Diosgenin was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China), with a purity of >98% as determined by high-performance liquid chromatography. Minimum essential medium (MEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation and Cytotoxicity assay kit, 3-methyladenine (3-MA), monodansylcadaverine (MDC) Autophagy Detection kit, and annexin V-allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) Apoptosis Detection kit were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Fetal bovine serum (FBS), Hoechst 33,342/propidium iodide (PI) Double Staining kit, TRIzol^® reagent, First-Strand cDNA Synthesis kit and Taq DNA Polymerase were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Bradford Protein Content assay kit (cat. no. KGA801), SDS-PAGE Gel Preparation kit (cat. no. KGP113), Coomassie Blue Staining kit (cat. no. KGP1001), Ponceau S staining solution (cat. no. KGP105) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

The DU145 human prostate cancer cell line was obtained from Nanjing KeyGen Biotech Co., Ltd. The cells were cultured in MEM containing 10% FBS at 37°C in a 5% CO₂ incubator. Cells in the logarithmic growth phase were used for subsequent experiments.

A cell suspension of 5×10⁴ cells/ml was prepared, and 100 µl of this was added to each well of a 96-well culture plate, which was incubated at 37°C in a 5% CO2 incubator for 24 h. Complete medium was used to dilute diosgenin to the desired concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0.1953125 µg/ml), and 100 µl of the corresponding diosgenin-containing medium was added per well. 3-MA was added to cells at a final concentration of 5 nM. Untreated cells were the negative control group, while cells treated with 10 µg/ml paclitaxel were the positive control group. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 48 h. The plate was then subjected to MTT staining, according to the procedures described previously (32), and the optical density of the samples was measured at a wavelength of 490 nm. The inhibition rate and IC50 value of each group were calculated using the following formula: Inhibition rate (%) = [(Negative control group - Experimental group) / Negative control group] × 100.

Cells growing in the logarithmic phase were trypsinized and seeded at a density of 5×10⁴ cells/well in a 6-well plate. The following day, once cells had adhered to the plate, the corresponding diosgenin-containing medium was added (0.6, 3 or 15 µg/ml). A negative control group, consisting of untreated cells was included. Following incubation for 48 h at 37°C and 5% CO₂, 0.25% trypsin [without ethylenediaminetetraacetic acid (EDTA)] was used to remove the cells. The cells were washed twice with phosphate-buffered saline (PBS) and centrifuged at 447.2 × g for 5 min at 20°C. A total of 5×10⁵ cells were resuspended in 500 µl binding buffer, following which 5 µl of annexin V-APC was added and mixed well, and 5 µl 7-AAD was added and mixed well. The reaction was performed at room temperature for 5–15 min in the dark, and a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA), together with the BD CellQuest software (BD Biosciences) was used to detect apoptosis.

DU145 cells (1×10⁵ cells/ml) in the logarithmic growth phase were incubated in diosgenin-containing medium (15, 3 or 0.6 µg/ml). A negative control group consisting of untreated cells was included. Cells were harvested 24 h later, using trypsin (0.25%) to remove the cells from the plate. Cells were then centrifuged at 111.8 × g for 10 min at 20°C. The supernatants were discarded, cells were washed twice with PBS and 2.5% glutaric acid was added. The cells were fixed for 90 min at 4°C and then embedded, sectioned and stained with uranyl acetate and lead citrate. Autophagosomes were observed under a transmission electronic microscope (JEM-1011; JEOL, Ltd., Tokyo, Japan).

