The affinity-purified rabbit anti-P5 antibody used in this study was kindly provided by Dr T. Komiya (Nagahama Institute of Bio-Science and Technology, Nagahama, Japan) (6). The rabbit anti-PDI polyclonal antibody was prepared as previously described (12). Protein G PLUS-agarose was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Recombinant human vimentin protein was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Anacardic acid, ribostamycin, bovine serum albumin (BSA) and purified PDI were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Temozolomide (TMZ) was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Pre-stained protein markers for SDS-PAGE and thapsigargin (Tg) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The other reagents were mostly obtained from Nacalai Tesque, Inc. All reagents used were of research grade.

The normal and cancer cell lines used in the present study are presented in Table I. A172, SNB19, T98G, BT20, MDA-MB-231, T47D, H322, H460, H526, Panc-1, SU86.86, Caki-1, LNCaP, PA-1, HeLa and WI38 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). BxPC3, HCT116, SK-OV-3 and OE19 cell lines were obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK). HepG2, HuCCT-1, KMRC-1, DLD-1, LoVo, SW837, PC-3, U937, HL-60, K562 and THP-1 cell lines were obtained from Health Science Research Resources Bank (Osaka, Japan). SW48 and SW480 cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Astrocytes (ACBRI371), pancreatic epithelial (PE) cells (ACBRI515), and hepatocytes (ACBRI3716) were purchased from Cell Systems (Kirkland, WA, USA) via DS Pharma Biomedical Co., Ltd. U251 and 293T cell lines were obtained from the National Cancer Institute, Frederick Cancer Research Facility, Division of Cancer Treatment Tumor Repository Program (Frederick, MD, USA) and RIKEN BioResource Center (Tsukuba, Japan), respectively. The ML-RCC cell line was kindly provided by Dr R. Puri (Center for Biological Evaluation, Food and Drug Administration, Silver Spring, MD, USA); the cell line was established as previously described (13). The cells were cultured in RPMI-1640 (Nacalai Tesque, Inc.) (U251, A172, SNB19, BT20, MDA-MB-231, T47D, H322, H460, H526, BxPC3, SU86.86, HuCCT-1, ML-RCC, DLD-1, SW837, LNCap, OE19, U937, HL-60, K562 and THP-1), McCoy's 5A (Thermo Fisher Scientific, Inc., Waltham, MA, USA) (Caki-1, HCT-116 and SK-OV-3), Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque, Inc.) (Panc-1, KMRC-1, HeLa and 293T), MEM (Nacalai Tesque, Inc.) (T98G, HepG2, PC-3, PA-1 and WI38), DMEM/F12 (Nacalai Tesque, Inc.) (LoVo, SW48 and SW480) or Complete medium kit (cat no. 4Z0-500-R; Cell Systems) (astrocytes, PE cells and hepatocytes) containing 10% fetal bovine serum (cat no. 172012-500ML; Sigma-Aldrich; Merck KGaA), 100 µg/ml penicillin and 100 µg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂ and 95% ambient air. All of the cell lines were used for flow cytometry and western blotting to assess the expression levels of P5. U251/Luc, SNB19, PE and astrocytes were used for the cell growth assay post-transfection with siRNA. U251, PE, BT20, H322, DLD-1, PA-1 and U937 cells were used for immunoprecipitation. U251/Luc cells were used for all other experiments described in this study.

Recombinant human P5 protein was purified using a Ni²⁺-chelating Resin Column (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), as previously described (11,12).

PDI, P5 and BSA proteins (1.6 µg) were separated by 12.5% SDS-PAGE, and western blotting was performed using rabbit anti-PDI (1:6,000) or anti-P5 (1:6,000) antibodies as primary antibodies, and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) (1:2,000; cat. no. NA934-1ML; GE Healthcare Bio-Sciences) as a secondary antibody. The staining of gel and membranes following SDS-PAGE and western blotting was performed using Coomassie Brilliant Blue (Nacalai Tesque, Inc.) and the Peroxidase Stain DAB kit (Nacalai Tesque, Inc.), respectively.

