Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma

  • Authors:
    • Ryota Kondo
    • Kousuke Ishino
    • Ryuichi Wada
    • Hideyuki Takata
    • Wei‑Xia Peng
    • Mitsuhiro Kudo
    • Shoko Kure
    • Yohei Kaneya
    • Nobuhiko Taniai
    • Hiroshi Yoshida
    • Zenya Naito
  • View Affiliations

  • Published online on: February 4, 2019     https://doi.org/10.3892/ijo.2019.4710
  • Pages: 1409-1421
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Protein disulfide‑isomerase A3 (PDIA3) is a chaperone protein that modulates folding of newly synthesized glycoproteins and responds to endoplasmic reticulum (ER) stress. Previous studies reported that increased expression of PDIA3 in hepatocellular carcinoma (HCC) is a marker for poor prognosis. However, the mechanism remains poorly understood. The aim of the present study, therefore, was to understand the role of PDIA3 in HCC development. First, immunohistochemical staining of tissues from 53 HCC cases revealed that HCC tissues with high PDIA3 expression exhibited a higher proliferation index and contained fewer apoptotic cells than those with low expression. In addition, the knockdown of PDIA3 significantly inhibited cell proliferation and induced apoptosis in HCC cell lines. These results suggest that PDIA3 regulates cell proliferation and apoptosis in HCC. An examination of whether PDIA3 knockdown induced apoptosis through ER stress revealed that PDIA3 knockdown did not increase ER stress marker, 78 kDa glucose‑regulated protein, in HCC cell lines. Furthermore, the association between PDIA3 and the signal transducer and activator of transcription 3 (STAT3) signaling pathway were investigated in vitro and in vivo. Immunofluorescence staining and co‑immunoprecipitation experiments revealed colocalization and binding, respectively, of PDIA3 and STAT3 in HCC cell lines. The knockdown of PDIA3 decreased the levels of phosphorylated STAT3 (P‑STAT3; Tyr705) and downstream proteins of the STAT3 signaling pathway: The anti‑apoptotic proteins (Bcl‑2‑like protein 1, induced myeloid leukemia cell differentiation protein Mcl‑1, survivin and X‑linked inhibitor of apoptosis protein). In addition, PDIA3 knockdown provided little inhibitory effect on cell proliferation in HCC cell lines treated with AG490, a tyrosine‑protein kinase JAK/STAT3 signaling inhibitor. Finally, an association was demonstrated between PDIA3 and P‑STAT3 expression following immunostaining of 35 HCC samples. Together, the present data suggest that PDIA3 promotes HCC progression through the STAT3 signaling pathway.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortalities worldwide (1). The principal treatment for HCC is surgical resection or liver transplantation (2). However, in the period between 2001 and 2008, the cumulative recurrence rate following complete resection of the tumor remained high (40-60% after 3 years) (3-5). Surgical treatment is often unavailable for patients with HCC who were diagnosed at an advanced stage. Multikinase inhibitors (sorafenib, regorafenib and lenvatinib) are approved for the systemic treatment of inoperable HCC. However, such drugs prolong the survival of patients with inoperable HCC for only a few months (6,7). Therefore, it is necessary to investigate novel therapeutic targets for this disease.

The signal transducer and activator of transcription 3 (STAT3) pathway is one of the major signaling pathways that promote tumor progression in HCC (8). This pathway is stimulated by inflammatory cytokines and growth factors (9-11). Activation of STAT3 through phosphorylation of tyrosine 705 leads to the formation of dimers that subsequently move from the cytosol to the nucleus to bind DNA, and thereby regulate gene expression and promote cell proliferation and survival (9-11). In the case of the liver, the STAT3 signaling pathway is activated by chronic hepatitis (hepatitis B or C infections, alcoholic hepatitis and non-alcoholic steatohepatitis) (11). It has been demonstrated that activated STAT3 is associated with tumor invasiveness, metastasis and poor prognosis in HCC (12-14).

Protein disulfide-isomerase A3 (PDIA3), also known as endoplasmic reticulum (ER) resident protein 57 or 58 kDa glucose-regulated protein, is a thiol oxidoreductase with protein disulfide isomerase activity. PDIA3 modulates the folding of newly synthesized glycoproteins and misfolded proteins in the ER (15). This protein also protects cells from ER stress-induced apoptosis (15). Furthermore, it has a variety of functions in the cytosol and nucleus (15). In several types of cancer, PDIA3 forms a complex with STAT3 in the nucleus (16-18). A high frequency of PDIA3-STAT3 complex formation is a marker of poor prognosis and increases resistance to radiotherapy in laryngeal cancer through modulation of STAT3 activity (19). We have previously reported that the PDIA3 expression levels in HCC tissues are higher than those in adjacent non-cancerous tissues, and that they are associated with overall survival time (20). However, the biological role of PDIA3 in HCC remains unclear.

