Diosgenin, purity >90% was identified in Nanjing Zelang Technology Co., Ltd. by HPLC and was purchased from Nanjing Zelang Medical Technology Co., Ltd. (cat. no. ZL20170702014). A First Strand cDNA Synthesis kit was obtained from Thermo Fisher Scientific, Inc., fetal bovine serum (FBS) was purchased from ExCell Biology, Inc., TRIzol^® was purchased from Invitrogen; Thermo Fisher Scientific, Inc., chloroform and isopropanol were purchased from Nanjing Chemical Reagent Co., Ltd. and 0.25% trypsin-EDTA, PBS, total protein extraction kit, Braford assay kit, 5X SDS-PAGE protein loading buffer solution, SDS-PAGE gel preparation kit, prestained protein molecular weight ladder, 10X Tris-glycine protein electrophoresis buffer, Coomassie blue staining protein detection kit, phosphorylated p38 (pP38) inhibitor SB203580, 10X electrotransfer buffer solution, Ponceau staining solution, western blotting primary antibody diluent, a western blotting secondary antibody diluent, enhanced chemiluminescent detection kit, internal reference primary antibody (anti-GAPDH; cat. no. KGAA002-1; dilution, 1:200), secondary antibody, and developing and fixing reagents were all purchased from KeyGen Biotechnology Co., Ltd.

Human osteosarcoma MG63 and U2OS cells were donated by Jiangsu Health Vocational College (Nanjing, China). The MG63 and U2OS cells were treated with 90% minimal essential medium supplemented with 10% FBS or 90% complete DMEM supplemented with 10% FBS, respectively. The cells were cultured at 37°C in 5% CO₂. The culture medium was replaced every 2 days.

Cells in the logarithmic growth phase were collected, and the two cell lines were prepared in cell suspensions at a concentration of 5×10⁴ cells/ml. The cells were added to 96-well cell culture plates (100 µl per well) and placed in a 37°C, 5% CO₂ incubator for 24 h. Diosgenin diluted to various concentrations in complete medium (200, 100, 50, 25, 12.5 and 6.25 µM) was added to the 96-well medium. Untreated cells were the negative control group. The culture plates were placed in a 37°C, 5% CO₂ incubator for 24 h after which MTT staining was performed. DMSO was used to dissolve the purple formazan and the optical density (OD) value was measured at λ=490 nm using a BioTek ELx800 plate reader (BioTek Instruments, Inc.). The inhibition rate and 50% inhibitory concentration (IC₅₀) of diosgenin at each concentration was calculated. Inhibition rate and IC₅₀ were calculated using the following formula: Inhibition rate (%) = [(Negative control group-Experimental group)/Negative control group] × 100.

Cells in the logarithmic growth phase were prepared at 1×10⁵ cells/ml and transferred to a 6-well plate, and the corresponding diosgenin containing medium, MG63 (80 µM) and U2OS (40 µM), was added. The next day, when the cell confluence was >60%, a sterile pipette tip was used to evenly scratch the 6-well plate. The floating cells were washed away with PBS, and serum-free medium was used for culture in a cell culture incubator. After 24 h, the cells were imaged (magnification, ×100), and the cell wound healing area was measured using Adobe Photoshop CS6 (Adobe Systems Europe, Ltd.) using an Olympus IX51 light microscope (Olympus Corporation). Relative wound closure was calculated using the following formula: relative wound closure (%)= (0 h time point area-each time point by the area)/0 h time point area × 100.

Diosgenin was added to cells in the logarithmic growth phase (MG63, 80 µM; U2OS 40 µM). Matrigel was thawed at 4°C overnight and diluted 1:2 using serum-free medium. In the upper chamber of the Transwell insert, 30 µl diluted Matrigel was added at 37°C for 120 min. The cell density was adjusted to 5×10⁵ cells/ml using serum-free medium. A total of 100 µl of the cells at a concentration of 5×10⁵ cells/ml was added to the upper chamber of a Transwell insert and 500 µl complete medium supplemented with 20% FBS was added to the lower chamber. The cells were then cultured in an incubator at 37°C and 5% CO₂. After 24 h, the chamber was removed, washed with PBS, fixed in absolute ethanol for 20 min at 37°C, stained with crystal violet for 20 min at 37°C, washed with PBS and air-dried. An inverted microscope was used for observation, and the transmembrane cells in each group were counted using an Olympus IX51 light microscope.

