Hep-2 human laryngeal cancer cells were obtained from Beinglay Biotech (Wuhan, China) and cultured at 37°C in a 5% CO₂ atmosphere in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin). rH was purchased from Combination Botai Biotechnology (Dalian, China) and used at final concentrations of 0.5, 1, 2 mg/ml or 25, 50, 100 μg/ml in the experiments. The drugs were prepared in RPMI-1640 medium before addition to the cell cultures. Cells treated with phosphate-buffered saline (PBS) were used as the negative controls, and cells treated with doxorubicin hydrochloride (DOX) (4 μg/ml) as the positive controls.

Hep-2 cells were seeded in 96-well plates at a density of 10⁴ cells/well in RPMI-1640 containing 10% FBS for 24 h and then treated with various concentrations of rH (0.5, 1 or 2 mg/ml) or DOX (4 μg/ml). Following treatment for 24 h, 10 μl/well of water-soluble tetrazolium salt (WST) (Beyotime, Beijing, China) was added, and the plates were incubated for an additional 4 h. The spectrometric absorbance at a wavelength of 570 nm was measured on a microplate reader. This experiment was repeated in triplicate.

Fibronectin (BD Biosciences, San Jose, CA, USA) was diluted to a final concentration of 100 μg/ml in PBS and 40 μl was added to each well of 96-well plates at 4°C overnight. Hep-2 cells were pretreated with various concentrations of rH (25, 50 or 100 μg/ml) or DOX (4 μg/ml) for 0.5 h. After treatment, 1×10⁵ cells were added to each well of 96-well plates coated with fibronectin and incubated for 2 h. The cells that did not adhere to the fibronectin were removed. The bound cells were fixed with 4% (w/v) paraformaldehyde and stained with crystal violet, followed by gentle rinsing with PBS and drying. Afterwards, 1% SDS was added, followed by mixing. Spectrometric absorbance at a wavelength of 540 nm was measured on a microplate reader. This experiment was repeated in triplicate.

A Transwell assay was performed using polycarbonate Transwell filters (Corning Costar, Cambridge, MA, USA). Briefly, cells were suspended in culture medium containing various concentrations of rH (25, 50 or 100 μg/ml) or DOX (4 μg/ml) and plated to the upper chamber with the bottom filled with culture medium containing basic fibroblast growth factor (bFGF) (Roche, Basel, Switzerland) (3 ng/ml). After incubation for 24 h, the cells on the upper side of the filters were removed mechanically, while cells that migrated to the bottom side were fixed in 4% (w/v) paraformaldehyde. The migrated cells were photographed and counted using Image-Pro Plus. Three independent experiments were performed.

For the invasion assay, modified polycarbonate Transwell filters (Corning Costar) coated with Matrigel (Matrigel basement membrane matrix; BD Biosciences) were used. The upper surface of the filter was coated with 60 μl Matrigel (4 mg/ml) at 37°C for 30 min. Cells were suspended in culture medium containing various concentrations of rH (25, 50 or 100 μ/ml) or DOX (4 μg/ml) and added to the upper chamber. The bottom chambers were filled with culture medium containing bFGF (3 ng/ml). After incubation for 16 h, cells on the upper surface of the filter were removed by scraping, and the invaded cells on the bottom side were fixed in 4% (w/v) paraformaldehyde. The invaded cells were photographed and counted using Image-Pro Plus software. Three independent experiments were performed.

For detection of cell apoptosis, the Hep-2 cells were stained with Hoechst 33324 (Beyotime). In brief, cells were plated on coverslips and treated with various concentrations of rH (0.5, 1 or 2 mg/ml) or DOX (4 μg/ml) for 24 h, followed by washing in PBS (pH 7.4) and fixing with 4% (w/v) paraformaldehyde at room temperature for 10 min. Following fixation, cells were washed three times in PBS (pH 7.4) and stained with Hoechst 33324 according to the manufacturer’s instructions. Stained nuclei were observed and photographed under a fluorescence microscope. Cells undergoing apoptosis demonstrated blue fluorescent nuclei (intact or fragmented). Three independent experiments were performed.

