The PC-3 and LNCaP cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 medium (DC/CIK 05-080-1A/B; Bioind, Israel) supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS; 04-001-1A/B-AUS; Bioind). The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

Cells were trypsinized, and 1.0×10⁴ cells were plated in individual wells of a 24-well plate containing RPMI-1640 medium with 10% FBS. Cells were treated with 0.1, 0.5 or 1 mg/ml ubenimex. Every 24 h, the medium was removed, adherent cells were trypsinized, and the total number of adherent cells in each well was quantified using a hemocytometer. Cell counts for 3-wells/time point were averaged for each group, and the data were used to generate growth curves.

The levels of lactate dehydrogenase (LDH) release were assessed as a method for determining the extent of cell death irrespective of the type of death. A 200-µl volume of cell suspension in complete medium (5×10³ cells/well) was dispensed in each well of a 96-well plate. Ubenimex was added at different doses. The 96-well plates were centrifuged for 5 min at 400 × g, and 120 µl of the supernatant from each well was then transferred into a new plate. The plates were incubated at room temperature for 30 min in the dark, and the absorbance was spectrophotometrically measured at a wavelength of 562 nm.

PC-3 cells in an exponential phase of growth were harvested and seeded into 96-well plates at a density of 3,000 cells/well in RPMI-1640 medium supplemented with different concentrations of ubenimex. After 24 or 48 h of culture, 10 µl WST-8 solution (WST-8 cell proliferation and cytotoxicity assay kit; Dojindo, Japan) was added into each well. The plates were then incubated for an additional 1 h at 37°C, and the absorbance was determined using a microplate reader (EL340; Bio-Tek Instruments, Hopkinton, MA, USA) at 450 nm.

PC-3 cells were plated in 6-well culture plates and grown to ~90% confluency. Next, a sterile P200 pipette tip was used to create a scratch across the monolayer. Cell debris was removed by washing with phosphate-buffered saline (PBS), and the cells were cultured in RPMI-1640 medium and 2% FBS supplemented with different concentrations of ubenimex. The area of the scratch was measured at 0, 8, 16 and 24 h. Quantification was performed by measuring the area of cell migration at different time points compared to the scratched area at 0 h. Each experiment was repeated 3 times.

Invasion assays were performed using Transwell chambers and PC-3 cells were used. Control untreated cells or cells treated with ubenimex (0.1, 0.5 or 1 mg/ml for 16 h) were trypsinized, and 1.5×10⁵ cells were plated in the upper wells in serum-free medium, while medium with 10% FBS was added to the lower well as a stimulus. After 36 h of incubation, the cells on the Matrigel side of the chambers were removed using a cotton swab. The inserts were fixed in methanol and stained using hematoxylin and eosin (H&E) staining. The number of invading cells attached to the other side of the inserts was quantified under a light microscope using 8 random fields at a magnification of ×200. The experiment was performed in triplicate.

We used PC-3 cells to form the invasion assay. In addition, invasion assays were performed using Transwell chambers that were pre-coated with 40 µl of 1 mg/ml Matrigel matrix (BD Biosciences, Bedford, MA, USA). Control untreated cells or cells treated with ubenimex (0.5 or 1 mg/ml for 16 h) were trypsinized, and 2.0×10⁵ cells were plated in the upper wells in serum-free medium, while medium with 10% FBS was added to the lower well as a stimulus. After 36 h of incubation, the cells on the Matrigel side of the chambers were removed using a cotton swab. The inserts were fixed in methanol and stained using H&E staining. The number of invading cells attached to the other side of the inserts was quantified under a light microscope using 8 random fields at a magnification of ×200. The experiment was performed in triplicate.

Cells were cultured in 24-well plates for 24 h, and were then treated with different doses of ubenimex (0, 0.1, 0.5 and 1 mg/ml) for 24 h. After the indicated treatment times, the cells were stained with AO (200 µg/ml) and EB (200 µg/ml) for 10 min, and then washed with PBS to remove background staining. Next, the cells were observed under a fluorescence microscope (Nikon, Inc., Japan).

To determine LC-3B and CD13 expression levels, proteins were extracted from the cells or tissues by suspension in radioimmunoprecipitation assay (RIPA) buffer. Samples were centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid (BCA) protein assay kit (Sigma, St. Louis, MO, USA). Protein (40 µg) was electrophoresed on a pre-cast bis-Tris polyacrylamide gel (12%), and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blotted with rabbit anti-APN (1:1,000), rabbit anti-LC3B (1:500) (both from Abcam, USA), and mouse anti-actin (1:5,000; BL005A; Biosharp, Beijing, China), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ZB2306; ZB2301; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).

We performed cell concentration smears and fixed the cells with 4% paraformaldehyde for 20 min. We thoroughly rinsed the cells with 0.01 M PBS (5 min × 3) and incubated the cells in 0.3% Triton at room temperature for 20 min, followed with a wash in 0.01 M PBS (5 min × 3). Next, 30 µl/sample goat serum blocking solution was added at room temperature for 60 min, followed by 1:100 dilution of primary antibody at 30 µl/sample (diluted in 0.01 M PBS). The cells were placed in a wet box at 4°C overnight and then washed in 0.01 M PBS (5 min × 3). On the next day, 4′,6-diamidino-2-phenylindole (DAPI) was added for 10 min, and the cells were thoroughly rinsed with 0.01 M PBS (5 min × 3), followed by the addition of 1:100 fluorescent secondary antibody at 30 µl/sample (0.01 M PBS diluted) at room temperature and in the dark for 60 min. Next, the cells were washed in 0.01 M PBS (5 min × 3) and mounted with an anti-fluorescent quencher.

