Taxifolin was purchased from Shanghai Huicheng Technology Ltd. (Shanghai, China). MTT, cell culture dishes and Hoechst 33258 were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The primary antibodies, mouse monoclonal anti-Akt serine/threonine kinase 1 (Akt1; cat. no. sc-135829; 1:1,200), rabbit polyclonal anti-phosphorylated (p-Ser473) Akt (cat. no. sc-7985-R, 1:1,000), mouse monoclonal anti-v-myc avian myelocytomatosis viral oncogene homolog (c-myc; cat. no. sc-70469, 1:800), mouse monoclonal anti-S-phase kinase associated protein 2 [SKP-2; cat. no. sc-74477 (p45), 1:1,000] and mouse and rabbit monoclonal anti-β-actin (cat. no. sc-8432/sc-130656, 1:1,000) as well as secondary antibody, rabbit anti-mouse IgG HRP (cat. no. sc-358914, 1:10,000) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The human osteosarcoma cell lines, U2OS and Saos-2, were obtained from the American Type Cell Culture Collection (Manassas, VA, USA). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator with 5% CO₂ and 95% air at 37°C.

Cells (U20S/Saos-2) were cultured at a density of 4×10³ cells/well in 96-well plates for 24 h. Increasing concentrations of taxifolin (0, 5, 10, 25 and 50 µM) were then added to the cells in 100 µl additional medium, and cells were cultured for a further 96 h. The medium was removed by aspiration and 100 µl water was added into the wells and stored overnight at −80°C. Plates were then thawed at 37°C, and cells were stained by the addition of buffer containing 10 mM Tris (pH 7.4) and 1 mM EDTA with Hoechst 33258 (0.2%) dye at 37°C for 1 h. The relative DNA content was assayed by measuring Hoechst 33258 fluorescence using a Wallac 1420 Victor2 Multilabel microplate reader (Perkin Elmer Inc., Waltham, MA, USA) at excitation and emission wavelengths of 355 and 460 nm, respectively (16).

Cell viability was measured using the MTT assay. U2OS and Saos-2 cells were seeded at a density of 4×10³ cells/well in 96-well plates for 24 h, and then treated with increasing concentrations of taxifolin (0–50 µM) for a further 48 h. A total of 50 µl MTT solution (2 mg/ml) was added to each well, and plates were incubated for 4 h. The plates were then centrifuged at 1,000 × g for 5 min at 37°C, to remove unconverted MTT and to leave the resulting formazan crystals at the bottom of the plates. The crystals were then dissolved in 100 µl dimethyl sulfoxide (DMSO), and the absorbance was read at 570 nm using a Titertek Multiskan MCC/340 plate reader (Titertek-Berthold, Berthold Detection Systems GmbH, Pforzheim, Germany). The results were presented as a relative percentage of the untreated control cells (17).

U2OS and Saos-2 cells were seeded at 6×10⁶ cells/dish in 100 mm dishes and cultured for 24 h prior to taxifolin treatment. Cells were treated with taxifolin (50 µM) and incubated for 48 h. Cells were then fixed using 70% ice-cold ethanol diluted in phosphate-buffered saline (PBS) for ~12 h at 37°C. Following fixation, cells were washed with PBS and incubated with PBS containing RNase A (0.1 mg/ml; Sigma-Aldrich; Merck KGaA) for 30 min at 4°C, before they were resuspended in 50 µg/ml propidium iodide (Sigma-Aldrich; Merck KGaA). The cell cycle distribution was determined using a BD FACScan flow cytometer (BD Biosciences) and the data were analyzed using the BD CellQuest Pro software program (version 5.1; BD Biosciences).

In order to determine soft agar colony formation, 6×10³ U2OS and Saos-2 cells were diluted in complete DMEM medium containing agar (0.3%) in 35-mm cell culture plates. The cells were cultured for 3 weeks in a 5% CO₂ incubator at 37°C and treated with different concentration of taxifolin (0, 25 and 50 µM). The colonies were stained with crystal violet (0.5 mg/ml) for 10 h at 37°C.