The aim of the present study was to understand the role of PDIA3 in HCC. PDIA3 expression levels, the effects of PDIA3 knockdown and the association between PDIA3 and STAT3 in HCC were examined. The expression of PDIA3 in HCC tissues was associated with cell proliferation, survival and expression of phosphorylated STAT3 (P-STAT3) in HCC. PDIA3 knockdown in HCC cell lines inhibited cell proliferation and induced apoptosis by suppression of the STAT3 signaling pathway. In the presence of the tyrosine-protein kinase JAK/STAT3 signaling inhibitor AG490, PDIA3 knockdown provided little additional inhibition of cell growth. These data suggest that PDIA3 promotes tumor development in patients with HCC through the STAT3 signaling pathway.

Materials and methods

Clinical samples

A total of 53 patients with HCC who underwent hepatectomy without preoperative therapy at the Nippon Medical School Hospital (Tokyo, Japan) between January 2016 and February 2018, were enrolled in this study. The tumor and adjacent normal tissues from these patients were fixed with 10% formalin at room temperature for 24 h within 2 h of resection and embedded in paraffin. Among them, 35 HCC samples were formalin-fixed within 30 min of resection and were used for immunostaining of P-STAT3. The baseline characteristics of the patients are summarized in Table I. This study was conducted according to the Declaration of Helsinki and the Japanese Society of Pathology, and was given official approval by the Ethics Committee of the Nippon Medical School Hospital (approval no. 29-03-908). Written informed consent was obtained from all patients.

Table I

Associations between clinicopathological factors and PDIA3 or P-STAT3 levels in hepatocellular carcinoma.

Table I

Associations between clinicopathological factors and PDIA3 or P-STAT3 levels in hepatocellular carcinoma.

CharacteristicsPDIA3 expression
P-STAT3 status
nHigh (n=29)Low (n=24)P-valuenPositive (n=15)Negative (n=20)P-value
Sex0.1270.372
 Male442222261016
 Female972954
Age, years0.2260.599
 <6513941147
 ≥65402020241113
HBsAg0.8050.605
 Positive633624
 Negative472621291316
HCV infection0.7070.767
 Positive2513121569
 Negative28161220911
Cirrhosis0.680.599
 Yes171071147
 No361917241113
AFP, ng/ml0.3150.486
 <20361818211011
 ≥20171161459
DCP, mAU/ml0.150.762
 <402310131367
 ≥4030191122913
Tumor sizea, cm0.990.503
 <5422319261214
 ≥51165936
Tumor number0.190.834
 1512724331419
 ≥2220211
Vascular invasion0.4420.419
 Positive1789826
 Negative362115271314
UICC stageb0.6240.245
 I352015261313
 II1899927
 III000000
 IV000000
Differentiation0.6970.827
 Good191091055
 Moderate30161423914
 Poor431211

a The largest diameter.

b Tumor-Node-Metastasis Classification of Malignant Tumors, 8th Edition (44). AFP, α-fetoprotein; DCP, des-γ-carboxy prothrombin; HBsAg, hepatitis B surface antigen; HCV, hepatitis C virus; PDIA3, protein disulfide-isomerase A3; P-STAT3, phosphorylated signal transducer and activator of transcription 3; UICC, Union for International Cancer Control.

Cell culture

Human hepatoma cell lines (Huh-7 and HuH-1) were obtained from the Japanese Collection of Research Bioresources cell bank (Osaka, Japan). A normal human hepatocyte cell line (THLE-2) was obtained from the American Type Culture Collection (Manassas, VA, USA). Huh-7 and HuH-1 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Nichirei Biosciences, Inc., Tokyo, Japan) at 37°C in a humidified 5% CO2 atmosphere.

The THLE-2 cell line was maintained in Bronchial Epithelial Cell Growth Medium (BEGM; Lonza Group Ltd., Basel, Switzerland) without gentamycin/amphotericin and epinephrine but with added 5 ng/ml epidermal growth factor (Corning, Inc., Corning, NY, USA), 70 ng/ml phosphoethanolamine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10% FBS. The THLE-2 cells were cultured in RPMI-1640 medium with 10% FBS for 7 days at 37°C in a humidified 5% CO2 atmosphere prior to protein extraction. A JAK/STAT3 signaling inhibitor, AG490 (Merck KGaA), dissolved in dimethylsulfoxide (DMSO; Wako Pure Chemical Industries, Ltd., Osaka, Japan), was used for the suppression of STAT3 signaling at 25 or 100 µM in cell growth or western blotting, respectively.