Cells in the logarithmic growth phase were trypsinized and seeded into a 6-well plate at a density of 5×10⁵ cell/ml/well. The following day, once cells had adhered, diosgenin-containing medium was added (15, 3 or 0.6 µg/ml). A negative control group consisting of untreated cells was included. Cells were harvested using 0.25% trypsin (without EDTA) following a 48-h incubation. Wash buffer (1X; 300 µl) was used to wash the cells once, and cells were then resuspended in 1X wash buffer at 1×10⁶ cells/ml. A total of 90 µl of cell suspension was transferred to a new microfuge tube, and 10 µl of MDC staining solution was added and gently mixed. Following staining at room temperature for 15–45 min in the dark, cells were collected by centrifugation at 800 × g for 5 min. The cells were washed three times with wash buffer and resuspended in 100 µl of collection buffer. The cell suspension was dropped onto a slide and covered with a coverslip. Cells were observed under a fluorescence microscope (IX51; Olympus Corporation, Tokyo, Japan).

Cells in the logarithmic growth phase were trypsinized and seeded onto a 6-well plate at a density of 5×10⁵ cell/ml/well. The following day, once cells had adhered to the plate, diosgenin-containing medium was added (15, 3 or 0.6 µg/ml). A negative control group consisting of untreated cells was included. Pre-chilled lysis buffer (200 µl), consisting of 20 nM Tris (pH 7.5), 150 mM NaCl, Triton-X-100 (cat. no. KGP701; KayGen Biotech, Co., Ltd), was added to each group and incubated on ice for 30 min. Following vortexing, the lysate was centrifuged at 13,000 × g for 10 min at 4°C. The supernatant was retained, and the Bradford Protein Content assay kit was used to measure the protein concentration of the samples. Proteins (30 µg) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Following blocking overnight with 5% non-fat milk, the membranes were incubated overnight at 4°C in a sealed bag with the following primary antibodies: Mouse anti-human light chain 3 (LC3)-I (cat. no. KGATG007; dilution, 1:500); mouse anti-human LC3-II (cat. no. KGATG007; dilution, 1:500); rabbit anti-human phosphatidylinositol 3 kinase (PI3K; cat. no. KG22639; dilution, 1:500); rabbit anti-human phosphorylated (p)-PI3K (cat. no. KG22638-2; dilution, 1:500); rabbit anti-human protein kinase B (Akt; cat. no. KG21502; dilution, 1:200); rabbit anti-human p-Akt (cat. no. KG11054-2; dilution, 1:200); rabbit anti-human mammalian target of rapamycin (mTOR; Ser 2448; cat. no. KGYT2914-7; dilution, 1:200); rabbit anti-human p-mTOR (cat. no. KGYP0176-6; dilution, 1:200); and anti-GAPDH (cat. no. KGAA002-2; dilution, 1:200). All primary antibodies were obtained from KayGen Biotech Co., Ltd. Tris-buffered saline and Tween 20 was used to wash the membrane three times for 10 min before it was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. KGAA37; dilution, 1:4,000) or goat anti-rabbit IgG (cat. no. KGAA35; dilution, 1:4,000; both from KayGen Biotech, Co., Ltd.) secondary antibodies for 1 h at 37°C. Finally, the membrane was visualized using an enhanced chemiluminescence method (cat. no. KGP1201; KayGen Biotech Co., Ltd.) and exposed to film. Protein band densities were quantified using Quantity One analysis software (version, V4.4.0.36; Bio-Rad, Inc., Hercules, CA, USA).

Cells in the logarithmic growth phase were trypsinized and seeded into a 6-well plate at a density of 1×10⁵ cell/ml/well. The following day, once cells had adhered, diosgenin-containing medium was added. A negative control group consisting of untreated cells was included. Cells were harvested with 0.25% trypsin (without EDTA) following incubation for 48 h. Cells (10⁵-10⁶) were resuspended in 1 ml of medium and 10 µl Hoechst 33,342 staining solution was added to the cells and incubated at 37°C for 5–15 min. The cells were centrifuged at 111.8 × g for 5 min at 4°C, and the supernatant was discarded. Buffer A (1 ml) was used to resuspend the cells, and 5 µl PI staining solution was added and incubated at room temperature for 5–15 min in the dark. The suspension was mixed well and observed under a fluorescence microscope. The number of apoptotic cells was determined by counting the number of PI-positive cells in 3 random fields of view for each sample.