The chicken chorioallantoic membrane (CAM) assay is an established in vivo model for investigating the process of new blood vessel formation and vessel responses to anti-angiogenic agents (13). We thereby applied the CAM assay to assess the effect of rH on angiogenesis. All experiments were performed on day 6 of chick embryo development, when CAM and its vasculature are well-developed. A 1 cm² window was made in the shell under aseptic conditions. Next, a sterile methylcellulose disc was added with 20 μl of recombinant human bFGF (10 ng/μl) (Roche) or vehicle PBS and placed on the CAM. The window was sealed with sterile scotch tape and the egg was kept in an incubator at 37°C with 60% humidity for 24 h. Then, various concentrations of rH (0.5, 1 or 2 mg/ml) or PBS was pipetted onto the methylcellulose disc and the window was sealed again with sterile scotch tape. The egg was incubated for an additional 24 h, observed and photographed with a Nikon digital camera.

After the rH treatment, Hep-2 cells were washed with ice-cold PBS (pH 7.4) and scraped into lysis buffer (KeyGen Biotech, Nanjing, China). The lysate was collected by centrifugation at 12,000 × g for 15 min at 4°C, and the supernatant (total cell lysate) was stored at −80°C. Protein concentrations were determined with a BCA protein assay reagent (Beyotime). Proteins (60 μg) were separated using SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked with Tris-buffered saline (TBS; 137 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing 0.1% Tween-20 and 5% dried milk powder. Rabbit anti-mouse VEGF-R antibody (diluted 1:1,000), rabbit anti-mouse FAK antibody (diluted 1:1,000) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse Bcl-2 antibody (diluted 1:1,000; Bioworld Technology, USA) and rabbit anti-mouse Bad antibody (diluted 1:1,000; Proteintech Group, Chicago, IL, USA) were used to detect the corresponding proteins. Signals were developed with the enhanced chemiluminescence detection system (Beyotime). Relative intensities of the specific bands were quantified using Gel-Pro Analyser 4.0 software.

Statistical analysis was performed on SPSS version 16.0 (version 16.1; SPSS, Inc., Chicago, IL, USA). All values are expressed as means ± standard deviation (SD) of at least three independent experiments. p-values were determined by the Student’s t-test and one-way ANOVA, with p<0.05 considered to indicate a statistically significant result.

To investigate the effects of rH on the cell viability of human laryngeal cancer cells, Hep-2 cells were treated with varying concentrations of rH (0.5, 1 or 2 mg/ml) in parallel with a standard widely used clinical chemotherapy drug (DOX) for 24 h, and subjected to a WST assay. As shown in Fig. 1A, compared to the negative control group, treatment of cells with rH resulted in a significant reduction in cell viability in a dose-dependent manner. The inhibitory rate increased from 12.7 to 39.8% at 24 h as the concentration of rH was escalated from 0.5 to 2 mg/ml. The inhibitory effect of rH at 2 mg/ml was similar to that of DOX at a concentration of 4 μg/ml. Thus, rH inhibits the cell viability of Hep-2 human laryngeal cancer cells.

To ascertain the mechanism of the loss of cell viability following rH treatment, we detected the effect of rH on the apoptosis of Hep-2 cells. We first used Hoechst 33324 staining to observe the change of morphology after rH treatment. As shown in Fig. 1B–F, cells exposed to rH for 24 h showed apparent cell shrinkage, chromatin compaction and nuclear fragmentation, all of which are typical apoptotic morphological changes. Similar morphological alterations were observed in the Hep-2 cells treated with DOX (Fig. 1F). In contrast, no obvious apoptosis was observed in the negative control cells. These results indicated that induction of cell apoptosis is one of the mechanisms by which rH inhibits the proliferation of Hep-2 cells.

To further support the above conclusion, we treated Hep-2 cells with rH and DOX, and determined the expression of the apoptosis-associated proteins Bad and Bcl-2 by western blot analysis. One of the cytotoxic effects of DOX is the induction of apoptosis (14). As expected, DOX decreased the anti-apoptotic protein Bcl-2 (Fig. 1G), while it increased the pro-apoptotic protein Bad (Fig. 1H). Similarly, rH dose-dependently decreased Bcl-2 (Fig. 1G), yet increased Bad (Fig. 1H). These data suggest that rH inhibits cell viability and induces apoptosis in Hep-2 cells, at least partly, by regulating the levels of pro-apoptotic and anti-apoptotic proteins.

