Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l glucose, L-glutamine and sodium pyruvate and antibiotics [100 µg/ml penicillin and 100 µg/ml streptomycin (P/S)] were purchased from Corning (Mediatech, Inc. Manassas, VA, USA). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Lipofectamine reagent was obtained from Promega (Madison, WI, USA). Tumor necrosis factor-α (TNF-α) was from R&D Systems (Minneapolis, MN, USA). Sodium butyrate, roscovitine, sulforaphane, wortmannin, PD98059, staurosporine, 5, 6-dichloro-1-β-D-ri bofuranosylbenzimidazole (DRB), lipopolysacchaide (LPS), caspase-3 inhibitor and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Gemcytabine was obtained from Hospira, Inc. (Lake Forest, IL, USA). Gemcitabine and caspase-3 inhibitor were diluted in phosphate-buffered saline (PBS) and the other reagents were dissolved in 100% ethanol prior to use.

A curated gene expression dataset comprising 23 normal and 29 tumor samples of the kidney cortex tissues of patients with clear cell RCC were obtained through the GEO database (GSE36895) for the analysis of regucalcin expression (36). These datasets contained gene expression data derived from the Affymetrix U133_plus2 platform. For microarray analysis, the expression and raw expression data (CEL files) were summarized and normalized using the Robust Multi-array Average algorithm and the Bioconductor package affy (http://www.bioconductor.org/packages/2.0/bioc/html/affy.html). The Spotfire Decision Site for Functional Genomics software package (TIBCO Software, Palo Alto, CA, USA) was used for visualization of the microarray data. For protein expression analysis, a dataset of immunohistochemistry was obtained from the Human Protein Atlas (HPA; www.proteinatlas.org), which is a database of proteins in human normal tissues and cancers (37,38). These were non-normally distributed data. We also evaluated regucalcin expression in 3 normal tissues and 12 renal adenocarcinoma tissues from the kidneys of patients by using the dataset of 2 antibodies (HPA029102 and HPA029103) for regucalcin. Moreover, we used the TCGA dataset of 468 patients with clear cell RCC (39) for outcome analysis. The data for regucalcin expression and clinical annotation were obtained by SurvExpress (40).

We used A498 cells obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). A498 cells are a renal proximal tubular epithelial cell line originating from a male adult patient with clear cell renal cell carcinoma (RCC), and are non-metastatic cells (41). The A498 cells are suitable as a transfection host. The cells were cultured in a DMEM containing 10% FBS and 1% P/S.

The A498 cells were transfected with the empty pCXN2 vector or pCXN2 vector expressing cDNA encoding human full length (900 bp) regucalcin (regucalcin cDNA/pCXN2). These vectors were prepared as used in our previous study (42). For transient transfection assays, the A498 cells were grown on 24-well plates to approximately 70–80% confluence. The regucalcin cDNA/pCXN2 or empty pCXN2 vector were transfected into A498 cells using the synthetic cationic lipid, Lipofectamine reagent, according to the manufacture’s instructions (Promega, Madison, WI, USA) (42). Following overnight incubation, Geneticin (600 µg/ml G418; Sigma-Aldrich) was added to the wells for selection, and the cells were cultured for 3 weeks. The surviving cells were plated at limiting dilution to isolate transfectants. Multiple surviving clones were isolated, transferred to 35-mm dishes, and grown in medium without geneticin. We obtained transfectant clones 1 and 2 exhibiting stable expression of regucalcin. The regucalcin levels in these clones were markedly increased as compared with those in the wild-type cells as shown in Fig. 2A and B. The regucalcin levels in clone 1 was higher than that of clone 2. Clone 1 was used in the following experiments.

The A498 wild-type cells or transfectants were seeded into 6-well dishes at a density of 1×10³/well and cultured in medium containing 10% FBS and 1% P/S under conditions of 5% CO₂ and 37°C for 8 days, when visible clones were formed on the plates (43). The colonies were washed with PBS and fixed with methanol (0.5 ml per well) for 20 min at room temperature, and then washed 3 times with PBS. The colonies were then stained with 0.5% crystal violet for 30 min at room temperature. Stained cells were washed 4 times with PBS. The plates were air-dried for 2 h at room temperature. The colonies containing >50 cells were counted under a microscope (Olympus MTV-3; Olympus Corporation, Tokyo, Japan).