Pre-cooled TRIzol (1 ml) was added to DU145 cells (1×10⁶) growing in logarithmic phase, and the solution was repeatedly retro-pipetted until it was transparent. The solution was then transferred to a 1.5 ml centrifuge tube, and incubated at room temperature for 5 min. Chloroform (0.2 ml) was subsequently added and the cover was fastened. The tube was shaken for 15s until the solution was white in color, before it was incubated at room temperature for 3 min. The solution was then centrifuged at 16099.2 × g for 15 min at 4°C, before 0.6 ml of the supernatant was transferred to a fresh centrifuge tube. Ispropyl alcohol (0.6 ml) was subsequently added and, after gentle vortexing, the sample was incubated at room temperature for 10 min and centrifuged at 16099.2 × g for 10 min at 4°C. The supernatant was removed and 1 ml 70% ethanol was added along the tube wall, before it was vortexed gently and centrifuged at 16099.2 × g for 10 min at 4°C. The supernatant was removed, air-dried and precipitated at room temperature for approximately 5 min, before 30–50 µl RNase-free water was added to dissolve the RNA precipitate. Following complete dissolution, the solution was preserved at −70°C until required. The RNA was reverse transcribed into cDNA using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), and RT-qPCR was performed using SYBR Green Realtime PCR Master Mix (cat. no. QPK-201; Toyobo, Co., Ltd., Osaka, Japan) and the StepOnePlus software program (version 2.0; Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers were synthesized by Gen Script (Nanjing) Co., Ltd. (Nanjing, China) and had the following sequences: Sense, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and antisense, 5′-GCCATCACGCCACAGTTTC-3′ for GAPDH (101 bp); sense, 5′-ATGTCCACAGAAAGTGCCAA-3′ and antisense, 5′-GGGTGATCCACATCTGTCTG-3′ for Beclin 1 (140 bp); and sense, 5′-AAATCCGACCACTAATTGCC-3′ and antisense, 5′-TGCTCTTCAGATGGTGATCC-3′ for B-cell lymphoma 2 (Bcl2; 114 bp). The amplification conditions were 95°C pre-denaturation for 5 min followed by 40 cycles of 95°C denaturation for 15 sec, 60°C annealing for 20 sec, and 72°C extension for 40 sec. The specificity of the amplified products was monitored by melting curves. ABI StepOne software (version, 2.3; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to calculate the relative expression (using 2-ΔΔCq) of the target genes in each group, and GAPDH served as an internal reference to assess the expression of target genes (33).

All data are presented as the mean ± standard deviation. Statistical analyses were performed in SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance was used to compare differences between groups. P<0.05 was considered to indicate a statistically significant difference.

DU145 cells were treated with diosgenin (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 or 0.1953125 µg/ml) for 48 h. Diosgenin inhibited DU145 cell proliferation in a dose-dependent manner (Fig. 1). At 48 h, the IC50 was 6.757 µg/ml, therefore 0.6, 3 and 15 µg/ml diosgenin concentrations were used for subsequent experiments.

To assess whether the inhibition of proliferation by diosgenin was associated with apoptosis, DU145 cells were treated with diosgenin for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry. Diosgenin induced apoptosis in DU145 cells in a dose-dependent manner (P<0.01 for 0.6, 3 and 15 µg/ml diosgenin; Fig. 2).

To verify whether the cytoplasmic vacuoles observed by inverted microscopy were associated with autophagy, transmission electron microscopy was used to observe autophagosomes in DU145 cells treated with diosgenin. As presented in Fig. 3, untreated cells contained normal nuclei, cytoplasm, and organelles, whereas diosgenin-treated cells exhibited a large number of autophagosomes of various sizes. In addition, autophagosomes containing mitochondria were observed. This suggests that autophagy occurred in the cells following diosgenin treatment.