Western blotting was conducted as previously described (14). Briefly, total protein extracts were prepared from cells lysed with reporter lysis buffer (Promega Corporation, Madison, WI, USA). Subsequently, total protein concentration was quantified by measuring the absorbance at 280 nm using a NanoDrop 1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Proteins (20 µg/lane) were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes using the iBlot system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The membranes were blocked with 5% skim milk (w/v) in PBS for 90 min at room temperature, after which, the membranes were probed with anti-P5 (1:6,000), anti-Bip (1:500; cat. no. MAB4846; R&D Systems, Inc. Minneapolis, MN, USA), anti-vimentin (1:800; cat. no. ab20346; Abcam, Cambridge, UK) or anti-β-actin (1:4,000; cat no. A5316-.2ML; Sigma-Aldrich; Merck KGaA) as primary antibodies overnight at 4°C, and with a horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:2,000; cat. no. NA934-1ML; GE Healthcare Bio-Sciences) or sheep anti-mouse IgG (1:2,000; cat. no. NA931-1ML; GE Healthcare Bio-Sciences) secondary antibody for 3 h at room temperature. The blots were then analyzed with Chemi-Lumi One Super reagent (Nacalai Tesque, Inc.) using a LAS-3000 LuminoImage analyzer (Fujifilm, Tokyo, Japan). Densitometric analysis of the bands obtained from western blotting was performed using Multi Gauge software V3.0 (Fujifilm).

Flow cytometry was performed as previously described (15). Briefly, the affinity-purified rabbit anti-P5 antibody was labeled with 6-(fluorescein-5-carboxamido) hexanoic acid succinimidyl ester (FAM-X) using a FAM-X labeling kit (KPL, Inc., Gaithersburg, MD, USA), according to the manufacturer's protocol. Subsequently, 1.0×10⁵ normal or cancer cells were incubated with the labeled antibody for 2 h at room temperature. After incubation, the cells were washed twice with PBS and flow cytometry was performed using FACSCalibur (BD Biosciences, San Jose, CA, USA). The data were analyzed using CellQuest software version 6.0 (BD Biosciences).

Stealth RNA interference (RNAi) duplexes, negative control medium GC (cat. no. 12935300; Thermo Fisher Scientific, Inc.) and P5 siRNA (5′-CCAUAUCCUUGAUACUGGAGCUGCA-3′ and 5′-UGCAGCUCCAGUAUCAAGGAUAUGG-3′) (Invitrogen; Thermo Fisher Scientific, Inc.) were used for P5 knockdown experiments, as previously described (16). Briefly, U251/Luc, SNB19, PE cells and astrocytes were grown to 40–50% confluence on a 6-well plate or 35 mm glass-bottomed dish; the cells were and transfected with 0.125 or 0.25 µM siRNA in incomplete medium without FBS, penicillin and streptomycin. siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol (6,16). After transfection, cells were kept at 37°C for 3 h, and medium was replaced with complete medium. A total of 24 h post-transfection, bioluminescence imaging was performed, and 48 or 72 h post-transfection, cell samples were prepared for western blotting or reverse transcription-polymerase chain reaction (RT-PCR). A total of 72 h post-transfection, cells were seeded in 96-well plates for cell growth and migration assays.

Cell growth was assessed using the WST-8 assay, as previously described (15). Briefly, cells were seeded in 96-well plates at a concentration of 3,000 cells/well, and the number of living cells was measured using Cell Count Reagent SF (Nacalai Tesque, Inc.). Absorbance was measured at a wavelength of 450 nm using a microplate reader (GE Healthcare Bio-Sciences). Cell viability was also assessed using the WST-8-assay, as previously described (15). Briefly, U251/Luc cells were seeded in 96-well plates as aforementioned, and were treated with various concentrations (0, 25, 50, 100 and 200 µM) of TMZ in the presence or absence of 25 µM anacardic acid, 1 mM ribostamycin, or a combination of these compounds. Cell viability was calculated after 48 h; the viability of untreated control cells was set to 100%. Cell migration was assessed using an Oris™ Cell migration assay (Platypus Technologies, LLC, Madison, WI, USA), according to the manufacturer's protocol.