The A498 wild-type cells (1×10⁵/ml per well) and A498 cells transfected with the regucalcin cDNA (1×10⁵/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 1, 2, 3, 4 or 6 days in a water-saturated atmosphere containing 5% CO₂ and 95% air at 37°C (42,44). In separate experiments, the A498 wild-type cells or transfectants were cultured in DMEM containing 10% FBS and 1% P/S in the presence of either sodium butyrate (10 and 100 µM), roscovitine (10 and 100 nM), sulphoraphan (1 and 10 nM), wortmannin (0.1 or 1 µM), PD98059 (1 or 10 µM), staurosporine (10 or 100 nM), TNF-α (0.1 or 1 ng/ml), DRB (0.1 or 1 µM), or gemcitabine (1 or 10 nM) for 3 days. Following culture, the cells were detached from each culture dishes by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg –free PBS (Thermo Fisher Scientific, Waltham, MA, USA) with incubation for 2 min at 37°C. Each well was then supplemented with 0.9 ml of DMEM containing 10% FBS and 1% P/S. The cell number in the cell suspension was counted as described below in the section ‘Cell counting’.

The A498 wild-type cells (1×10⁵/ml per well) cells and A498 cells transfected with the regucalcin cDNA (1×10⁵/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 3 days. Upon reaching subconfluency, they were cultured for an additional 24 h in the presence or absence of either TNF-α (0.1 or 1 ng/ml) or LPS (0.1 or 1 µg/ml) (45). In separate experiments, the A498 wild-type cells (1×10⁵/ml per well) or transfectants were cultured for 3 days, and upon reaching subconfluency, then cultured for an additional 24 h in the presence or absence of either TNF-α (1 ng/ml) or LPS (1 µg/ml) with or without caspase-3 inhibitor (10 µM) for 24 h (45). Following culture, the cells were detached by the addition of a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg²⁺-free PBS per well as described above in the section of ‘Cell proliferation assay’, and the cell number was counted as described below in the section ‘Cell counting’.

To detach cells on each well, the culture dishes were incubated for 2 min at 37°C following the addition of a solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca²⁺/Mg²⁺-free PBS, and the cells were detached through pipetting after the addition of DMEM (0.9 ml) containing 10% FBS and 1% P/S (31,42,44,45). Medium containing the suspended cells (0.1 ml) was mixed by the addition of 0.1 ml of 0.5% trypan blue staining solution. The number of viable cells was counted under a microscope (Olympus MTV-3; Olympus Corporation) with a hemocytomete (Sigma-Aldrich) using a cell counter (Line Seiki H-102P, Tokyo, Japan). For each dish, we took the average of 2 counts. Cell numbers are shown as number per well.

