Antibodies against HIF-2α, GAPDH and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The anti-VEGFR2 antibody was obtained from Abcam (Cambridge, MA, USA). Antibodies against HIF-1α, CBP and Oct-4 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Horseradish peroxidase-linked goat anti-mouse and goat anti-rabbit IgG antibody for immunoprecipitation and immunoblotting analysis, fluorescein isothiocyanate-linked goat anti-mouse and goat anti-rabbit IgG antibody for immunofluorescence and confocal microscopy were purchased from Jackson ImmunoResearch (Newmarket, UK). Small interfering (si)RNAs were obtained from Gene Pharma (Shanghai, China). Endostatin and N-4 endostatin (Δ2-5 endostatin) were obtained from Protgen Ltd. (Beijing, China).

Wild type mouse nucleolin (mNCL^wt; NM_005381) was subcloned into the pCMV6-AN-GFP vector (Origene, Rockville, MD, USA) and NLS-deficient mutant nucleolin (mNCL^mut) was constructed using the Quick Change Mutagenesis kit (Stratagene, Santa Clara, CA, USA). All the plasmids were purified from Escherichia coli using PowerPrep Plasmid Purification kits (Origene, Rockville, MD, USA).

The HUVEC cells in 24-well plates were transfected with the mNCL^wt or the mNCL^mut plasmid using a TurboFect reagent (Thermo Fisher Scientific, Waltham, MA, USA), then co-transfected twice (at 0 and 24 h) with 200 pmol scramble siRNA (or siRNA pool) or nucleolin siRNA.

Apo and holo samples (lab stock) preperation was performed as previously described (16) Briefly, HUVECs were incubated with bovine serum albumin, apo-endostatin (non-zinc-binding), BSA+ZnCl₂ or holo-endostatin (zinc-binding) for 12 h, all of which were lab stock.

CRL-1730 human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured with 5% CO₂ in endothelial cell medium (Sciencell, Carlsbad, CA, USA) as previously described (17). For RNAi, oligofection of siRNA duplexes was performed using Oligofectamine (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. In brief, HUVECs were transfected twice (at 0 and 24 h) with 200 pmol of scramble siRNA or importin α1, importin β1 or nucleolin siRNAs. Following 24 h, the mRNA expression levels of target genes in transfected cells were detected by quantitative polymerase chain reaction (qPCR). The oligonucleotide sequences were as follows: importin α1, CGUUGUACCAGAAACUACC; importin β1, UCGGUUAUAUUUGCCAAGA; and nucleolin, GGCAAAGCAUUGGUAGCAA.

HUVECs were plated in 12-well plates at 4×10⁵ cells/well and transfected with pGL3 (empty vector) or pGL3-HIF-1α luciferase plasmid (lab stock) (15) using TurboFect (Thermo Fisher Scientific Inc., Waltham, MA, USA). Following 24 h, mNCL^wt and NLS deficient mNCL^mut were ectopically expressed in HUVECs cotransfected with si-nucleolin siRNA (avoiding the interference of endogenous nucleolin). The luciferase activity was measured in triplicate using the Bright-Glo Luciferase assay system (Promega Corporation, Madison, WI, USA).

HUVECs were incubated with bovine serum albumin (BSA), apo-endostatin (non-zinc-binding), BSA+ZnCl₂ or holo-endostatin (zinc-binding) for 12 h. Total RNA was isolated using an RNeasy kit (Qiagen, Hilden, Germany) and reverse transcribed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific Inc.). qPCR amplification was performed using the SYBR-Green qPCR Master mix kit (Stratagene, Santa Clara, CA, USA). The primers used were as follows: Forward: 5′-CGTTCC TTCGATCAGTTGTC-3′ and reverse: 5′-TCAGTG GTGGCAGTGGTAGT-3′ HIF-1α; forward: 5′-CAAGCT ACTCAAGCTGCCAG-3′ and reverse: 5′-CAACAG AGAAATGAATGCTG-3′ for importin α1; forward: 5′-CAA GGCACAATATCAGC-3′ and reverse: 5′-GCAGTC AGAATCTCATTGG-3′ importin β1; forward: 5′-CCTTCT GAGGACATTCCAAGACA-3′ and reverse: 5′-ACGGTA TTGCCCTTGAAATGTT-3′ for nucleolin; and forward: 5′-CGGCTACCACATCCAAGGAA and reverse: 5′-ACC ACCCTGTTGCTGTAGCC-3′ for GAPDH. Independent experiments were performed in triplicates.

HUVECs were fixed with 4% paraformaldehyde, permeabilized with phosphate-buffered saline (PBS) containing 0.2% Triton X-100, washed twice with PBS and blocked with PBS containing 10% normal goat serum for 15 min. Cells were stained with primary antibodies for 2 h, washed three times with PBS for 15 min and incubated with fluorescein isothiocyanate FITC-linked anti-mouse and anti-rabbit IgG antibodies for 30 min at room temperature. Nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Haimen, China). All immunofluorescence images were analyzed with a Nikon A1 laser scanning confocal microscope (63x/1.49 NA oil objective) and NIS-Elements AR software (Nikon Corporation, Tokyo, Japan).

Immunoprecipitation and immunoblotting assays were performed as previously described (15) and experiments were repeated at least twice.