Immunoprecipitation was performed using an anti-P5 antibody, as previously described (14). Briefly, cancer and normal cells were washed with ice-cold PBS and were lysed with radioimmunoprecipitation assay buffer (Nacalai Tesque, Inc.) on ice for 15 min. Cell lysates were collected after centrifugation at 300 × g for 5 min at 4°C, and the total protein concentration of the supernatant was determined spectrophotometrically using a NanoDrop 1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.). The supernatant from the cell lysate containing 100 µg of protein was incubated with 50 µl Protein G PLUS-agarose solution at 4°C for 1 h, and following centrifugation at 19,000 × g for 3 min, the supernatant was transferred to a new tube. The supernatant was incubated at 4°C for 1 h following addition of an anti-P5 antibody (1:100), and was further incubated at 4°C overnight following the addition of 50 µl protein G PLUS-agarose solution. The agarose was collected by centrifugation at 19,000 × g for 3 min, washed at least three times with cold PBS, and boiled in SDS-PAGE sample buffer at 98°C for 5 min. The samples were then separated by 12.5% SDS-PAGE, and the bound proteins were visualized by silver staining using Sil-Best Stain One (Nacalai Tesque, Inc.), according to the manufacturer's protocol. To examine the binding of vimentin with P5 in cells by immunoprecipitation, total cell lysates from U251, PE, BT20, H322, DLD-1, PA-1 and U937 cells were prepared, and immunoprecipitation was performed using the anti-P5 antibody as aforementioned. The samples were separated by 12.5% SDS-PAGE, and then western blotting was performed using anti-P5 and anti-vimentin antibodies.

The bands of interest were excised from the gel using a box cutter after silver staining, and in-gel digestion was performed using the In-gel tryptic digestion kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Gel trypsin digestion and protein identification by liquid chromatography-tandem mass spectrometry (LC/MS/MS) were performed as previously described (14) at the Medical Research Support Center, Kyoto University (Kyoto, Japan). Acquired datasets were analyzed using ProteinPilot Software v. 4.5 Beta (SCIEX, Tokyo, Japan) with the Paragon algorithm and a combined database of UniProtKB/Swiss-Prot data and known contaminants (SCIEX).

Surface plasmon resonance experiments were performed using the Biacore T100 system (GE Healthcare Bio-Sciences), as previously described (11). Briefly, recombinant human vimentin protein was immobilized on the surface of a CM5 sensor chip with N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide activation chemistry, according to the manufacturer's protocol. As an analyte, purified recombinant P5 protein was injected over the flow cell, and HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% Tween-20, 3 mM EDTA, pH 7.4) was used as a running buffer to inhibit nonspecific binding. An approximate equilibrium dissociation constant (Kd) value was obtained as previously described using Biacore T100 evaluation software ver. 2.0.2 (GE Healthcare Bio-Sciences) (17).

