The 786-O and OS-RC-2 RCC cell lines were purchased from the cell bank of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 (HyClone Biotechnology, Carlsbad, CA, USA) supplemented with penicillin, streptomycin and 10% FBS. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

To determine the expression of APN, proteins were extracted from the cells or tissues by suspension in RIPA buffer. Samples were centrifuged at 15,000 rpm at 4°C for 10 min and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the BCA protein assay kit (Sigma, St. Louis, MO, USA). Protein (40 μg) was electrophoresed on a pre-cast Bis-Tris polyacrylamide gel (8%) and then transferred to a PVDF membrane. The membranes were blotted with rabbit anti-APN (1:1,000; Epitomics Biotechnology, Burlingame, CA, USA), rabbit anti-LC3B (1:1,000; Sigma) rabbit anti-Caspase3 (1:1,000, Abgent; San Diego, CA, USA)or mouse anti-GAPDH (1:3,000 TA08; ZsBio), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ZB2306 and ZB2301; both from ZsBio, Beijing, China). Immunoblots were visualized by enhanced chemiluminescence (LAS4000).

The APN activity in the RCC cells was detected spectrophotometrically using L-leucine-p-nitroanilide (Peptide Institute, Inc., Osaka, Japan) as an APN substrate. Cells (5×10⁴) were incubated in a 96 well microtiter plate with 0.1, 0.25 or 0.5 mg/ml ubenimex at 37°C for 24 h. After culture, the medium was aspirated, the cells were washed with PBS and then 200 μl of 1 mM alanine-p-nitroanilide was added per well. Each well was then incubated at 37°C for 60 min. The APN enzyme activity was estimated by measuring the absorbance at 405 nm using a microplate reader (Labsystems, Multiskan Bichromatic, Helsinki, Finland).

Cells were trypsinized and 1.0×10⁴ cells were plated in individual wells of a 24-well plate containing RPMI-1640 with 10% FBS. Cells were treated with 0.1, 0.25 or 0.5 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 hematocytometer. The cell counts for 3 wells/time-point were averaged for each group and the data were used to draw growth curves.

Cells in an exponential phase of growth were harvested and seeded in 96-well plates at a density of 3,000 cells/well in RPMI-1640 supplemented with different concentrations of ubenimex. After a 24 or 48 h culture, a 10 μl WST-8 solution (WST-8 cell proliferation and cytotoxicity assay kit; Dojindo, Kumamoto, Japan) was added into each well. 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.

The RCC cells were plated in 6-well culture plates and grown to ~100% confluency before scratching with a sterile P200 pipette tip across the monolayer. The cell debris were removed by being washed with PBS and the cells were cultured in RPMI-1640 and 2% FBS supplemented with different concentrations of ubenimex. The area of the scratch was measured at 0, 12 and 18 h and quantification was performed by measuring the area of cell migration at different time points compared to the scratch area at 0 h. Each experiment was repeated 3 times.

Invasion assays were performed using Transwell chambers that were pre-coated with 40 μl of 1 mg/ml Matrigel matrix (BD Bioscience, Bedford, MA, USA). Control untreated cells or cells treated with ubenimex (0.25 mg/ml for 24 h) were trypsinized and 1.0×10⁵ cells were plated in the upper wells in a serum-free medium, while medium with 10% FBS was added to the lower well as a stimuli. After 12 h of incubation, the cells on the Matrigel side of the chambers were removed with a cotton swab. The inserts were fixed in methanol and stained with H&E staining. The number of the invading cells attached to the other side of the inserts was counted under a light microscope using 8 random fields at ×200 magnification. The experiment was performed in triplicate.

The levels of LDH release were assessed as a method for determining the extent of cell death irrespectively 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 (0.25 or 0.5 mg/ml), 3-MA (2 mM) and/or rapamycin (0.1 μM) was added for 24 h. The 96-well plates were centrifuged for 5 min at 400 × g and then 120 μl of the supernatant from each well was transferred to a new plate. The plates were incubated at room temperature for 30 min in the dark and then the absorbance was spectrophotometrically measured at a wavelength of 560 nm.

The RCC cells were treated with 0.25 mg/ml ubenimex for 12 h. They were then fixed with 3% glutaraldehyde and 2% paraformaldehyde in a 0.1 M PBS buffer for 30 min, postfixed with 1% osmium tetroxide for 1.5 h, washed and stained in 3% aqueous uranyl acetate for 1 h, then dehydrated in an ascending series of ethanol and acetoned and embedded in Araldite. Ultrathin sections were cut on a Reichert ultramicrotome, double stained with 0.3% lead citrate and examined on a JEOL-1200EX electron microscope (Jeol, Tokyo, Japan).

RCC tissues were obtained from 76 patients diagnosed with RCC (median age, 63.8 years; range, 19–85) who were treated by radical nephrectomy at Shandong Provincial Hospital (Jinan, China) between July 2009 and January 2011. Formalin-fixed and paraffin-embedded specimens were used in our analysis. Ten specimens of non-neoplastic renal tissues were also obtained from these patients. This study was approved by the Ethics Committee of the Shandong Provincial Hospital.