The adhesion of cancer cells to the extracellular matrix and cell surface molecules is a key step during metastasis in vivo (15). We next aimed to ascertain whether rH promotes the adhesion of Hep-2 cells to the extracellular matrix and cell surface molecules by testing the adhesion of Hep-2 cells to fibronectin. rH (50–100 μg/ml) significantly inhibited the adhesion of Hep-2 cells to fibronectin, while 25 μg/ml did not (Fig. 2). The reduction in cell adhesion capabilities following rH treatment at concentrations of 25 to 100 μg/ml was increased from 13.4 to 46.5%. The inhibitory effect of rH at 100 μg/ml was approximately equal to that of DOX at 4 μg/ml.

We further assessed the in vitro migration and invasion of Hep-2 cells following treatment with rH using the Boyden chamber model. Results from the migration assay showed that the number of cells that migrated to the lower side of the membrane was significantly reduced in the rH-treated groups in a dose-dependent manner, as compared to the untreated cells (Fig. 3A–D). Treatment of the cells with 25, 50 or 100 μg/ml rH for 24 h inhibited cell migration by 15.5, 24.9 and 43.1%, respectively (Fig. 3F). Treatment of Hep-2 cells with 4 μg/ml of DOX led to a 36.0% reduction in cell migration (Fig. 3E and F).

Movement of cells through Matrigel-coated Boyden chambers mimics the early steps of tumor invasion (16). We thus applied a Matrigel assay to test whether rH affects the invasion of Hep-2 cells and found that rH significantly decreased bFGF-induced cell invasion through the Matrigel in a dose-dependent manner (Fig. 4A–D and F). Relative to the untreated control, treatment of Hep-2 cells with 25, 50 or 100 μg/ml rH for 24 h inhibited the number of cells invading the lower chamber by 17.1, 29.0 and 39.8%, respectively. The inhibitory effect of 100 μg/ml of rH was approximately equal to that of 4 μg/ml DOX (Fig. 4E and F). These results demonstrated that rH significantly decreased the migratory and invasive capabilities of the Hep-2 cells.

Rapidly proliferating tumor cells rely on sustained angiogenesis, a hallmark of cancer cells (17). To determine whether rH suppresses microvessel formation in vivo, we performed a CAM assay using day 6 fertilized eggs. PBS-treated CAMs showed normal vascularization with primary, secondary and tertiary vessels and dendritic branching (Fig. 5A). In sharp contrast, rH-disc implanted CAMs demonstrated significantly reduced formation of new microvessels in a dose-dependent manner (Fig. 5B–D). rH significantly decreased the vessel area, vessel length and number of dendrites. Impressively, the addition of rH at 2 mg/ml totally blocked the microvessel formation (Fig. 5B–D). These results indicate that rH possesses the capacity for anti-angiogenesis.

VEGF and its corresponding receptor (VEGF-R) are important regulators of tumor angiogenesis. Moreover, VEGF-Rs are not only expressed on endothelia, yet also on different types of solid tumor cells and leukemic cells (18–20). FAK is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor-mediated signaling, and plays critical roles in cell migration and proliferation, processes vital for angiogenesis (21). The increased expression of FAK in cancer cells has been suggested to play a role in the tumor angiogenic switch to promote aggressive tumor progression and metastasis (22). To assess the mechanism of the inhibition of angiogenesis and explore the possible molecules involved in rH-induced reduction of migration and invasion in Hep-2 cells, we determined the expression of VEGF-R and FAK by western blot analysis. Compared to PBS, DOX apparently reduced the protein levels of VEGF-R and FAK. Exposure of Hep-2 cells to rH for 24 h resulted in decreased expression levels of VEGF-R and FAK in a dose-dependent manner (Fig. 5E and F), suggesting that rH reduces adhesion, migration, invasion and angiogenesis of cancer cells through suppressing VEGF-R and FAK expression.