Cells in the logarithmic growth phase were harvested and plated at a density of 5×10⁴ cells/ml. Diosgenin was added to each cell line (MG63, 80 µM; U2OS, 40 µM) and cultured for 24 h, after which the cells were air dried at room temperature (20°C), fixed with 4% paraformaldehyde for 30 min at 20°C, and subsequently washed with PBS three times. Two drops of a 3% H₂O₂-methanol solution and 75 µl ready-to-use goat serum (cat. no. AR0009; Wuhan Boster Biological Technology, Ltd.) were added dropwise and then incubated for 20 min at room temperature. The primary antibodies used were rabbit anti-human TGF-β1 (cat. no. KG22744-1; dilution, 1:100; Nanjing KeyGen Biotech Co., Ltd.) and incubated for 2 h at 37°C, after which the samples were washed with PBS three times. A FITC-conjugated secondary antibody (1:200 dilution) was added and incubated for 1 h in the dark at 37°C, and the samples were washed with PBS three times. After dropwise addition of a DAPI solution 0.2 µg/ml, antiquench gel (cat. no. KGF028; Nanjing KeyGen Biotech Co., Ltd.) was used for mounting. A total of three high-expression regions of the cells were observed under a fluorescence microscope and photographed for preservation. The integrated optical density (IOD) under the field of view was calculated using Image-Pro Plus version 6.0 (Media Cybernetics, Inc.), and the mean density was calculated as mean density = IOD/area of the field.

Cells in the logarithmic growth phase were harvested and plated at a density of 5×10⁵ cells/ml. The following day, after the cells had adhered to the wells, the medium was replaced and diosgenin was added to each cell line (MG63, 80 µM; U2OS, 40 µM). In the SB203580 group, SB203580 (cat. no. KGR0067 Nanjing KeyGen Biotech Co., Ltd.) was co-cultured with cells for 1 h. After 24 h, total cellular protein was extracted using a total protein extraction kit (cat. no. KGP250; Nanjing KeyGen Biotech Co., Ltd.), and the protein concentration was measured with a bicinchoninic acid protein concentration assay kit. Proteins were resolved for 90 min by 10% SDS-PAGE (30 µg/well). The resolved proteins were transferred to PVDF membranes using a Bio-Rad semidry transfer system (Bio-Rad Laboratories, Inc.). The PVDF membrane was subsequently blocked with 5% skimmed milk powder overnight at 4°C and then incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: Rabbit anti-human E-cadherin (cat. no. KG22195-2; 1:200); rabbit anti-human vimentin (cat. no. KG22794-2; 1:200); rabbit anti-human ERK1/2 (cat. no. KG30107-2; 1:200); rabbit anti-human jun N-terminal kinase (JNK) 1/2 (cat. no. KG22481-2; 1:200); rabbit anti-human P38 (cat. no. KG30244-2; 1:200); rabbit anti-human pERK1/2 (cat. no. KG30246-2; 1:200); rabbit anti-human pJNK1/2 (cat. no. KG11504-2; 1:200); and rabbit anti-human pP38 (cat. no. KG11253-2; 1:200). The secondary antibodies used were goat anti-rabbit immunoglobulin G (IgG; cat. no. KGAA25; 1:200) and goat anti-mouse IgG (cat. no. KGAA37; 1:4,000). Both the primary and secondary antibodies were obtained from Nanjing KeyGen Biotech Co., Ltd. The following day, the membrane was incubated with the corresponding secondary antibody (1:4,000) for 1 h at room temperature, and the signal was visualized using enhanced chemiluminescent reagent (cat. no. KGP1121; Nanjing KeyGen Biotech Co., Ltd.). A gel imager and Gel-Pro32 version 4.4.0.36 (Bio-Rad, Inc.) were used for collecting images and analyzing the gray scale values relative to the GAPDH signal.

Cells in the logarithmic growth phase were harvested and digested with 0.25% trypsin and a trypsin digestive solution containing 0.02% EDTA to prepare a single-cell suspension. Subsequently 1 ml TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and 200 µl chloroform were added, incubated at 4°C for 10 min, mixed gently and centrifuged at centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was transferred to a new microcentrifuge tube. A total 800 µl precooled methanol (4°C) was added, and the samples were placed at 4°C for 5 min with gentle agitation. The supernatant was aspirated and discarded after centrifugation at 12,000 × g for 10 min at 4°C. Subsequently, 1 ml precooled 75% ethanol (4°C) was added, and the sample was centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was aspirated and discarded, and 30 µl diethyl pyrocarbonate water was added and dissolved at 4°C for 5 min. RNA was prepared for storage in a −70°C freezer. A total of 5 µl RNA sample and 495 µl 1X Tris-EDTA buffer were mixed. The concentration and purity of the RNA were determined by measuring the absorption values at 260 and 280 nm. An OD260/OD280 of 1.8–2.0 was considered to indicate RNA of good quality that could be used for subsequent experiments. The extracted RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) using the following incubation conditions: 25°C for 5 min, 42°C for 60 min, and on ice for 5 min. Next, qPCR was performed using a fluorescence qPCR instrument (ABI StepOne Plus; Thermo Fisher Scientific, Inc.). Primers were synthesized by Jiangsu Health Vocational College. The primer sequences were: GAPDH (90 bp) forward, 5-AGATCATCAGCAATGCCTCCT-3 and reverse, 5-TGAGTCCTTCCACGATACCAA-3; and E-cadherin (108 bp) forward, 5-CCAAGCAGCAGTACATTCTACA-3 and reverse, 5-CATTCACATCCAGCACATCCA-3. The amplification conditions were as follows: Predenaturation at 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 35 sec. The specificity of the amplified products was monitored by a dissolution curve. Relative gene expression was analyzed and calculated using the 2^−ΔΔCq method (30), and the expression levels of the target genes were normalized to GAPDH (ABI StepOne version 2.3; Applied Biosystems; Thermo Fisher Scientific, Inc.).

