S6k/p-ERK, MMP-2/MMP-9/laminin 5γ2 and vimentin/fibronectin were determined by western blotting. Doxazosin markedly decreased the cellular protein expression of VEGF-A monomer and dimer (Fig. 5A). Furthermore, doxazosin significantly decreased the protein expression of VE-cadherin and EphA2, and significantly reduced the phosphorylation levels of PDK1, AKT, mTOR, P70S6K and ERK in the 3D Matrigel cell culture model (Fig. 5A). The aforementioned results revealed the doxazosin-mediated suppression of VM formation. MMP-2 and MMP-9 are reported to degrade collagen in the basement membrane, supporting remodeling of the ECM and the regulation of VM formation (40). Although the laminin 5γ2 chain is mainly cleaved by activated MMP-2 (not MMP-9) to produce the 5γ2′ and 5γ2× cleaved fragments (which subsequently trigger the migration and invasion of tumor cells), both MMP-2 and MMP-9 colocalize with VM networks to assist VM formation (41). In the present study, doxazosin significantly reduced the protein expression levels of MMP-2 and MMP-9 and downregulated the protein expression levels of the cleaved forms of laminin, 5γ2′ and 5γ2× (Fig. 5B). EMT is reported to be implicated in VM formation and is associated with the tumor invasion-metastasis cascade (42). Doxazosin also significantly downregulated the protein expression levels of the two EMT markers, vimentin and fibronectin, in the 3D Matrigel-cultured A549 model (Fig. 5C). Collectively, these results confirmed that doxazosin displayed anti-VM activity in the NSCLC cell model.