The A498 wild-type cells, control vector cDNA-transfected cells, or regucalcin cDNA-transfected cells were plated in 100 mm dishes at a density of 1×10⁶ cells/well in 10 ml of DMEM containing 10% FBS and 1% P/S. Following culture for 3 days, the cells were washed 3 times with cold PBS and removed from the dish by scraping using cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with the addition of protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). The lysates were then centrifuged at 17,000 × g, at 4°C for 10 min. The protein concentration of the supernatant was determined for western blotting using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard. The supernatant was stored at −80°C until used. Samples of 40 µg of supernatant protein per lane were separated by SDS polyacrylamide gel electrophoresis (12%, SDS-PAGE) and transferred onto nylon membranes for immunoblotting using specific antibodies against various proteins obtained from Cell Signaling Technology, Inc. including Ras (#14429 rabbit), PI3 p1100 α (#4255, rabbit), Akt (#9272, rabbit), phosphor-Akt (#9271, rabbit), mitogen-activated protein kinase (MAPK; #4695, rabbit), phosphor-MAPK (#4370, rabbit), Rb (#9309, mouse), p21 (#2947, rabbit), c-jun (#9165, rabbit), signal transducer and activator of transcription 3 (Stat3; #12640, rabbit), phospho-Stat3 (#9131, rabbit) and β-actin (#3700, mouse) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) including p53 (sc-126, mouse), c-fos (sc-52, rabbit), nuclear factor (NF)-κB p65 (sc-109, rabbit) and β-catenin (sc-39350, mouse). Rabbit anti-regucalcin antibody was obtained from Abcam (Cambridge, MA, USA; ab213459, rabbit), as has been used previously (22,28,32). Target proteins were incubated with one of the primary antibodies (1:1,000) overnight at 4℃, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305; diluted 1:2,000). The immunoreactive blots were visualized with a SuperSignal West Pico Chemiluminescent Substrate detection system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. β-actin (diluted 1:2,000; Cell Signaling Biotechnology, Inc.; #3700, mouse) was used as a loading control. Three blots from independent experiments were scanned on an Epson Perfection 1660 Photo scanner, and bands quantified using Image J software.

Statistical significance was determined using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc., La Jolla, CA, USA). Multiple comparisons were performed by one-way analysis of variance (ANOVA) with the Tukey-Kramer multiple comparisons post test for parametric data as indicated. Survival curves were constructed by Kaplan-Meier analysis and were compared with the log-rank test as performed with IBM SPSS. The non-normally distributed data in Fig. 1B were analyzed by the Mann-Whitney test and the rest of the data were analyzed with the Student’s t-test to compare the means as performed with IBM SPSS Statistics 18 software (IBM, Chicago, IL, USA; http://www.ibm.com). P<0.05 was considered to indicate a statistically significant difference.

To generate regucalcin-overexpressing cells, human clear cell RCC A498 cells were transiently transfected with the empty pCXN2 vector or the vector containing full length (33 kDa protein) human regucalcin using lipofection. We obtained transfectant clone 1 or 2 with stable expression of regucalcin. The regucalcin levels in these clones were increased 11.2- or 6.1-fold as compared with wild-type cells, respectively (Fig. 2A and B). To determine the effects of the overexpression of regucalcin on the growth of A498 cells in vitro, clone 1 was used in the following experiments. First, to determine the effects of the overexpression of regucalcin on colony formation, the A498 wild-type cells and transfectants were cultured for 8 days when colony formation clearly appeared (Fig. 2C). The number of colonies was found to be decreased in the regucalcin-overexpressing trans-fectants (vector, clone 1 or 2) as compared with that of the wild-type cells (Fig. 2D). The mass growth of the wild-type A498 cells was enhanced with the increasing days of culture periods (Fig. 2E). This enhancement was clearly suppressed in the transfectants (Fig. 2E). Thus, the overexpression of regucalcin suppressed the colony formation and proliferation of human renal cancer A498 cells.

The effect of the overexpression of regucalcin on the death of A498 cells was then investigated. The A498 wild-type cells and transfectants were cultured for 3 days to reach subconfluency. They were then cultured for a further 24 h following the addition of various factor known to induce apoptotic cell death (27,45). The number of wild-type cells was decreased by culture with TNF-α (0.1 or 1 ng/ml) or LPS (0.1 or 1 µg/ml) (Fig. 3A). The overexpression of regucalcin did not lead to the death of the wild-type cells, and the apoptotic cell death-inducing factors did not cause the cell death of the transfectants (Fig. 3B). This suggests that the suppressive effects of regucalcin over-expression on cell growth do not result from the death of A498 cells.

