The human MG-63 OS cell line (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was cultured in Eagle's minimum essential medium (Gibco Life Technologies, Grand Island, NY, USA), supplemented with 1% non-essential amino acids, 10% fetal bovine serum, 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/ml streptomycin (Sigma-Aldrich), and incubated at 37°C with 5% CO₂ in a humidified incubator.

A total of ~5×10² cells were seeded into each well of a 96-well plate and incubated at 37°C with 5% CO₂ in a humidified incubator. Following incubation for 24 h, the botanical extracts (diosmin, lemon bioflavonoids, neosperidin dihydrochalcone, Melissa rosemary acid, oleanolic acid, tartaric acid, ellagic acid, neohesperidin, phillyrin, betaine anhydrous, panax quinquefolium saponin, paeoniflorin, solanesol and resveratrol) that were purchased from Dalian Zhuoer Hightechnology Co., Ltd. (Dalian, China) were added and incubated for 48 h. The final concentration of each botanical extract was 10 µg/ml. Finally, the expression of β-catenin in the MG-63 cells treated with botanical extract was assessed by immunofluorescent staining. In brief, the cells were fixed with −20°C methyl alcohol for 20 min. Following 10 min of natural drying, the cells were washed with phosphate-buffered saline (PBS) three times, 0.2% Triton X-100 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was used for cell permeabilization (3 min). Following an additional wash with PBS, the samples were sealed with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 30 min. The monoclonal rabbit anti-human anti-β-catenin primary antibody (1:400; ab32572; Abcam, Cambridge, MA, USA) was diluted in 1% BSA, added into the samples and incubated at 4°C overnight. The next day, polyclonal bovine anti-rabbit aminomethylcoumarin-coupled secondary antibodies (IgG; 1:4,000; Wuhan Amyjet Scientific Co., Ltd., Wuhan, China) were added for another 30 min of incubation in the dark. The samples were washed with PBS three times and sealed with 95% glycerinum (Sinopharm Chemical Reagent Co., Ltd.), then observed and photographed under a ×100 magnification using a fluorescence microscope (TFM-680; Shanghai Tuming Optical Instrument Co., Ltd., Shanghai, China).

The cells were seeded into a 96-well plate at a density of 5×10² cells/well in 100 µl culture medium. The cells were cultured for 24 h at 37°C in 5% CO₂ in a humidified incubator. The botanical extract, resveratrol, was added to the cells at a final concentration of 10, 20 or 40 µg/ml. Following treatment for 24, 48, 72 or 96 h at 37°C, 100 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into the culture well and the cells were incubated for 4 h at 37°C with 5% CO₂ in a humidified incubator. Following incubation, the supernatant was discarded and 200 µl dimethyl sulfoxide was added and incubated for 5 min on an oscillator (HZP-250; Shanghai Jinghong Laboratory Equipment Co., Ltd.). Finally, the optical density at 450 nm (OD₄₅₀) was measured using a microplate reader (iMark Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a cell growth curve was produced.

The Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime Institute of Biotechnology) was used to detect cell apoptosis, as previously described (24). The cells were harvested subsequent to treatment with resveratrol alone or in combination with the GSK3β inhibitor chir99021, which was used to investigate whether resveratrol may target the Wnt signal pathway, and a single cell suspension was prepared using trypsin (0.25%; Gibco-BRL, Grand Island, NY, USA). The samples were washed with PBS, and subsequently, centrifuged at 157 × g and 4°C for 5 min. The cells were resuspended in binding buffer (BD Biosciences, San Diego, CA, USA), which consisted of 10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl and 5 mM CaCl₂, and the cell density was adjusted to 5×10⁵ cells/ml. A 95-µl aliquot of the cell suspension was mixed with 5 µl Annexin V-FITC prior to incubation in the dark at room temperature for 10 min. Subsequently, the suspension was washed with PBS and resuspended in 190 µl binding buffer, 10 µl propidium iodide (PI; Sigma-Aldrich) was added. The samples were examined using a flow cytometer (BD FACSVantage; BD Biosciences) with a 488 nm excitation source. The test wavelength was set at 515 nm for FITC and 560 nm for PI. The results were analyzed using CellQuest software (version 5.2.1; BD Biosciences) to determine apoptosis rate.

The total protein from the cells was extracted using radioimmunoprecipitation lysis buffer, containing 1 mM phenylmethylsulfonyl fluoride (Sinopharm Chemical Reagent Co., Ltd.), and the concentration was determined using the Bradford Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The protein sample (20 µg) was separated on 10% SDS-PAGE gels (Life Technologies, Gaithersburg, MD, USA) and was subsequently transferred onto nitrocellulose membrane (Sigma-Aldrich). The membrane was blocked in PBS, containing Tween-20 (PBST) and 5% non-fat milk for 1 h at 4°C. The membrane was subsequently incubated in primary monoclonal rabbit anti-human antibodies from Abcam against c-Myc (1:100; ab32072), β-catenin (1:400; ab32572) and GAPDH (1:4,000; ab181602), for 12 h at 4°C. Following antibody incubation, the membrane was washed three times with PBST and incubated for 30 min at 4°C with the goat anti-rabbit polyclonal secondary antibody (dilution 1:5,000; Beijing Boer Xi Technology Co., Ltd., Beijing, China) labeled with horseradish peroxidase. Finally, the membrane was washed three times with PBST and the protein bands were visualized with SuperSignal (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were re-probed with a monoclonal rabbit anti-human β-actin antibody (1:1,000; A2066; Sigma-Aldrich) as a loading control at 37°C.