For statistical analysis, the data are expressed as the mean ± standard deviation and compared using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

Abdollahi et al (8) have reported that the impact of endostatin on genetic expression is widespread in 12% of the genome. Notably, a number of hypoxia-associated genes are significantly downregulated by endostatin. Since HIF-1 signaling has been well-documented as an angiogenic inducer (18–20), the endostatin-induced repression of several HIF-1 or HIF-2-associated genes were examined by qPCR. As shown in Fig. 1A, the expression of all HIF-1α associated genes in HUVECs, including hif-1α, egln1, vegfr2, endothilin-1 and cyclin D1 was significantly suppressed by endostatin, while the HIF-2α-associated genes, including hif-2α and oct-4, were not affected. The protein levels of HIF-1α, VEGFR2, HIF-2α and Oct-4 were further detected. Consistently, the expression of HIF-1α and VEGFR2, but not HIF-2α and Oct-4, were suppressed by endostatin (Fig. 1B). To further confirm whether endostatin may directly repress the transcriptional activity of HIF-1α, HIF-1α-luciferase activity was detected following endostatin treatment. As shown in Fig. 1C and D, endostatin suppressed HIF-1α-luciferase activity in a time- and dose-dependent manner, while having little effect on HIF-2α-luciferase activity (Fig. 1E and F). These results indicate that endostatin inhibits HIF-1α expression at the transcriptional level.

A number of studies have reported that nuclear translocation is essential for the antitumor effects of endostatin (13,15). As treatment of human umbilical vein endothelial cells (HUVECs) with endostatin for 30 min leads to endostatin in cell nucleus (15), endostatin in the cell nucleus was defined as nuclear-tanslocated endostatin. Downregulation of HIF-1α was hypothesized to be modulated by nuclear-translocated endostatin. The importin α1β1/nucleolin complex has been observed to mediate endostatin nuclear translocation (15). In the current study, siRNAs were used to block importin-dependent translocation. The results showed that siRNAs that targeted importin α1 or β1 significantly eliminated the endostatin-mediated suppression of HIF-1α expression (Fig. 2A and B). In addition, endostatin exhibited little effect on HIF-1α-luciferase activity upon knock-down of importin α1 or β1 (Fig. 2C). To further verify that the downregulation of HIF-1α was modulated by the nuclear-tanslocated endostatin, wild-type mouse nucleolin (mNCL^wt) and NLS-deficient mutant nucleolin (mNCL^mut) were ectopically expressed in HUVECs co-transfected with si-nucleolin siRNA. Compared with mNCL^wt, the ectopic mNCL^mut in HUVECs significantly attenuated endostatin-mediated suppression of the HIF-1α mRNA level (Fig. 2D). These observations demonstrate that nuclear-translocated endostatin is critical in HIF-1α suppression.

The crystal structure of endostatin shows that endostatin is a Zn(II)-dependent protein (21) and other studies have reported that its Zn(II)-binding capacity is responsible for its anti-angiogenic activity (16,22). Moreover, the Zn(II) dissociation constant of endostatin was measured to be 6.7 nM (16), suggesting a marked Zn(II)-binding capacity. Thus, Zn(II) homeostasis in the nucleus was hypothesized to be regulated by the nuclear-translocated endostatin. To verify this hypothesis, HUVECs were incubated with BSA, apo-endostatin (non-zinc-binding), BSA+ZnCl₂ or holo-endostatin (zinc-binding) for 12 h. The cells were collected and analyzed, as shown in Fig. 3A–C. Compared with apo-endostatin, holo-endostatin had a reduced ability to suppress the mRNA and protein levels of HIF-1α and marginally downregulated the HIF-1α-luciferase activity. Previously, Fu and Luo (22) reported that the N-terminal 2–5 amino acid residues (HSHR) of endostatin were critical for its zinc binding. N-4 endostatin (Δ2-5 endostatin) was used to further confirm this observation. Consistently, N-4 endostatin treatment had little effect on HIF-1α expression and HIF-1α-luciferase activity (Fig. 3D–F). In addition, no significant differences among BSA, apo-N-4 endostatin, BSA+ZnCl₂ or holo-N-4 endostatin groups in regulating HIF-1α expression or HIF-1α-luciferase activity were identified (Fig. 4A–C). These results indicate that the competition for Zn(II) by nuclear-translocated endostatin results in the transcriptional inactivation of HIF-1α.

In this study, the inhibitory effect of endostatin on HIF-1α expression was investigated. Although HIF-1α itself is not a Zn(II)-binding protein, it activates the transcription of adaptive genes by recruiting a well-known co-activator, CBP/p300 in a Zn(II)-dependent manner (23). As a result of a reduction in free Zn(II) induced by nuclear-translocated endostatin, the interaction between HIF-1α and CBP/p300 may be interrupted and the HIF-1α-mediated transcription is likely to be further disturbed. Since HIF-1α is a self-regulated gene, the disrupted association of CBP/p300 with HIF-1α may also be caused by endostatin-induced HIF-1α suppression. To exclude this possibility, HUVECs were treated with endostatin for the indicated times and mRNA were detected by qPCR. As shown in Fig. 5A, endostatin treatment for 3 h showed little effect on HIF-1α mRNA. Therefore, HUVECs were treated with BSA, apo-endostatin, BSA+ZnCl₂ or holo-endostatin for 3 h and the cell immunofluorescence imaging was captured by confocal microscopy. As shown in Fig. 5B and C, compared with apo-endostatin, holo-endostatin only marginally interfered with the co-localization of CBP/p300 and HIF-1α. To further confirm this result, HUVECs were treated as described in Fig. 4B and the interaction between HIF-1α and CBP/p300 was evaluated by immunoprecipitation assay. Consistently, holo-endostatin treatment exhibited a marginal effect on the association of CBP/p300 with HIF-1α compared with the apo-endostatin group (Fig. 5D). These findings demonstrate that competition for Zn(II) by nuclear-translocated endostatin disrupts the interaction between CBP/p300 and HIF-1α.