Prostate cancer cells were treated with 1 mg/ml ubenimex for 1 h, and then fixed with 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS buffer for 30 min, post-fixed with 1% osmium tetroxide for 1.5 h, and washed and stained in 3% aqueous uranyl acetate for 1 h. Next, the cells were dehydrated in an ascending series of ethanol and acetone and embedded in Araldite. Ultrathin sections were cut using a Reichert ultramicrotome, double-stained with 0.3% lead citrate and examined on a JEOL 1200EX electron microscope (Japan).

Male nude mice (6–8 weeks old) were used in our experiments. The animals were housed (five animals/cage) at 24±2°C and 50±10% relative humidity and were subjected to a 12-h light/12-h dark cycle. The animals were acclimatized for 1 week prior to the start of the experiments and were provided with a Purina chow diet and water ad libitum. PC-3 cells (5×10⁶ cells in 0.1 ml of PBS) were injected at one site of the right flank. Seven days after injection, the animals were randomly distributed into each group (10/1 group). Mice were treated with: i) vehicle (corn oil); ii) 15 mg/kg ubenimex; or iii) 30 mg/kg ubenimex. These treatments were administered 3 times/week for 4 weeks. Mouse body weights were measured once/week and were used as an indicator of systemic toxicity of the treatment. Tumor growth was measured every two days, and the tumor volume was calculated according to the formula: Tumor volume (mm³) = large diameter (mm) x small diameter (mm)²/2.

Data were statistically analyzed using the Student's t-test, the χ² or Fisher's exact tests. Statistical tests were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Western blot and immunofluorescence analyses were used to examine APN expression. As shown in Fig. 1A and B, the expression level of APN in the metastatic PC-3 cell line was obviously higher compared to the level in the non-metastatic LNCaP prostate cancer cells. Thus, we concluded that APN is associated with the metastasis of prostate cancer cells.

Western blot and immunofluorescence analyses were used to examine APN expression in the prostate cancer cells following treatment with ubenimex (Figs. 1A and C; 6C and I). After a 24 h treatment of ubenimex, in both LNCaP and PC-3 cells, APN expression was robustly inhibited in a dose-dependent manner. Thus, we concluded that ubenimex functions as an APN inhibitor in both metastatic and non-metastatic prostate cancer cells.

Western blot analyses, cellular immunohistochemistry, electron microscopy and AO-EB staining were used to examine the autophagy level in the prostate cancer cells. We chose to examine the cells following exposure to ubenimex for 16 h. Next, we observed that LC3B and Beclin 1 levels increased with increasing doses of ubenimex in the LNCaP and PC-3 cells (Fig. 6). This finding indicated that a higher dose of ubenimex induced a higher level of autophagic cell death. LDH assays were used to examine the cytotoxicity of ubenimex (Fig. 2E and F). In addition, an LDH cytotoxicity assay was performed after pretreating the prostate cancer cells with rapamycin (an inducer of autophagy) or 3-methyl-adenine (an inhibitor of autophagy). Rapamycin enhanced the levels of ubenimex-induced cell death while 3-methyladenine reversed the effect in both cell lines (Fig. 2G and H). Next, we performed electron microscopy to confirm these results. Similar findings were obtained in the PC-3 and LNCaP cells (Fig. 5A). Furthermore, AO-EB staining indicates the level of DNA damage, which can reflect autophagic cell death (13). Thus, we examined AO staining, at 16 h. In addition, AO staining levels, which indicate autophagic cell death, increased in a dose-dependent manner in response to ubenimex treatment (Fig. 5C and F). Thus, we concluded that ubenimex induced autophagic cell death in the prostate cancer cells.

To examine the effects of ubenimex on the proliferation of prostate cancer cells, we performed a growth curve analysis and WST-8 cell proliferation assay (Fig. 2A–D). LNCaP and PC-3 cells were treated with different doses of ubenimex, and cell growth was assessed over a 6-day time course. Cell growth was significantly decreased in both cell lines in a concentration-dependent manner, although the effect was more obvious in the PC-3 cells (Fig. 2A). These results were confirmed using the WST-8 assay after 24- and 48-h exposure to ubenimex (Fig. 2C and D). Moreover, we examined these effects in vivo. All of the data demonstrated that ubenimex inhibited proliferation of prostate cancer cell lines.

Wound-healing migration and Transwell assays were performed to determine whether ubenimex affects the migration and invasion capacity of PC-3 cells. The migration capacity of the PC-3 cells was significantly suppressed by ubenimex in a concentration-dependent manner after 16 or more hours of exposure (Figs. 3 and 4A). Moreover, the dose of ubenimex used was also an important factor affecting prostate cancer cells. Thus, we further examined the effect of ubenimex on the invasive activity of PC-3 cells using Matrigel invasion assays (Fig. 4C). Pretreatment with ubenimex markedly inhibited the invasive capacity of the PC-3 cells. Taken together, these results suggest that ubenimex inhibited the migration and invasion of metastatic prostate cancer cells.

Next, we evaluated the antitumor growth effect of ubenimex in vivo. Tumors were induced by the injection of PC-3 cells into nude mice. While ubenimex has been clinically used previously, we measured the body weights of the mice weekly. Fig. 7A shows that none of the treatments produced any loss in body weight, which would constitute a sign of toxicity. The tumor weight in the nude mice was significantly reduced in a dose-dependent manner following ubenimex treatment (Fig. 7B and C). Thus, we demonstrated that ubenimex inhibited PC-3 cell proliferation in vivo.