The lentiviral vector pGC-FU and pGC-FU-HIF-1α were used for HIF-1α overexpression studies. A eukaryotic vector plasmid containing small interfering (si)R-Mimic was used as a control and three plasmids containing different siRNA (siR) sequences targeting HIF-1α were used for knockdown experiments. All plasmids were purchased from Shanghai GeneChem Co., Ltd.

Human CRC HCT-116 cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% fetal calf serum. HUVECs, were obtained from The Experiment Center of Putuo Hospital affiliated to Shanghai University of Chinese Medicine and were cultured in endothelial cell medium containing 15% fetal calf serum. Both cell lines were incubated at 5% CO₂ at 37°C.

Tan IIA (98% pure) was purchased from Xi'an Guanyu Bio-Tech Co., Ltd. and cobalt chloride (CoCl₂) was obtained from Sigma-Aldrich; Merck KGaA.

HCT-116 cells were routinely cultured and during the logarithmic phase were plated into a 24-well plate at a density of 1×10⁵ cells/ml. Cells were divided into the following four conditions: Blank group, CoCl₂ group, pGC-FU vector group and pGC-FU-HIF-1α group; with three wells per condition. Cells were incubated until confluence had reached 30–50%. For infection, 1×10⁸TU/ml pGC-FU vector or pGC-FU-HIF-1α lentivirus was added to 60 µl culture medium and this mix was added to each well. After 8–12 h, the state of the cells was observed, and the culture medium was replaced with fresh medium. A total of 48 h after infection, fluorescence was observed and subsequent experiments were performed.

A total of 5×10⁵ cells/ml HCT-116 cells were plated per well in a 6-well plate. Cells were divided into the following six conditions: Blank group, CoCl₂ group, siR-mimic group, siR-HIF-1α sequence 1 + CoCl₂ group, siR-HIF-1α sequence 2 + CoCl₂ group and siR-HIF-1α sequence 3 + CoCl₂ group; with three wells per condition. Cells were incubated until confluence had reached 40–60%. A mixture containing 4 µg of DNA and 12 µl of HilyMax was added to 100 µl serum-free medium and sufficiently mixed. The mixture was incubated at room temperature for 15 min and subsequently added to the wells, which were cultured for 8–12 h. The medium was replaced with fresh medium and incubated for a further 48 h. Transfection was confirmed by observing fluorescence expression under a fluorescent microscope.

HCT-116 cells transfected with the various siRNAs were used for RT-qPCR. The medium from cells treated with CoCl₂ was replaced with fresh medium containing 200 µM CoCl₂ and incubated for 48 h. Subsequently, a total RNA extraction kit was used to extract RNA according to the manufacturer's protocol. RNA purity and concentration were determined. For reverse transcription, 4 µl 5Χ PrimeScript Buffer, 1 µl PrimeScript RT Enzyme Mix I, 1 µl Oligo dT Primer (50 µmol/l), 1 µl random hexamers (100 µmol/l) and 4 µl of total RNA were added and brought to a final volume of 20 µl using Rnase-free water. The reverse transcription temperature protocol was as follows: 37°C for 15 min and then 85°C for 5 sec. The primer sequences used were: HIF-1α forward, 5′-CGAAGTAGTGCTGACCCTGC-3′ and reverse, 5′-AACTTTGTCTAGTGCTTCCATCG-3′; HIF-1α probe, 5′-AGGTGTCTGATCCTGAATCTGGGGCA-3′; GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and reverse, 5′-ACCCTGTTGCTGTAGCCA-3′, GAPDH probe; 5′-TTGCCCTCAACGACCACTTTGTC-3′. All primers were synthesized by Sangon Biotech Co., Ltd. For qPCR, a solution containing 0.4 µl each of forward and reverse primers, 0.8 µl probe, 2 µl cDNA, 0.4 µl Rox Reference Dye and 10 µl Premix EX Taq was prepared. The solution was brought to a final volume of 20 µl using dH₂O. The thermocycling conditions were as follows: Pre-denaturation, 94°C for 10 sec; followed by 40 cycles of denaturation, 95°C for 5 sec, annealing at 60°C and extension for 34 sec. PCR was performed using an ABI 7300 SDS. GAPDH was used as an internal reference. HIF-1α relative mRNA expression levels were calculated using the 2^−ΔΔCq method (DCq=HIF-1α Cq-GAPDH Cq) (12).