The total RNA was extracted from the tissue/cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The total RNA (0.5 µg) was used as a template to prepare the cDNA in a 10 µl reaction, which included 1 µl (50 µM) Oligo d (T), 0.5 µl dNTP mixture (10 mM of each; Takara, Bio, Inc., Otsu, Japan), 0.25 µl RNase inhibitor (40 U/µl, Takara, Bio, Inc.) and 0.5 µl RTase M-MLV (200 U/µl; Takara, Bio, Inc.). The reaction conditions were as follows: 42°C for 1 h, followed by 85°C for 5 sec. The cDNA was diluted five-fold and was used as the PCR template. A SYBR^® Premix Ex Taq™ kit (Takara, Bio, Inc.) was used to detect gene expression and was performed using a PCR system (qTOWER 2.2, Analytik Jena, Jena, Germany), according to the manufacturer's instructions. Briefly, 2 µl cDNA was added to reaction system with 10 µl SYBR^® Premix Ex Taq (2X), 0.4 µl PCR primer mixture (10 µM each) and 7.6 µl dH₂O. The primer sequences from Sangon Biotech Co., Ltd. (Shanghai, China) used were as follows: β-catenin, forward 5′-TTCGCACAGTTCTACGTGCT-3′ and reverse 5′-GGTGTGCACGAACAA GCA AT-3′; c-Myc forward 5′-CTTCTCTCCGTCCTCGGATTCT-3′ and reverse 3′-GAAGGTGATCCAGACTCTGACCTT-3′; β-actin forward 5′-GCACCACACCTTCTACAA-3′ and reverse 5′-TGCTTGCTGATCCACATCTG-3′. The cycling conditions were as follows: 95°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C for 30 sec; followed by a melting curve stage in which the temperature increased from 60–95°C at a rate of 5°C/sec. The actin gene was used as a control to normalize the relative expression levels of the target gene. The 2^−ΔΔCt method was performed to calculate the relative expression of the target gene (25).

All data are presented as the mean ± standard deviation. Statistical analysis of the data was performed using Student's t-test with SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.01 was considered to indicate a statistically significant difference.

β-catenin is one of the members of the canonical Wnt signaling pathway. The dysregulation of the canonical Wnt signaling pathway has been demonstrated in a variety of tumors. In order to screen for drugs, which effectively inhibit the expression of β-catenin, high content screening was performed in the human MG-63 OS cell line. The present study assessed 14 botanical extracts. The results demonstrated that, of the 14 botanical extracts, resveratrol markedly inhibited the protein expression of β-catenin, whereas no pronounced changes were observed in the remaining 13 botanical extracts (Fig. 1). Therefore, it was hypothesized that resveratrol may be used as a potential drug for the treatment of OS.

In order to confirm the hypothesis that resveratrol may benefit OS treatment, a CCK-8 assay was performed to detect the proliferation rate of the human MG-63 OS cells treated with resveratrol. The results demonstrated that resveratrol significantly (P=0.043, 20 µg/ml treatment group, 3 days; P=0.006, 40 µg/ml treatment group, 3 days; P=0.037, 10 µg/ml treatment group, 4 days; P=0.002, 20 µg/ml treatment group, 4 days; P=0.0009, 40 µg/ml treatment group, 4 days; treatment v.s. control). inhibited the proliferation of the human MG-63 OS cells. Furthermore, the inhibition rate was dose-and time-dependent (Fig. 2).

The apoptotic changes in the human MG-63 OS cells treated with resveratrol were determined using flow cytometry. The results revealed that the apoptotic rate of the cells increased significantly (P=0.046) following 72 h drug treatment, and the apoptotic rate reached 27.5% in the group treated with 40 µg/ml resveratrol (Fig. 3D). In the Wnt signaling pathway, GSK3β is a negative regulator, which is involved in the degradation of β-catenin (26). In the present study, chir99021, an inhibitor of GSK3β, was used to inhibit GSK3β. Following treatment with resveratrol and chir99021, cell apoptosis was detected using flow cytometry. As shown in Fig. 3, the apoptotic rate decreased markedly, compared with the control groups. These results suggested that resveratrol inhibited the proliferation of the human MG-63 OS cells through the apoptotic pathway.

To further understand the mechanism underlying the effects of resveratrol, western blotting and RT-qPCR were performed following treatment with resveratrol alone or combined with chir99021. As shown in Fig. 4, β-catenin was markedly (mRNA level: P=0.003, 20 µg/ml treatment group; P=0.0017, 40 µg/ml treatment group; resveratrol + DMSO, treatment v.s. control) downregulated at the protein and mRNA expression levels following treatment with resveratrol, however, the expression of β-catenin increased following treatment with resveratrol and chir99021. In addition, c-Myc, one of target genes of the Wnt signaling pathway, was also detected. c-Myc was markedly (mRNA level: P=0.003, 20 µg/ml treatment group; P=0.0023, 40 µg/ml treatment group; resveratrol + DMSO treatment v.s. control) downregulated at the protein and mRNA expression levels following treatment with resveratrol. When the inhibitor, chir99021, was added, the expression of c-Myc increased.