RT-PCR was performed as previously described (18). Briefly, following isolation of total RNA using a NucleoSpin RNA kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), RT was conducted using a ReverTra Ace qPCR RT kit (Toyobo Life Science, Osaka, Japan), according to the manufacturer's protocol. Subsequently, each 1-µl aliquot of cDNA was amplified in a final 50-µl PCR mixture containing Titanium taq DNA Polymerase (1:100; Takara Bio, Inc., Otsu, Japan) and 0.2 mM dNTP mixture solution from the Long Range PCR kit (Qiagen GmbH, Hilden, Germany). The PCR thermocycling conditions were as follows: Initial denaturation at 94°C for 1 min, followed by 30–35 cycles at 94°C for 30 sec, 60–61°C for 30 sec and 72°C for 30 sec, and a final extension step at 72 °C for 30 sec. For upregulation of the CHOP gene, which is a positive control for ER stress, U251/Luc cells were treated with 0.5 µM Tg for 6 h at 37°C, and cells were collected for the isolation of total RNA as aforementioned. Specific primers for RT-PCR were as follows: CCAAT-enhancer-binding protein homologous protein (CHOP), forward 5′-ATCAAAAATCTTCACCACTCTTGAC-3′, reverse 5′-ACTTTCCTTTCATTCTCCTGTTCTT-3′; vimentin, forward 5′-GGTACAAATCCAAGTTTGCTGACC-3′, reverse 5′-CTCAATGTCAAGGGCCATCTTAAC-3′; Snail, forward 5′-CAAGGCCATGTCCGGACCCACACTGGCG-3′, reverse 5′-CTTCCTGCTGGAGCTGGGGAAGGCTGTC-3′; Slug, forward 5′-GGCCAAACATAAGCAGCTGCACTGCG-3′, reverse 5′-CAGATGAGCCCTCAGATTTGACCTGTC-3′; Twist, forward 5′-GCAGACGCAGCGGGTCATGGCCAACG-3′, reverse 5′-CATCCTCCAGACCGAGAAGGCGTAGC-3′; N-cadherin, forward 5′-GACCCATCCACGCCGAGCCCCAGTATCCG-3′, reverse 5′-CACCCTGAAGTTCAGTCATCACCTCCACC-3′; E-cadherin, forward 5′-GCTAGTCTGAGCTCCCTGAACTCCTCAG-3′, reverse 5′-GGGGCCCGCCTCTCTCGAGTCCCCTAGTCG-3′; and GAPDH, forward 5′-GTCTTCACCACCATGGAGAAGGCT-3′ and reverse 5′-CATGCCAGTGAGCTTCCCGTTCA-3′. GAPDH was used as an internal control. PCR products were run on a 1% agarose gel, which was stained with GelRed (Biotium, Fremont, CA USA) for UV analysis.

U251 cells stably transfected with pBipPro-Luc (U251/Luc), in which the Bip promoter region was cloned into the pGL4.14 vector (Promega Corporation), were prepared. Successful transfection was confirmed using the LV200 imaging system (Olympus Corporation, Tokyo, Japan), through the observation of bioluminescence, as previously described (19). Luminescence images at the single-cell level were obtained using the LV200 imaging system, as previously described (19). Briefly, 24 h post-transfection of U251/Luc cells with negative control or P5 siRNA, images were captured using a ×40 objective lens at 10-min intervals, and Bip promoter activity was observed following the addition of D-luciferin. An expression vector of vimentin fused with PSmOrange (20) (vim/Orange) was provided by Addgene, Inc. (Cambridge, MA, USA). For the observation of vim/Orange, BP535-555HQ (Olympus Corporation) and 570-625RFP (Olympus Corporation) were used as excitation and emission filters, respectively. U251/Luc cells stably transfected with vim/Orange (U251/Luc/Orange) were also prepared in selective medium containing 200 µg/ml hygromycin B (Nacalai Tesque, Inc.) and 1,000 µg/ml G-418 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Successful transfection was confirmed using the LV200 system, through the observation of both bioluminescence and fluorescence using the filters. After the establishment of stable U251/Luc and U251/Luc/Orange cells, they were transfected with the negative control and P5 siRNAs, as aforementioned. All data analysis was performed using AQUACOSMOS ver. 2.6 software (Hamamatsu Photonics, Hamamatsu, Japan).