PDIA3 knockdown

Short-interfering RNAs (siRNAs) were purchased from Thermo Fisher Scientific, Inc. The two PDIA3 siRNAs used were designated PDIA3 si-1 and PDIA3 si-2 and known as Silencer® Select Pre-designed siRNA cat. no. 4392420, ID s6227 (5′-GGAAUAGUCCCAUUAGCAAtt-3′) and ID s6229 (5′-GCAACUUGAGGGAUAACUAtt-3′), respectively. Silencer® negative control #1 siRNA (cat. no. 4390844) was used as a negative control (Ctrl si). The transfection of the siRNA was performed using Lipofectamine® RNAiMAX Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The optimal concentration of siRNA for use in transfections was 5 nM. The medium was refreshed 24 h after cell seeding and the cells were cultured for a further 48 and 72 h for the apoptosis and cell cycle assay and western blotting, respectively. The cell transfection efficiency was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, as described below.

RT-qPCR assay

A total of 2.5×105 cells were seeded in 60-mm dishes and cultured for 48 h. Total RNA was extracted using the NucleoSpin RNA kit (Takara Bio Inc., Otsu, Japan), and 1 µg of total RNA was used for reverse transcription using the SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The reaction conditions were as follows: 25°C for 10 min, 42°C for 60 min and 85°C for 5 min. The qPCR was performed for PDIA3 and 18S rRNA (as an internal standard) using the StepOnePlus Real-Time PCR system with TaqMan probes and primers (18S, cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: 20 sec at 95°C, followed by 40 cycles of 1 sec at 95°C and 20 sec at 60°C. The RT-qPCR results are expressed as the ratio of target mRNA to 18S rRNA and calculated using the 2-ΔΔCq method (21). The gene expression levels were measured in triplicate.

Protein extraction and western blotting

Total protein was extracted using urea/thiourea buffer containing 7 M urea (Wako Pure Chemical Industries, Ltd.), 2 M thiourea (Nacalai Tesque, Inc., Kyoto, Japan), 3% 3-(3-[cholamidopropyl]-di methylammonio)-1-propanesulfonate (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and 1% Triton X-100 (Sigma-Aldrich; Merck KGaA) from the cells after 96 h of siRNA transfection, as described previously (22). The protein concentration was determined using Pierce® 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Inc.). A total of 10 µg protein from each cell extract was loaded onto and separated by 5-20% SDS-PAGE (e-PAGEL®; ATTO Corporation, Tokyo, Japan) and then transferred to a polyvinylidene difluoride membrane in a Trans-Blot® Turbo™ Transfer Pack using a Trans-Blot Turbo transfer system (both Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for 1 h with 5% skimmed milk in TBS containing 0.2 M Tris-HCl, 150 mM NaCl and 0.01% Tween-20, and then incubated with specific primary antibodies overnight at 4°C. The antibodies to the following proteins were used: PDIA3 (cat. no. ab13506; 1:2,000), induced myeloid leukemia cell differentiation protein Mcl-1 (cat. no. ab32087; 1:2,000), 78 kDa glucose-regulated protein (GRP78; cat no. ab21685; 1:2,000) (all Abcam, Cambridge, UK), STAT3 (cat. no. 9139; 1:2,000), P-STAT3 (Tyr705; cat. no. 9145; 1:2,000), survivin (cat. no. 2808; 1:2,000), X-linked inhibitor of apoptosis protein (XIAP; cat. no. 14334; 1:2,000), Bcl-2-like protein 1 (Bcl-XL; cat. no. 2764; 1:2,000), cyclin D1 (cat. no. 2978; 1:1,000) (all Cell Signaling Technology, Inc., Danvers, MA, USA), B-cell lymphoma 2 (Bcl-2; cat. no. sc-492; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), cellular tumor antigen p53 (cat. no. M7001; 1:1,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and β-actin (cat. no. A5316; 1:10,000; Sigma-Aldrich; Merck KGaA). Following washing in TBS with 0.01% Triton X-100 for 30 min, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. A106PU; 1:10,000; American Qualex Scientific Products, San Clemente, CA, USA) for 1 h at room temperature. The immunoreactive products were visualized using SuperSignal West Dura Extended Duration Substrate for each protein, and SuperSignal West Pico Chemiluminescence substrate (both Thermo Fisher Scientific, Inc.) for β-actin. The data were quantified by Quantity One 1-D analysis software version 4.6.2 (Bio-Rad Laboratories, Inc.). The experiments were performed ≥3 times.