A total of 5×10³ HCT-116 cells/well were plated in a 96 well plate (12 wells per condition) and cultured overnight. Once the cells had adhered, 2, 4, 8, 16, 32, and 64 µM of Tan IIA were added to the cells cultured under normal conditions. Additionally, 1, 5, 10, 15 and 20 µM of Tan IIA were added to the cells cultured under hypoxic conditions (simulated using 200 µM of CoCl₂). Cells were cultured for 24, 48 and 72 h, and HCT-116 medium was routinely collected. ECM containing 15% FBS was used to produce 5 different volume concentrations (6.25, 12.5, 25, 50 and 75% HCT-116 medium in the cell culture). Subsequently, 1×10³ cells/ml were plated per well in a 96 well plate. Once the cells had adhered, the tumor supernatant cultures were replaced with 5 gradient concentrations of HCT-116 medium. The cells were cultured for 48 h after which 20 µl CCK-8 solution was added to each well, and cultured for a further 4 h. Absorbance was measured using a microplate reader at 450 nm/630 nm. Growth inhibition rate (GIR) was calculated using the following equation: GIR=[1-(ODn-OD0)/(ODc-OD0)]x100. OD₀ was the absorbance in the blank group. OD_c was the absorbance of the normal control group. OD_n was the absorbance of cells treated with the various doses of Tan IIA. The IC₅₀ of Tan IIA was calculated. Experiments were repeated three times independently.

A total 0.5 ml 1×10⁵ cells/ml suspension was plated per well. Cells were divided as follow: Blank group, CoCl₂ group, pGC-FU vector group, pGC-FU HIF-1α group, siR-mimic group, siR-HIF-1α group, siR-HIF-1α + CoCl₂ group, Tan IIA-L group (2.5 µM), Tan IIA-M group (5 µM), Tan IIA-H group (10 µM) and Tan IIA-M + CoCl₂ group. Cells were treated 5 gradient concentrations of HCT-116 medium. To simulate hypoxia, 200 µM of CoCl₂ was added in the CoCl₂ group and cells were incubated. After 48 h, the supernatant from each well was collected in a sterile area. The supernatant was centrifuged at 626 × g for 10 min to remove the cell debris. Specific ELISA kits were used measure the expression levels of VEGF and bFGF according to the manufacturer's protocol and experiments were repeated three times.

Total proteins were extracted from cells with Nuclear and Cytoplasmic Protein Extraction kit (P0027; Beyotime Institute of Biotechology) and protein detected with Enhanced BCA Protein Assay kit (P0010; Beyotime Institute of Biotechology). Lysates were diluted to a protein concentration of 5 µg/µl and 60 µg of protein samples were resolved on a 10% gel using SDS-page. Proteins were transferred to PVDF membranes (EMD Millipore) using a Bio-Rad Trans-Blot (Bio-Rad Laboratories, Inc.) at 100 mV and 135 mA. Membranes were blocked in 5% skimmed milk at room temperature for 2 h or at 4°C overnight. Subsequently, the membranes were incubated in 5% BSA solution with a HIF-1α primary antibody (cat. no. 3716S; dilution 1:500; Cell Signaling Technology, Inc.) for 2 h at room temperature or overnight at 4°C. Subsequently, the membranes were washed with TBS-Tween four times, 10 min each, after which the membranes were incubated with the secondary horseradish peroxidase-conjugated anti-goat antibody (cat. no. HAF109; dilution 1:1,000; R&D Systems China Co., Ltd.) or anti-rabbit antibody (cat. no. HAF008; dilution 1:1,000; R&D Systems China Co., Ltd.) at room temperature for 2 h. The membranes were washed six times using TBS-Tween. Signals were visualized using by adding enhanced chemiluminescent reagent (EMD Millipore) for 1–2 min, and X-ray films were exposed to the membranes and developed. Densitometry analysis was performed using ImageJ (National Institutes of Health).

A total of 1.5×10⁶ HCT-116 cells were plated well in a 6-well plate and allowed to adhere. Medium containing 200 µM of CoCl₂ and 2.5, 5 or 10 µM Tan IIA was added. Cells were further cultured for 24 h, after which the medium was removed, the cells were trypisinized, resuspended and fixed. A total of 200 µl of cells at a density of 5×10⁵ were incubated with phycoerythrin-labeled anti-HIF-1α antibody (cat. no. 79233; dilution 1:1,600; Cell Signaling Technology, Inc.). As the blank control, 200 µl PBS was used. Cells were incubated on ice for 40 min in the dark with the antibody and subsequently fixed with 500 µl of 70% ethanol. Fluorescence was measured using a flow cytometer (BD Biosciences).