A total of 16 male BALB/c nude mice (age, 6–8 weeks; weight, 15–18 g) were procured from the National Cancer Institute at Frederick, Laboratory Animal Sciences Program (Frederick, MD, USA). BALB/c nude mice were maintained in a steel cage, under a 12-h dark/light cycle with free access to food and water. All the mice were subcutaneously injected with 5×10³ U2OS cells suspended in Matrigel (0.5 ml), and tumor growth was monitored weekly from 1 week post-injection. Mice were divided into control (8 mice with 14 tumors) and drug-treated (8 mice with 13 tumors) groups. At 21 days post-inoculation, mice in the drug-treated group were administered with 25 mg/kg taxifolin intraperitoneally (i.p) once every 2 days for remaining 24 days. The control mouse group was administered with an equal volume of saline (vehicle). The treatment was terminated at 45 days post-inoculation. Tumor size was measured using calipers and tumor volume was calculated using the following formula: Volume = length × width × height × 0.52 (18). Mouse body weight and tumor volumes were expressed as the mean ± standard deviation (SD). The present study was approved by the Ethics Committee of Hanzhong Centre Hospital (HCH-4665; Hanzhong, China).

U2OS and Saos-2 cells (1×10⁴) were treated with different concentrations of taxifolin (0, 25 and 50 µM) for 72 h at 37°C and lysed using lysis buffer containing 0.5% sodium deoxycholate, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% nonidet P-40, 0.1% SDS, 1 mM dithiothreitol, 5 mM EDTA, 50 mM sodium fluoride, and 10 µg/ml aprotinin by incubating for 1 h at 37°C. The resultant cell lysates were centrifuged at 3,000 × g for 10 min at 37°C and total cellular protein concentration was determined using a BCA protein assay kit (BioVision, Inc., Milpitas, CA, USA). Protein extracts (~60 µg) were separated by 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. The membrane was blocked with TBS containing Tween-20 and 5% skimmed milk for 2 h at 37°C and incubated with primary antibodies against Akt, p-S473-Akt, c-myc, SKP-2 and β-actin at 4°C overnight, followed by probing with secondary antibody for 2 h at 37°C. Detection of bands was performed using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, Chalfont, UK), and band densities were measured using Scion Image software (version 4.0) from Scion Co. (Frederick, MD, USA).

U2OS cells (5×10⁵) were plated in 6-well plates for 24 h prior transfection and then U2OS cells were transfected with SKP-2 and AKT overexpression plasmids (1 µM) like pcDNA3.1-SKP2, pcDNA3.1-AKT (designed by Nanjing Keygen Biotech Co., Ltd., Nanjing, China) and an empty vector (pcDNA3.1) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), for 24 h. Finally the transfected cells were treated with 0 or 25 µM of taxifolin for 48 h. Transfection of U2OS cells with the empty pcDNA3.1 vector served as a negative control (19).

Briefly, U2OS cells (1×10⁴) were plated in 6-well plates for 24 h prior to transfection. Cells were treated with 0, 25 and 50 µM taxifolin for 48 h, then trypsinized (centrifuged at 600 × g for 10 min at 37°C) and washed with PBS to remove adherent cells. A total of 1×10³ treated cells diluted in 200 µl serum-free DMEM were added to the upper compartment of transwell migration chambers (BD Biosciences), and DMEM containing 10% FBS was added to the lower compartment. The chambers were then incubated at 37°C overnight. Cells that had migrated to the underside of the transwell filters were fixed with 70% methanol and stained with 10% Giemsa solution at room temperature for 1 h. Filters were washed with water and quantification was performed by analyzing 6 random fields and counting the number of cells on the filter (lower side) under phase contrast microscopy at a magnification of ×200.

For the invasion assays, Matrigel-coated transwell chambers were used (BD Biosciences). A total of U2OS cells (1×10⁵) were plated in 6-well plates for 24 h prior to transfection. Cells were then treated with 0, 25, and 50 µM taxifolin for 48 h, and trypsinized (by centrifuging at 600 × g for 10 min at 37°C to remove adherent cells) and washed with PBS. Then the 1×10⁴ taxifolin-treated U2OS cells were diluted in 200 µl serum-free DMEM and added to the upper compartment of Matrigel-coated transwell chambers (BD Biosciences). DMEM containing 10% FBS was added to the lower chamber before the plates were incubated overnight. The non-invaded cells were removed and the invaded cells on the underside of the filters were fixed with 70% methanol at 37°C for 1 h. Filters were washed with water and stained with 10% Giemsa solution for 1 h at 37°C. The number of invaded cells was quantified by counting the number of cells on the filter (6 random fields) under phase contrast microscopy at a magnification of ×200.