Tissue microarrays were constructed using a manual tissue arrayer (Beecher, Silver Spring, MD, USA). Three cylindrical core biopsies (0.6-mm in diameter) were taken from different sites of each tumor and precisely arrayed using a recipient paraffin tissue microarray block. Ten specimens of the non-neoplastic renal tissues were also resected from adjacent regions of the RCCs and analyzed for comparison. To assess APN expression, 4-μm tissue microarray sections were deparaffinized, rehydrated and subsequently incubated with monoclonal rabbit antibodies targeting human APN (1:500; Epitomics Biotechnology). Hematoxylin served as a counter-stain. Incubation without the primary antibody was used as a negative control. The expression of APN was evaluated by two independent assessors at ×200 magnification and scored as follows: −, negative; +, weak; ++, moderate and +++, strong.

Data were analyzed statistically by the Student’s t-test, χ² test or Fisher’s exact test and analysis was performed using the Statistical Package for Social Science (SPSS for Windows package release 10.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered statistically significant.

Ubenimex is known as an inhibitor of APN, which is identical to the cell surface molecule CD13 (2). To confirm the expression of APN in two RCC cell lines, 786-O and OS-RC-2, and to evaluate the effects of ubenimex on APN expression and activity, we performed western blotting and enzymatic activity assays. Both cell lines expressed APN, although expression appeared to be consistently higher in the 786-O cells. Furthermore, the expression remained high after a 24-h treatment with ubenimex (Fig. 1A). Conversely, APN activity was reduced by ubenimex in a dose-dependent manner (Fig. 1B). These results verify that APN is expressed in the RCC cells and suggest that ubenimex targets the activity, but not the expression of APN.

To examine the effects of ubenimex on the proliferation of RCC cells, the 786-O and OS-RC cells were treated with a range of concentrations of ubenimex and the cell growth was assessed over a 6-day time course. The cell growth was significantly decreased for both the cell lines in a concentration-dependent manner, although the effect was more obvious in the 786-O cells (Fig. 2A). These results were verified by the WST-8 assay after a 24- and 48-h exposure to ubenimex (Fig. 2B).

To determine whether ubenimex affects the migratory ability of RCC cells, we performed scratch wound-healing migration assays. The migratory abilities of the 786-O and OS-RC-2 cells were significantly suppressed by ubenimex in a concentration-dependent manner after a 12- or 18-h exposure (Fig. 3). We further examined the effect of ubenimex on the invasion activity of RCC cells using Matrigel invasion assays. Pretreatment of ubenimex markedly inhibited the invasive abilities of the 786-O and OS-RC-2 cells, although once again, the effects appeared more dramatic in the 786-O cells (Fig. 4). Collectively, these results suggest that ubenimex inhibits the tumorigenic properties of RCC cells, including proliferation, migration and invasion of RCC cells, although the extent of the effect may vary according to the RCC cell line.

To determine whether ubenimex induces the cell death of RCC cells, we performed LDH cytotoxicity assays following ubenimex treatment. The LDH assay determines the extent of cell death irrespectively of the type of cell death. The mortality of both the RCC cells was significantly increased after treatment with 0.5 mg/ml ubenimex for 18 or 24 h; the mortality of the 786-O cells was also significantly increased after treatment with 0.25 mg/ml ubenimex for 18 or 24 h (Fig. 5).

To investigate the mechanism of this effect, we assessed the levels of an apoptosis marker (Caspase3) and an autophagy marker (LC-3B). LC-3B expression increased after ubenimex treatment, while the expression of Caspase3 remained unchanged (Fig. 6A) These results indicated that autophagic cell death, rather than apoptotic cell death may be the predominate mode of ubenimex-induced RCC cell cytotoxicity. To verify this hypothesis, an LDH cytotoxicity assay was performed after pretreating the RCC cells with rapamycin (an inducer of autophagy) or 3-methyladenine (an inhibitor of autophagy). Rapamycin enhanced the levels of ubenimex-induced cell death while 3-methyladenine reversed the effect in both cell lines (Fig. 6B). Western blotting confirmed the relationship between ubenimex and autophagy in 786-O cells (Fig. 6C). These results indicate that ubenimex promotes significant levels of cell death in RCC cells and that the death occurs via an autophagic mechanism.

In order to confirm this theory, electron microscopy was used to visualize the cell morphology after both cell lines were treated with ubenimex compared to the control cells. Ubenimex treatment increased the presence of autophagosomes filled with debris in both the cell lines; only a few vacuoles were observed in the control cells (Fig. 7A). Quantification of the autophagosomes indicated higher autophagy levels after ubenimex treatment (Fig. 7B).

Immunohistochemical analysis showed strong expression of APN in the epithelial cells of the renal proximal tubules of the normal kidney tissue. Most of the RCC tumor tissues (59/76, 77.6%) also showed positive staining, although the staining was comparably weak (Fig. 8A and B). Reduced expression of APN in the RCC tissue was verified by western blotting of 4 representative normal adjacent kidney tissues/RCC tissue pairs (Fig. 8C). The intensity of APN was not significantly different between the clear cells and the papillary RCC and was not associated with tumor size grade or stage (Table I). These results suggest that APN was expressed in the RCC, but the levels of expression was reduced as compared to the normal tissues. The differential expression of APN may be suggestive of a function of this protein in RCC. Furthermore, the expression of APN in some RCC tumors provides a rationale for considering ubenimex for the treatment of those tumors that express APN, although the extent of expression may limit the efficacy.