MDC-labeled autophagic vacuoles were observed under an inverted fluorescence microscope and exhibited clear vesicles in the cytoplasm and perinuclear region, and the changes of the particles inside the cell were used to determine the level of autophagy. As presented in Fig. 4, diosgenin-treated cells exhibited increased fluorescence intensity and numbers of autophagic vacuoles compared with the control group. The number and staining intensity of the vacuoles increased in a dose-dependent manner. This suggests that diosgenin induces autophagy.

Western blotting was used to detect changes in the protein expression levels of LC3 and caspase 9 following treatment of DU145 cells with diosgenin. On SDS-PAGE gels, LC3-II ran faster than LC3-I, producing two bands by western blot. Fig. 5A indicates that untreated cells exhibited only a faint LC3-I band, whereas LC3-II was not detected. By contrast, following treatment with diosgenin, the protein expression levels of LC3-II increased significantly in a dose-dependent manner (0.6 µg, P=0.006; 3 µg, P<0.001; 15 µg, P<0.001; Fig. 5A). In addition, diosgenin treatment significantly increased the protein expression levels of caspase 9 compared with the control group, in a dose-dependent manner (0.6 µg/ml, P<0.001; 3 µg/ml, P=0.002; 15 µg/ml, P<0.001; Fig. 5B).

The PI3K/Akt/mTOR signaling pathway is the canonical pathway that negatively regulates the initiation of autophagy. It has been reported that inhibition of this signaling pathway induces cell autophagy. As presented in Fig. 6, western blotting demonstrated that diosgenin inhibited the phosphorylation of PI3K, Akt, and mTOR in the PI3K/Akt/mTOR signaling pathway.

Preliminary experiments determined that diosgenin treatment induced autophagy and apoptosis in DU145 cells and that the inhibition of DU145 proliferation by diosgenin occurred in a dose-dependent manner. To determine whether the cytotoxicity exhibited by diosgenin was mediated by autophagy, the autophagy inhibitor 3-MA was used, and MTT assays were performed to assess cytotoxicity. Compared with cells treated with diosgenin alone, the addition of 3-MA increased the percentage of non-viable DU145 cells (P<0.01; Fig. 7).

Hoechst 33342 and PI double staining distinguish live and dead cells. The nuclei of cells in the late apoptotic or early necrotic stages stain red, whereas the nuclei of live cells stain blue. No significant differences in the percentages of apoptotic cells were observed between control cells (Fig. 8A) and cells treated with 3-MA alone (Fig. 8B). However, cells treated with diosgenin (Fig. 8C) for 48 h exhibited nuclei with a bead-like shape, forming apoptotic bodies. Diosgenin treatment increased the percentage of apoptotic cells compared with control cells. Cells treated with diosgenin and 3-MA (Fig. 8D) exhibited an increased percentage of apoptotic cells compared with cells treated with diosgenin alone. These results demonstrated that apoptosis increased significantly following the inhibition of diosgenin-induced autophagy.

As cytotoxicity of diosgenin was increased following the inhibition of autophagy, it was investigated whether autophagy inhibited the effect of diosgenin on apoptosis. RT-qPCR was performed to analyze mRNA expression levels in DU145 cells treated with diosgenin. Using Beclin 1 and Bcl2 as markers, the effect of diosgenin on autophagy and apoptosis was examined following the inhibition of autophagy. As presented in Fig. 9, following treatment with 5 mM 3-MA alone, the mRNA expression levels of Beclin 1 decreased compared with the control cells. Following treatment with 6.8 µg/ml diosgenin, the mRNA expression levels of Beclin1 increased significantly compared with control cells (P<0.01). However, when diosgenin was added following simultaneous treatment with 3-MA, Beclin 1 mRNA expression levels significantly decreased compared with cells treated with diosgenin alone (P<0.01). Following treatment with diosgenin alone, Bcl2 mRNA expression levels decreased significantly compared with control cells (P<0.01). Simultaneous treatment with 3-MA significantly decreased Bcl2 mRNA expression levels compared with cells treated with diosgenin alone (P<0.01). These results suggest that when autophagy was inhibited, the apoptotic effect of diosgenin was increased.