HUVECs were divided into separate groups as follows: HCT-116 group, pGC-FU vector group, pGC-FU-HIF-1α group, siR-mimic group, siR-HIF-1α group, CoCl₂ group, siR-HIF-1α + CoCl₂ group and 1.0, 2.5, 5.0 or 10 µM Tan IIA groups, with three wells per condition. A suspension of HUVECs at a density of 1×10⁵ cells/ml single-cell suspension in serum-free ECM, were plated in a 48-well plate pre-coated with 200 µl Matrigel. After the cells had adhered, medium from one of the groups above or 200 µl of tumor medium was added and cells were cultured. Tube formation of HUVECs was observed and imaged under an Olympus CKX41 inverted microscope (magnification ×100; Olympus Corp.) and three images were obtained per well.

A total of 200 µl single-cell suspension of HUVECs in ECM at a density of 6×10⁵/ml were added to the upper chamber of a Transwell insert. Tan IIA (1.0, 2.5, 5 or 10 µM) was added to the wells. In the bottom chamber, 600 µl of ECM containing 20% FBS was added. Cells were incubated for 24 h, after which the medium in the upper chamber was discarded. The insert was washed twice with phosphate-buffered saline (PBS), and the cells on the upper membrane were removed using a cotton swab. Cells which had migrated were fixed at room temperature for 30 min, stained with 0.1% crystal violet for 20 min, washed by PBS three times and observed and imaged under a Olympus CKX41 inverted microscope (magnification ×100; Olympus Corp.). Cells were extracted using 100 µl 10% acetic acid per pore for 10 min. The optical density (OD) values were measured at 590 nm, and 10% acetic acid in the blank pores was used as the reference to zero the OD. The inhibition of migration by Tan IIA was calculated as follows: Inhibition rate=(OD in the positive control group-OD in the Tan IIA dosing group)/OD in the positive control group ×100%.

Statistical analysis was performed using SPSS version 22.0 (IBM, Corp.). Data are presented as the mean ± standard deviation of three repeats. If the data passed tests for normality and homogeneity of variance, the one-way ANOVA was applied for statistical inference. If these conditions were not met, a non-parametric test (Wilcoxon rank sum test) was selected. The test used a standard α=0.05. P<0.05 was considered the threshold for a statistically significant difference.

In cells infected with the HIF-1α overexpression lentiviral vector, pGC-FU-HIF-1α, HIF-1α mRNA expression levels were 5.01±0.5 times higher compared with the control lentiviral group, and 4.33±0.15 times higher compared with the blank group. The protein expression levels of HIF-1α were also increased significantly (P<0.05; Fig. 1A and B). In cells infected with pGC-FU-HIF-1α, VEGF expression was 426.576±6.834 pg/ml compared with 246.129±8.948 pg/ml in the blank group. bFGF expression was 861.39±11.106 pg/ml in cells infected with pGC-FU-HIF-1α compared with 481.872±9.186 pg/ml in the blank group. Expression of both VEGF and bFGF were significantly increased compared to the blank group (both P<0.01; Fig. 1C and D) These results indicated that pGC-FU-HIF-1α increased HIF-1α expression in HCT-116 cells which in turn increased secretion of VEGF and bFGF.

HCT-116 cells were transfected with siR-mimic control plasmid or one of the three HIF-1α interference plasmids. Hypoxia was simulated using 200 µM CoCl₂ for 48 h. RT-qPCR and western blotting revealed that HIF-1α mRNA and protein expression levels were decreased in siR-HIF-1α-transfected cells compared with CoCl₂-treated cells and the blank vector group. siR-HIF1α sequence 3 demonstrated the greatest decrease on expression (P<0.01; Fig. 2A and B) and as such was used in all subsequent experiments. In HCT-116 cells transfected with siRHIF-1α, VEGF expression was 229.725±15.712 pg/ml compared with 246.129±8.948 pg/ml in the blank group under normal conditions, and this difference was not significant (P=0.216; Fig. 2C). Under hypoxic conditions, VEGF expression was significantly decreased from 495.176±13.668 pg/ml in the CoCl₂ group compared with 262.533±22.069 pg/ml in cells transfected with CoCl₂ + siR-HIF1α (P<0.05; Fig. 2C). bFGF expression under normal conditions was decreased from 478.93±11.676 pg/ml in the blank group to 450.981±11.106 pg/ml in the siR-HIF1α group, although the difference was not significant (P=0.216; Fig. 2D). However, under hypoxic conditions, expression of bFGF significantly decreased from 984.954±18.372 pg/ml in the CoCl₂ group compared with 514.234±13.239 pg/ml in cells transfected with CoCl₂ + siR-HIF1α (P<0.05; Fig. 2D). These results indicated that knockdown of HIF-1α reduced VEGF and bFGF expression under hypoxic conditions but not normal conditions.