Experimental results are expressed as the mean ± SD. Differences among groups were examined for statistical significance using one-way analysis of variance followed by Dunnett's multiple comparison post hoc test on SPSS software (version 21.0, IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The viability and proliferation of taxifolin-treated human U2OS and Saos-2 osteosarcoma cells were examined using MTT and Hoechst 33258 assays, respectively. Results from the MTT assay demonstrated that the viability of U2OS and Saos-2 cells was inhibited by taxifolin treatment in a dose-dependent manner at concentrations ≥25 µM compared with the untreated cells (Fig. 2A). In addition, taxifolin treatment suppressed the proliferation of U2OS and Saos-2 cells in a dose-dependent manner at concentrations ≥25 µM when compared with the untreated cells (Fig. 2B). The half maximal effective concentration of taxifolin was similar for cell viability and proliferation (data not shown), suggesting that the observed decrease in cell viability may have been due to the suppression of cell proliferation.

The anchorage-independent growth of U2OS and Saos-2 cells was examined using a soft agar colony formation assay. The colony formation results demonstrated that treatment with 25 and 50 µM taxifolin markedly suppressed the ability of U2OS and Saos-2 cells to form colonies when compared with the untreated cells (Fig. 3). These results suggest a potential anti-tumor effect of taxifolin.

In order to determine whether taxifolin treatment inhibits tumor growth in vivo, U2OS cells were used to generate tumor xenograft models in nude mice. As demonstrated in Fig. 4A, intraperitoneal administration of taxifolin (25 mg/kg once every 2 for 24 days) was associated with a 55% reduction (P<0.01) in the mean volume of U2OS xenograft tumors at the end of the experiment (45 days). The body weight of untreated and taxifolin-treated mouse groups was gradually reduced but not significantly altered (Fig. 4B). These results suggest that taxifolin treatment inhibited osteosarcoma tumor growth in vivo.

The effect of taxifolin on the cell cycle was examined in human osteosarcoma U2OS and Saos-2 cells by flow cytometry analysis. Treatment of U2OS and Saos-2 cells with 25 µM taxifolin for 48 h significantly increased the percentage of cells in the G₁ phase, and significantly reduced the percentage of cells in the S and G₂/M phases when compared with untreated cells (Fig. 5). In addition, taxifolin treatment increased the percentage of U2OS and Saos-2 cells in the sub-G₁ fraction (cells undergoing apoptosis) when compared with untreated cells (Fig. 5). These results demonstrated that taxifolin treatment induced G₁ cell cycle arrest and increased apoptosis in osteosarcoma U2OS and Saos-2 cells.

Western blot analysis was performed to determine whether taxifolin treatment altered the expression of proteins involved in cell cycle regulation. Taxifolin treatment markedly reduced the protein expression levels of AKT, p-Ser473-AKT, c-myc and SKP-2 in U2OS and Saos-2 cells when compared with untreated cells (Fig. 6A). Transfection of U2OS cells with empty pcDNA3.1 plasmids or the pcDNA3.1-AKT vector was performed in order to overexpress AKT and investigate its association with c-myc and SKP-2. As demonstrated in Fig. 6B, overexpression of AKT markedly reversed the taxifolin-induced repression of p-AKT, c-myc and SKP-2 protein expression levels, indicating that decreased AKT expression may mediate taxifolin-induced c-myc and SKP-2 inhibition. These results revealed that taxifolin reduced the expression levels of signaling factors implicated in cell cycle regulation, potentially via the AKT signaling pathway.

As demonstrated in Fig. 6A, taxifolin treatment resulted in a gradual reduction in SKP-2 expression levels in U2OS and Saos-2 cells. Therefore the effect of SKP-2 overexpression on taxifolin-induced anti-cancer properties was examined further. SKP-2 overexpression in U2OS cells (Fig. 7A) partially inhibited the taxifolin-induced decrease in cell viability when compared with cells transfected with empty vectors (Fig. 7B). The migration and invasion of U2OS cells transfected with empty vector plasmids were markedly reduced by taxifolin treatment when compared with untreated cells (Fig. 7C and D). By contrast, overexpression of SKP-2 significantly attenuated the taxifolin-mediated reduction in U2OS cell migration and invasion when compared with empty vector-transfected cells (Fig. 7C and D). As overexpression of SKP-2 was not observed to eliminate the inhibitory effects taxifolin completely, it is possible that additional mechanisms or signaling pathways, other than AKT/SKP-2, may be triggered by taxifolin in osteosarcoma cell lines.