Experiments were repeated at least two times. Statistical significance was determined using Student's t-test for pairwise comparisons, whereas multiple comparisons were evaluated by one-way analysis of variance followed by Dunnett's test using JMP Pro version 14 (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study examined the expression levels of P5 on the surface of normal and cancer cells by fluorescence-activated cell sorting (FACS) analysis using an affinity-purified anti-P5 antibody labeled with FAM-X, which was confirmed to be specific to P5 protein and did not cross-react with PDI or bovine serum albumin (Fig. 1A). The expression levels of P5 were detected on the cell lines shown in Table I using FACS analysis; the results revealed that P5 expression was markedly increased on the surface of several cancer cell lines, including U251, T98G, H322, DLD-1, HCT116, SW837, PA-1, U937, K562 and THP-1 cells compared with normal cell lines, and it was expressed at the highest levels on the surface of K562 and THP-1 cells, which are chronic myelogenous and acute monocytic leukemia cell lines (21,22), respectively (Fig. 1B). A constant increase in the surface expression of P5 was observed in glioblastoma (U251, A172, SNB19, and T98G), breast (BT20, MDA-MB-231, T47D), colon (DLD-1, HCT116, LOVO, SW480, SW837), and ovarian and uterine cervical (PA-1, SK-OV-3, HeLa) cancer, and leukemia (U937, HL-60, K562, THP-1) cell lines (Fig. 1B). In addition, the expression levels of P5 were compared between normal and cancer cell lines; its expression was significantly increased on the surface of cancer cell lines, compared with normal cells; leukemia cell lines were not included in this analysis (Fig. 1C). The present study also investigated the expression of P5 in these cell lines by western blotting using an anti-P5 antibody. P5 was expressed in all cell lines examined; however, its expression levels differed among the cell lines (Fig. 2A and B). When the expression levels of P5 in total cell lysates were compared between normal and cancer cell lines, it was revealed that its expression was not significantly different between normal and cancer cells; leukemia cell lines were not included in this analysis (Fig. 2B). These results suggested that P5 expression may be increased on the surface of several cancer cells compared with normal cells; however, the enzyme was expressed at a constant level within both normal and cancer cells.

Alongside a previous report regarding the role of P5 in cancer cells (4), the present study indicated that P5 may have a significant role in cancer cells. Therefore, the present study further investigated the functional roles of P5 in cancer cells. The effects of P5 knockdown in cancer cells on activation of the Bip promoter during cancer cell growth were analyzed, as our previous study indicated that the Bip promoter is periodically activated, according to real-time monitoring using bioluminescence at the single-cell level in U251 glioblastoma cells (19). Following siRNA-induced knockdown of P5, western blotting confirmed that its expression was reduced in cancer cells (Fig. 3A). Real-time bioluminescence monitoring at the single-cell level demonstrated that Bip promoter activation was observed in U251/Luc cells even post-transfection with negative control siRNA during cancer cell growth (Fig. 3B and C), as shown in our previous study (19); however, it was not activated post-transfection with P5 siRNA, although Bip promoter activity was maintained and luminescence was still observed in the cancer cells (Fig. 3B and C). Since the Bip promoter is periodically activated, particularly in dividing cells during cell growth (19), these results suggested that knocking down P5 in cancer cells may affect Bip promoter activity and cancer cell division during cell growth. The present study investigated the effects of P5 knockdown in U251/Luc cells on the expression of Bip and CHOP, which are markers of ER stress responses; there was no significant difference in their expression between the groups transfected with the negative control or P5 siRNAs (Fig. 3D). Therefore, knocking down P5 in cancer cells may not induce an ER stress response.