Under normal conditions, HCT-116 cell culture medium was used to prepare 6.25, 12.5, 25, 50 and 75% HCT-116 medium conditioned ECM to simulate the growth environment of a tumor. The media were used to culture HUVECs for 48 h. A CCK-8 assay was used to determine the effect of ECM on the proliferation of HUVECs. The results revealed that excluding the 6.25% medium, proliferation of HUVECs was significantly increased when conditioned medium was added. Among the various percentages of conditioned media used, the 50% media resulted in the largest increase in proliferation (P<0.01; Fig. 3A). ECM gel was used to assess the effects of HIF-1α on tube formation. The results revealed that in the cells grown in medium from HIF-1α overexpression cells (pGC-FU-HIF-1α), tube formation was significantly increased compared with cells grown in medium from either the blank group or pGC-FU-vector group (P<0.01). Furthermore, medium from the siR-HIF-1α group significantly decreased tube formation in HUVECs, and the difference was statistically significant (P<0.05; Fig. 3B and C).

Under both normal and hypoxic conditions, Tan IIA at different concentrations was added to the HCT-116 cells for 24, 48 and 72 h. The results revealed that Tan IIA decreased proliferation of HCT-116 cells in a concentration and time-dependent manner (Fig. 4A and B). Under normal conditions, the IC₅₀ of Tan IIA on HCT-116 cells for 24, 48 and 72 h was 30.7±1.52, 13.3±0.56 and 5.1±0.55 µM, respectively. Under hypoxic conditions, the IC₅₀ was 8.4±1.09, 2.1±0.18 and 0.99±0.17 µM, respectively (Fig. 4C and D). Therefore, Tan IIA exhibited a more notable effect on proliferation under hypoxic conditions.

Western blotting and flow cytometry revealed that Tan IIA can significantly decreased HIF-1α expression in HCT-116 under hypoxic conditions in a concentration dependent manner (P<0.01). Under hypoxic conditions, 10 µM Tan IIA almost completely abrogated CoCl₂-induced HIF-1α upregulation (P=0.489; Fig. 5A and B). Similarly, 10 µM Tan IIA significantly inhibited secretion of VEGF and bFGF in HCT-116 cells under normal conditions (P<0.05; Fig. 5C and D). Furthermore, the effects of Tan IIA on secretion of VEGF and bFGF were notably greater under hypoxic conditions; 10 µM of Tan IIA could completely abrogate the CoCl₂-induced effects on HIF-1α expression (P<0.01; Fig. 5C and D). siR-HIF-1α decreased the expression of VEGF and bFGF in cells grown under hypoxic conditions to levels similar to that observed under normal conditions. However, siR-HIF1α did not affect the expression of VEGF and bFGF under normal conditions (Fig. 5C and D). Tan IIA, significantly decreased the expression levels of VEGF and bFGF in HIF-1α-overexpressing and knockdown cells, (Fig. 5C and D). The results indicated that Tan IIA decreased VEGF and bFGF expression in HCT-116 cells under normal and hypoxic conditions.

A CCK-8 assay revealed that in HUVECs treated with 0.5, 1, 2, 5 and 10 µM Tan IIA HUVECs for 24, 48 and 72 h, proliferation was decreased in a time and concentration dependent manner compared with the control group (Fig. 6A). A tube formation assay revealed that Tan IIA inhibited tube formation in HUVECs in a dose-dependent manner, and 1 µM Tan IIA was sufficient to statistically reduce tube formation (P<0.01; Fig. 6B and C). A Transwell migration assay revealed that migration was decreased in cells treated with Tan IIA, and 1 µM Tan IIA was sufficient to statistically decrease migration. Tan IIA-mediated inhibition of migration was determined to be concentration-dependent (Fig. 6D and E).