The human osteosarcoma cell line Saos-2 was purchased from the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, China); McCOY’s 5A, fetal bovine serum (FBS), trypsin, methabenzthiazuron (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA); resveratrol was obtained from MP Biomedicals, Inc. (Aurora, OH, USA). Antibodies were purchased from the following sources: anti-human anti-HIF-1α, -E-cadherin and -vimentin from Cell Signaling Technology (Boston, MA, USA); anti-human anti-β-actin, goat-anti-mouse and anti-rabbit IgG, and rabbit anti-goat IgG from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HIF-1α small interfering (si)RNA and siRNA controls were obtained from Santa Cruz Biotechnology, Inc.; Transwell chambers from EMD Millipore (Billerica, MA, USA); Invitrogen^® Lipofectamine 2000 from Thermo Fisher Scientific (Waltham, MA, USA); PCR mix from Xi’an Runde Biotechnology (Xi’an, China); and PCR primer sets from Dingguo Biotechnology Co., Ltd. (Beijing, China).

The human osteosarcoma Saos-2 cell line was cultured in McCOY’s 5A medium supplemented with 15% FBS, 100 μg/ml ampicillin and 100 μg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂. In experiments designed to evaluate the role of hypoxia, cells were first incubated in normoxic conditions to acquire the desired subconfluence level (65–70%), and then cultured in controlled hypoxic conditions (3% O₂), as previously described (36,37) for up to 24 h. Osteosarcoma cells at the exponential phase were incubated in six orifice plates in McCOY’s 5A supplemented with 1% FBS for 24 h. The drugs (or solvent only) were then added to the medium containing 1% FBS, and cells were cultured for an additional 24 h prior to the Matrigel invasion assay.

Each group of cells was seeded onto a 96-well plate at a density of 5,000–10,000 cells/well for 24 h prior to serum starvation for 24 h. Following starvation, cells were incubated in McCOY’s 5A medium supplemented with 15% FBS. After 12, 24, 48 or 72 h, the medium was removed, and the MTT reagent (5 mg/ml) was added in each well for a 4-h incubation at 37°C. Optical densities (OD) were measured at 490 nm on a microplate reader (BioTek Instruments, Inc., Winooski, VA, USA). The proliferation rate was calculated as OD (sample)/OD (control). Reported values are the average of triplicate experiments.

The invasive ability of each group of cells was evaluated though a Transwell chamber-based invasion assay. Briefly, the upper surface of the Transwell filter was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Before treatment, cells at the exponential phase were incubated in McCOY’s 5A medium supplemented with 1% FBS for 24 h in 6-well plates. The cells (5×10⁴) were resuspended in serum-free medium and allowed to migrate towards a serum gradient (20%) in the lower chamber for 24 h, in a humidified tissue culture incubator. Non-invasive cells were removed from the top of the Matrigel by scraping with a cotton-tipped swab. Invasive cells at the bottom surface of the filter were fixed with 4% paraformaldehyde and stained with Crystal Violet dye (Boster Biological Technology, Ltd., Wuhan, China). The number of invasive cells was counted under a light microscope (Axio Observer A1; Carl Zeiss Microscopy GmbH, Jena Germany) at 10 random fields for each membrane, and pictures were acquired at ×100 magnification with a AxiocaCam MRc5 camera (Carl Zeiss Microscopy GmbH). The tumor cell invasion assay was performed in triplicate.

Saos-2 cells were washed with ice-cold phosphate-buffered saline and lysed in situ with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1.0% Triton X-100, 0.1% SDS), supplemented with protease inhibitors (Roche Diagnostics, Penzberg, Germany) and phosphatase inhibitors (Sigma-Aldrich). Following incubation on ice for 30 min, cell lysates were centrifuged at 12,000 × g for 15 min at 4°C to remove the debris. Proteins (100 μg) were separated by 10% SDS-PAGE and were transferred to polyvinylidene difluoride (PVDF) membranes (Roche Diagnostics). Then, the PVDF membranes were blocked with 5% non-fat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and incubated with the primary antibodies overnight at 4°C. After washing with TBST five times for 10 min each, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The membranes were washed again with TBST and an enhanced chemiluminescence (ECL) kit (USCN Life Science Inc., Wuhan, China) was used to visualize the immunoblots.