Since Bip promoter activation during cancer cell growth was not observed post-transfection with P5 siRNA, the present study examined the effects of siRNA-induced P5 knockdown in U251/Luc cancer cells on cell growth and migration. P5 knockdown in cancer cells significantly inhibited cell growth, as assessed using the WST-8 assay, compared with in cells transfected with or without negative control siRNA (Fig. 4A); this phenomenon was also observed when using another glioblastoma cell line, SNB19 (data not shown). However, transfection of normal PE or astrocyte cells with P5 siRNA, in which the knockdown of P5 expression was confirmed by western blotting, did not inhibit cell growth (Fig. 4A). The present study also investigated the effects of P5 knockdown in U251/Luc cancer cells on cell migration; P5 knockdown reduced cancer cell migration compared with in cells transfected with or without negative control siRNA (Fig. 4B). These results suggested that P5 may serve an important role in cancer cell growth and migration.

Since the present findings suggested that P5 serves important roles in cancer cells, further functional analysis of this enzyme was conducted by screening for specific proteins that bind to P5 in cancer cells compared with normal cells. Immunoprecipitation was performed using an anti-P5 antibody, and several proteins were revealed to specifically bind to P5 in cancer cells compared with normal cells (Fig. 5A). After performing the immunoprecipitation experiments several times, one of the protein bands visualized by SDS-PAGE and silver staining was identified as a specific protein that bound to P5 in cancer cells (Fig. 5A). To identify this binding protein, the band was excised from the gel, in-gel digested and analyzed by LC-MS/MS. The band was identified as human vimentin protein (data not shown). Subsequently, immunoprecipitation with an anti-P5 antibody using a total cell lysate from U251 cells, and western blotting using an anti-vimentin antibody, confirmed that vimentin could bind to P5. In addition, vimentin was revealed to bind to P5 predominantly in U251 glioblastoma cells (Fig. 5B). Subsequently, the present study performed a biomolecular interaction analysis with purified recombinant P5 and vimentin proteins using the Biacore T100 system; vimentin immobilized on the sensor chip was revealed to interact with P5 protein and the Kd value of this interaction was 1.13±0.26×10⁻⁵ M (Fig. 5C). These results suggested that vimentin may bind to P5 specifically and is a P5-binding protein predominantly in glioblastoma cells.

Since vimentin was revealed to predominantly bind to P5 in glioblastoma cells, the present study examined the effects of siRNA-induced P5 knockdown on the expression of vimentin in glioblastoma cells. P5 knockdown did not affect the expression of vimentin, as assessed by RT-PCR (Fig. 6A). Conversely, P5 knockdown affected the morphology of glioblastoma cells compared with cells transfected with negative control siRNA (Fig. 6B). The present study also conducted real-time monitoring, and simultaneously observed luminescence and fluorescence at the single-cell level using U251/Luc/Orange cells and the LV200 system; activity of the Bip promoter was decreased in cancer cells transfected with P5 siRNA and the expression pattern of vimentin was different from that in cells transfected with negative control siRNA (Fig. 6C). In addition, P5 knockdown inhibited the division of glioblastoma cells and induced death of cells that could not divide, as observed by the LV200 system (data not shown). These results suggested that P5 serves important roles in the division of glioblastoma cells and the localization of vimentin in these cells. Since vimentin is well known as a member of the intermediate filament family (23), and is widely used as a marker of EMT, which occurs during the metastasis of cancer cells (24,25), the effects of siRNA-induced P5 knockdown in glioblastoma cells on the expression of EMT marker proteins, such as Snail, Slug, Twist, N-cadherin and E-cadherin, were determined. P5 knockdown in glioblastoma cells decreased the expression of Snail and increased the expression of Slug; however, it had no effect on the expression of Twist, N-cadherin and E-cadherin, as assessed by RT-PCR (Fig. 6D).

The present study investigated the effects of anacardic acid on the cytotoxic activity of TMZ, in order to evaluate the availability of P5 for molecular targeting. In response to anacardic acid, the cytotoxic activity of TMZ against glioblastoma cells was not significantly affected (Fig. 7). However, a slight increase in the cytotoxic activity of TMZ was detected in the presence of both anacardic acid and ribostamycin; however, this finding was still not significant (Fig. 7).