All the data are presented as the mean ± standard deviation and experiments were performed in triplicate. Statistical analyses were performed in SPSS software version 16.0 (SPSS, Inc.). Statistical comparisons between two groups were performed using an unpaired Student's t-test and comparisons between multiple groups were performed using a one-way ANOVA followed by a post hoc Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

MG63 and U2OS cells were treated with diosgenin at different molar mass concentrations (200, 100, 50, 25, 12.5 and 6.25 µM) for 24 h. The results shown in Fig. 1 revealed that diosgenin had inhibitory effects on both cell lines in a dose-dependent manner. The 24-h IC₅₀ of this drug in MG63 and U2OS cells were 76.2 and 40.15 µM, respectively.

The cell scratch test is a method for detecting cell movement and can be used to detect the invasive and metastatic capacities of tumor cells (31). Fig. 2 shows that relative wound closure (%) of the MG63 cells in the control group were 34.03±3.42 and 77.45±4.50 after 12 and 24 h, respectively, while relative wound closure (%) of the U2OS cells in the control group were 48.16±3.18 and 67.71±4.22, respectively. However, upon treatment with the IC₅₀ dose of diosgenin, relative wound closure (%) of MG63 cells (80 µM) were 18.83±2.61 and 28.75±2.11 after 12 and 24 h, respectively. Similarly, when U2OS cells were treated with 40 µm diosgenin, relative wound closure (%) were 27.36±3.26 and 36.02±3.74, respectively. Compared to those of the cells in the control group, the in vitro migratory abilities of the two types of osteosarcoma cells in the group treated with diosgenin were decreased (P<0.01).

We next employed a Transwell test, which detects the invasive ability of cells based on their ability to pass through Matrigel (32). As shown in Fig. 3, the number of MG63 cells passing through the Matrigel after 12 and 24 h in the control group was 97±2.59 and 122±1.58/field of view, respectively. For U2OS cells, the counts were 84.2±3.27 and 204.6±2.51/field of view, respectively. However, the numbers of MG63 cells passing through the Matrigel after 12 and 24 h in the drug-treated group was only 41±2.07 and 53±1.82/field of view, respectively, and the number of U2OS cells was 45.4±2.7 and 55.6±2.3/field of view, respectively. Compared with that of the respective control for each cell line, the invasive ability of the diosgenin-treated osteosarcoma cells was decreased (P<0.05).

As shown in Fig. 4, following treatment with diosgenin, the ODs were 1.25±0.03 and 1.07±0.04/pixel in the MG63 and U2OS cells, respectively. Compared with the respective control group, the average TGF-β1 ODs of MG63 and U2OS cells were 1.33±0.02 and 1.25±0.04/pixel. TGF-β1 protein expression in the two treated osteosarcoma cell line groups was decreased (P<0.05).

To determine whether the effect of diosgenin on the invasion and migration of the two osteosarcoma cell lines was associated with EMT, western blotting was used to detect changes in TGF-β1, E-cadherin and vimentin protein expression in the osteosarcoma cell lines prior to and following diosgenin administration. As shown in Fig. 5, after diosgenin treatment, the protein expression levels of TGF-β1 were decreased, while those of E-cadherin were increased in both cell types (P<0.01), suggesting that the invasive and migratory capacities of the cells were inhibited. However, diosgenin had no effect on vimentin protein expression.

Western blot analysis was used to assess the ERK/JNK/P38 pathway in the osteosarcoma cell lines before and after diosgenin treatment. As shown in Fig. 6, among the ERK/JNK/P38 pathway molecules, only the pP38 protein exhibited a decreased level in both osteosarcoma cell lines following administration of diosgenin (P<0.01), while changes in the levels of other proteins in the pathway were not statistically significant.

Diosgenin treatment was combined with a pP38 protein inhibitor and the expression levels of a downstream indicator of EMT, E-cadherin was observed (33). As shown in Fig. 7, the cellular expression level of E-cadherin in the group treated with the pP38 inhibitor SB203580 (20 µM) was not statistically different compared with the control group; however, the expression level of E-cadherin in the osteosarcoma cell lines treated with diosgenin in combination with SB203580 group was decreased compared with the cells in the diosgenin group (MG63, P<0.05; U2OS, P<0.01).