Total RNAs were extracted from osteosarcoma cells with the Invitrogen^® TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol, and the RT reaction was performed using a PrimeScript RT Reagent kit (Takara, Dalian, China). cDNAs (1 μl for each sample) were amplified by PCR using the following primer sequences: HIF-1α forward (F), 5′-AAGTCTAGGGATGCAGCA-3′, and reverse (R), 5′-CAAGATCACCAGCATCATG-3′; E-cadherin F, 5′-ATTCTGATTCTGCTGCTCTTG-3′, and R, 5′-AGTCCT GGTCCTCTTCTCC-3′; vimentin F, 5′-AATGACCGCTTC GCCAAC-3′, and R, 5′-CCGCATCTCCTCCTCGTAG-3′; β-actin F, 5′-ATTCTGATTCTGCTGCTCTTG-3′, and R, 5′-AGTCCTGGTCCTCTTCTCC-3′. β-actin was used as the normalization control. The amplified products were observed by 1.5% agarose gel electrophoresis. Images of the gel were acquired and analyzed under a UV transilluminator. The RT-PCR assay was carried out in triplicate.

siRNAs targeting the HIF-1α gene (HIF-1α-Homo-2258: 5′-CCACCACUGAUGAAUUAAATT-3′ and 5′-UUUAAUUCAUCAGUGGUGGTT-3′) and negative control siRNAs (NC: 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from Santa Cruz Biotechnology, Inc. Cells (10⁵/well) seeded in 6-well plates were transfected with 100 nM siRNA using the Lipofectamine 2000 reagent according to the manufacturer’s instructions. The cells were used for further experiments at 48 h after transfection.

Data were expressed as the mean ± standard deviation. Differences were evaluated using unpaired two-tailed Student’s t-tests with unequal variance for multiple comparisons using the SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). P-values <0.05 were considered to indicate statistically significant differences. All experiments were independently repeated at least three times.

To investigate the potential role of resveratrol on osteosarcoma Saos-2 cell proliferation under hypoxic conditions, normal (grown under normoxia) cells, hypoxic cells and hypoxic cells pre-treated with resveratrol were seeded onto 96-well plates at different time-points, and the proliferative rate of in each group was investigated by the MTT assay. Hypoxia markedly increased the proliferation rate of Saos-2 cells as compared to the normal cell group, and resveratrol significantly reduced the proliferation rate of the hypoxic cell group at different time-points (Fig. 1).

We further tested the effect of resveratrol on the Saos-2 cell invasive ability under an hypoxic microenvironment. The results showed that hypoxia significantly increases the number of invasive Saos-2 cells as compared to normoxia, and resveratrol markedly reduced the invasive ability of hypoxic cells (Fig. 2).

To investigate whether Saos-2 cells undergo EMT as a result of exposure to hypoxia, we examined the expression of markers of epithelial and mesenchymal phenotypes by western blot analysis. Hypoxic cells displayed decreased E-cadherin and increased vimentin expression (Fig. 3). However, when hypoxic cells were pre-treated with resveratrol, the hypoxia-induced EMT process was reversed. Resveratrol treatment caused a marked increase in the expression of E-cadherin, and a significant decrease in the expression of vimentin (Fig. 3).

We further investigated whether resveratrol inhibits HIF-1α expression, which was induced in tumor cells upon exposure to hypoxic stress, a common condition in aggressive solid tumors. We observed that pretreatment of Saos-2 cells with resveratrol reduced the hypoxia-induced HIF-1α protein accumulation without affecting the HIF-1α mRNA level (Fig. 4). Quantitative data from the three repeated experiments, using β-actin as the normalized control, showed the relative HIF-1α mRNA expression level (± standard deviation) in each group was 0.136±0.042, 0.758±0.084 and 0.728±0.080 for the normal, hypoxia and hypoxia + resveratrol groups, respectively. Furthermore, quantitative data from the three repeated experiments revealed the relative HIF-1α protein expression levels (± standard deviation), using β-actin as the normalization control, were 0.323±0.050, 0.842±0.096 and 0.419±0.046 for the normal, hypoxia and hypoxia + resveratrol groups, respectively. These results indicate that the HIF-1α protein level was signincantly decreased in the hypoxia + resveratrol group, when compared with the hypoxia group (P<0.05), however, no significant difference in HIF-1α mRNA expression was identified (P>0.05).

Next, we investigated whether resveratrol attenuates the effects of hypoxia through downregulation of the HIF-1α gene in Saos-2 cells. We transiently silenced HIF-1α and analyzed its expression and cell growth under hypoxic conditions (Fig. 5A and B). In the si-control transfected group of cells exposed to hypoxic conditions, treatment with resveratrol significantly reduced the proliferation rate (Fig. 5C), increased the level of E-cadherin and decreased that of vimentin (Fig. 6A). Furthermore, resveratrol significantly decreased the invasive ability of Saos-2 cells, which was induced by hypoxia (Fig. 6B). In the si-HIF-1α-transfected group, however, resveratrol had no significant effect under hypoxic conditions (Figs. 5C and 6). These findings suggest that HIF-1α expression plays a vital role in the resveratrol-abrogated effects of hypoxia in Saos-2 cells.