We then investigated whether the effects of overexpressed regucalcin on cell death are mediated via caspase-3, which activates nuclear DNA fragmentation, inducing apoptotic cell death (16). Regucalcin has been demonstrated to suppress nuclear DNA fragmentation due to the inhibition of caspase-3 activity in isolated rat liver nuclei (16) and to induce apoptotic cell death in cloned rat hepatoma H4-II-E cells induced by TNF-α or thapsigargin (45). The A498 wild-type cells and transfectants, upon reaching subconfluency were cultured in the presence of TNF-α (1 ng/ml) or LPS (1 µg/ml) with or without caspase-3 inhibitor (10 µM) for 24 h (Fig. 3C and D). The effects of LPS or TNF-α on cell death were either not observed or diminished in the presence of caspase-3 inhibitor (Fig. 3C). The stimulatory effects of TNF-α or LPS on cell death were either not observed or diminished in transfectants cultured with or without caspase-3 inhibitor (Fig. 3D). These findings suggest that the suppressive effects of regucalcin overexpression on cell growth are likely due, at in least part, to the inhibition of caspase-3 activity in A498 cells. Thus, the suppressive effects of regucalcin overexpression on the proliferation of A498 cells are not mediated by cell death.

Suppressive effects of regucalcin overexpression on the proliferation of A498 cells are mediated through various signaling pathways.

To determine the mechanisms through which regucalcin overexpression suppresses the proliferation of A498 cells, we investigated whether the suppressive effects of regucalcin overexpression are attenuated in the presence of various inhibitors that induce cell cycle arrest in vitro (Fig. 4). Wild-type cells were cultured for 3 days in the presence of butyrate (10 and 100 µM) (46), roscovitine (10 and 100 nM) (47) or sulforaphane (1 and 10 nM) (48). The proliferation of the wild-type cells was suppressed in the presence of these inhibitors (Fig. 4A). The effects of these inhibitors were not potentiated in the transfectants (Fig. 4B). These results suggest that the overexpression of regucalcin induces G1 and G2/M phase cell cycle arrest in the A498 cells.

Subsequently, we determined the involvement of signaling factors in the suppressive effect on cell proliferation induced by the overexpression of regucalcin. The proliferation of the A498 wild-type cells was suppressed in the presence of wortmannin (0.1 or 1 µM), an inhibitor of PI3 kinase (50), PD98059 (1 or 10 µM), an inhibitor of extracellular signal-regulated kinase (ERK) and MAP kinase (51), and staurosporine (10 or 100 nM), a calcium signaling-related inhibitor (52) (Fig. 5A). The blocking of these pathways did not potentiate the suppressive effects of regucalcin overexpression of cell proliferation (Fig. 5B).

DRB is an inhibitor of RNA polymerase II-dependent transcriptional activity (53). Gemcitabine is a potent antitumor agent that induces nuclear DNA damage (54). In the current study, these inhibitors inhibited the proliferation of wild-type cells (Fig. 5C). However, these effects did not occur in the trans-fectants (Fig. 5D). These results suggest that the overexpression of regucalcin suppresses various signaling processes linked to cell proliferation, and that the regucalcin-overexpressing cells exhibit a lack of responses to the above-mentioned inhibitors of these pathways.

Overexpression of regucalcin regulates the expression of various proteins linked to cell signaling and transcriptional activity. We then investigated whether the overexpression of regucalcin affects the expression of key protein involved in signaling pathways and transcriptional activity. The results of western blot analysis revealed that the levels of Ras, PI3 kinase, Akt, phospho-Akt, MAP kinase and phospho-MAP kinase were diminished by the overexpression of regucalcin (Fig. 6A and B). These results suggest that the overexpression of regucalcin suppresses the activation of Ras-linked signaling pathways in A498 cells. By contrast, the overexpression of regucalcin elevated the protein levels of tumor suppressors, p53 and Rb, and p21, an inhibitor of the cell cycle (Fig. 6C and D). In addition, the overexpression of regucalcin diminished the levels of c-fos, c-jun, Stat3, β-catenin and NF-κB p65, which are transcription factors linked to the proliferation of A498 cells (23,25,55) (Fig. 6C and D). We determined the changes in the levels of 14 proteins, which may be major signaling proteins related to the proliferation of cancer cells, although various other proteins are also implicated in the proliferation of cancer cells.