Melatonin, trypsin, MTT and Triton X-100 were purchased from Sigma Chemical Co./Merck KGaA. Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco Laboratories (Thermo Fisher Scientific, Inc.); TGF-β1 was purchased from (R&D); YC-1 (cat. no. sc-202856) was purchased from Santa Cruz Biotechnology, Inc. Antibodies against MMP-9 (cat. no. sc-13520), E-cadherin (cat. no. sc-52327), N-cadherin (cat. no. sc-8424), vimentin (cat. no. sc-53464), Snail (cat. no. sc-10437), β-actin (cat. no. sc-69879) and HIF-1α (cat. no. sc-53546) were purchased from Santa Cruz Biotechnology, Inc. The ECL kit was purchased from Pierce/Thermo Fisher Scientific, Inc. RIPA buffer and the BCA protein assay kit were purchased from Beyotime. PVDF membranes were purchased from Millipore. All reagents used were trace element analysis grade. All water used was glass distilled.

OS MG-63 cells were purchased from the Shanghai Cell Bank (Shanghai, China). The cells were treated with DMEM containing 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO₂ with 95% humidity. Cells were passaged at ~80% confluency.

MG-63 cells were seeded into 96-well plates at a density of 2×10⁴ cells/well and exposed to 0–1,000 nmol/l) melatonin for 24 h. MTT reagent (10 µl) was added to each well and incubated for 4 h at 37°C. Reaction products were extracted with DMSO (150 µl) and absorbances were recorded at ~450 nm on a microplate reader (Bio-Rad Laboratories, Inc.).

MG-63 cells were lysed in RIPA buffer and BCA assays performed. Proteins (10 µg) were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in TBS (containing 0.5% Tween-20) and probed with primary antibodies at 4°C overnight. The antibodies included: Anti-β-actin (dilution 1:400), anti-HIF-1α (dilution 1:400), anti-E-cadherin (dilution 1:400), anti-N-cadherin (dilution 1:400), anti-vimentin (dilution 1:400), anti-Snail (dilution 1:400), and anti-MMP-9 (dilution 1:400). After washing three times with TBS/0.1% Tween 20, the membranes were labeled with HRP-conjugated secondary antibodies (cat. no. sc-2030; dilution 1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Immunoreactive bands were visualized using ECL. The intensity of the bands was quantified using Image Lab software (version 2.1, Bio-Rad Laboratories, Inc.). All blots were representative of three independent experiments.

MG-63 cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 for 5 min and blocked in 10% AB-serum in 1% bovine serum albumin (BSA) for 30 min. Cells were then washed and stained with anti-E-cadherin primary antibodies (dilution 1:400) for 2 h at 37°C and incubated with TRITC-conjugated fluorescent secondary antibodies (cat. no. BA1089; dilution 1:100) for 30 min at room temperature. Nuclei were stained with Hoechst 33342 for 10 min and cell morphology was examined under an optical microscopy (magnification, ×400; Olympus Corporation).

Snail was cloned into pcDNA3.1 (Genechem Co.) and transiently transfected into MG-63 cells using Lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific, Inc.). Cells were harvested 48 h post-transfection.

Total RNA was extracted using TRIzol (Invitrogen/Thermo Fisher Scientific, Inc.) and reverse transcribed using SYBR PrimeScript RT-PCR kits (Takara Inc.) according to the manufacturer's protocol. cDNAs were amplified by polymerase chain reaction (PCR) using the primers shown in Table I. PCR reactions were performed using a Gene Amp PCR system 9700 (PerkinElmer). Amplified products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. Images were quantified using FluoroImager SI (GE Healthcare). Representative results were shown (n=3).

All statistical analyses were performed using SPSS (version 19.0; IBM Corp.). Data are represented as the mean ± SD. One-way ANOVA test was used for statistical comparisons. If multigroup comparisons were made, then ANOVA was used together with Scheffe post-hoc test (n=5). P<0.05 was considered to indicate a statistically significant difference.

EMT is key to cancer progression and can be induced by TGF-β (14). MG-63 cells were cultured with TGF-β1 (20 ng/ml) to assess its ability to induce EMT in OA cells (15) through the expression of known EMT markers including E-cadherin, vimentin and N-cadherin by western blot analysis (Fig. 1A) and RT-PCR (Fig. 1B). E-cadherin was downregulated, while N-cadherin and vimentin were significantly induced by TGF-β1 in a time-dependent manner. These data suggested that TGF-β1 triggers EMT in OA cells.

Previous studies have reported that melatonin inhibits tumor invasion through EMT inhibition (16) but its effects on OA cells are unclear. Through MTT assays, no significant changes were observed in cell survival rates for the different concentrations (0, 50, 100, 200, 500 and 1,000 nM) of melatonin (Fig. 2A). In melatonin-containing media, the morphology of the MG-63 cells was unchanged, and no apoptosis occurred (Fig. 2B). In subsequent experiments, 200 nM (intermediate concentration) of melatonin was used which had minimal effect on MG-63 cell survival. However, immunofluorescence and western blot analysis suggested that melatonin partially reversed the loss of E-cadherin expression and increase in N-cadherin and vimentin expression in response to TGF-β1 (Fig. 2C and D). These results show for the first time that melatonin can reverse EMT processes in OA cells.

Extensive research indicates that the Snail/MMP-9 signaling plays a vital role in EMT and tumor metastasis (17). To further explore the underlying mechanism of the inhibitory effects of melatonin on TGF-β1-mediated EMT, Snail/MMP-9 signaling were analyzed using western blot analysis. Fig. 3A and B shows that the levels of Snail and MMP-9 were upregulated in response to TGF-β1 in a time-dependent manner. In addition, TGF-β1 activated Snail/MMP-9 signaling while melatonin alone had no effects on Snail/MMP-9 activation. The addition of melatonin to TGF-β1-stimulated cells reversed the activation of Snail/MMP-9 signaling (Fig. 3C and D). Similarly, the effects of melatonin on HIF-1α expression suggested that melatonin attenuated TGF-β1 signaling through HIF-1α (Fig. 3E and F). Snail expression in response to TGF-β1 was markedly downregulated in cells pretreated with melatonin. Taken together, these data indicate that melatonin exerts its inhibitory effects in part by antagonizing Snail/MMP-9 and HIF-1α pathways in OA cells.

The data obtained to this point suggested that Snail/MMP-9 signaling regulates EMT. To further investigate the effects of melatonin on Snail/MMP-9 signaling, Snail was overexpressed in MG-63 cells (Fig. 4A). Snail overexpression was coupled to a marked reduction in E-cadherin and increased expression of vimentin/N-cadherin. The melatonin-mediated suppression of EMT in MG-63 cells was attenuated through Snail overexpression (Fig. 4B). These data further confirmed that melatonin suppresses Snail/MMP-9 signaling to inhibit EMT in OA cells.

HIF-1α can induce EMT and metastasis in cancer cells (18). Next, it was ascertained whether a loss of HIF-1α negatively affects the Snail/MMP pathways. As shown in Fig. 5, the HIF-1α inhibitor YC-1 not only downregulated HIF-1α expression, but markedly inhibited the upregulation of Snail and MMP-9 in response to TGF-β1. These data provide evidence that HIF-1α activates Snail/MMP-9 expression and that inhibition of HIF-1α attenuates EMT in MG-63 cells.