Immunoprecipitation

The total protein was extracted from cells 96 h after seeding. The cells were lysed in Pierce™ IP Lysis Buffer (Thermo Fisher Scientific, Inc.) and protease inhibitor (P8340; Sigma-Aldrich; Merck KGaA) for 10 min on ice after washing in PBS. The cell lysates were centrifuged at 20,000 × g for 10 min at 4°C. The resulting supernatant was collected as a cell extract. Cell lysate (2 mg), antibody (3 µg), and protein A/G PLUS-Agarose (30 µl) were combined and incubated overnight at 4°C. The mixture was purified using Sigma Prep Spin Columns with Break-Away Tips (Sigma-Aldrich; Merck KGaA) and the proteins collected were used for immunoblotting with antibodies against PDIA3 and STAT3. Normal mouse IgG (3 µg; cat. no. SC-2025; Santa Cruz Biotechnology, Inc.) was used as a negative control for the immunoprecipitation experiments.

Cell proliferation assay

Cells were seeded in 96-well plates, at a density of 5,000 cells per well, and cultured at 37°C in a humidified 5% CO2 atmosphere following siRNA transfection with PDIA3 si-1, PDIA3 si-2 or Ctrl si, as described above. After 0, 24, 48, 72 and 96 h of proliferation, cells were incubated with the WST-8 cell-counting reagent (Dojindo Molecular Technologies, Inc.) for 2 h at 37°C. The optical density of the culture solution in each well was determined at 450 nm using a microplate absorbance reader (iMark™; Bio-Rad Laboratories, Inc.). The experiments were performed ≥3 times.

Apoptosis analysis

Cells (5×105) were seeded in a 75-ml flask and cultured in 10 ml RPMI-1640 medium. The medium was changed 24 h after PDIA3 siRNA treatment. Floating and attached cells were collected 72 h after seeding. Double staining with Annexin V and ethidium homodimer III was performed using the Apoptotic, Necrotic, and Healthy Cells Quantification kit (Biotium, Inc., Freemont, CA, USA), following the manufacturer's protocol. The apoptotic cells were counted by flow cytometry on a BD FACSCanto™ II flow cytometer and analyzed using BD FACSDiva™ software version 6.1.3 (both Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The experiments were performed ≥3 times.

Cell cycle analysis

Cell harvesting was performed in the same manner as for the apoptosis analysis. The collected cells were washed in PBS and fixed in 100% methanol for 10 min. The cells were incubated with RNase (0.25 mg/ml) for 30 min at 37°C. Propidium iodide (50 µg/ml) was then added and incubated for 30 min at 4°C in the dark. The stained cells were analyzed by flow cytometry on a BD FACSCanto II flow cytometer and analyzed using BD FACSDiva software version 6.1.3. The experiments were performed ≥3 times.

Hematoxylin and eosin staining

Tissue sections (4 µm thickness) were used for hematoxylin eosin staining. Following deparaffinization, the sections were stained hematoxylin (Merck KGaA) for 30 min at room temperature, and eosin (Wako Pure Chemical Industries, Ltd.) for 20 min at room temperature, following washing in water. The sections were then washed and dehydrated. These sections were evaluated manually under a light microscope (×100 magnification; Olympus Corporation, Tokyo, Japan).

Immunostaining and scoring

Tissue sections (4 µm thickness) were used for immunostaining. Following deparaffinization, sections were pretreated at 121°C for 15 min in 10 mM citrate buffer (pH 6.0) for PDIA3 and proliferation marker protein Ki-67 staining, and Histofine® Antigen Activation Liquid (pH 9.0; Nichirei Biosciences, Inc.) for P-STAT3 staining. Endogenous peroxidase was blocked in 100% methanol (Wako Pure Chemical Industries, Ltd.) containing 0.3% hydrogen peroxide (Wako Pure Chemical Industries, Ltd.) for 30 min at room temperature. The sections were then incubated with antibodies for PDIA3 (1:500), P-STAT3 (1:400) and Ki-67 (MIB-1; cat. no. M7240; 1:100; Dako; Agilent Technologies, Inc.) in PBS containing 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 16 h at 4°C. The sections were further incubated with Histofine Simple Stain™ MAX PO (rabbit or mouse; Nichirei Biosciences, Inc.) for 30 min at room temperature, and peroxidase activity was visualized by 3,3′ diaminobenzidine. The sections were then counterstained with hematoxylin at room temperature for 1 min.

For PDIA3, the intensity and proportion of stained tumor cells were semi-quantitated. If the tumor cell staining was not apparent, the intensity and proportion were scored as 0. If it was apparent, the intensity of tumor cell staining was categorized into three grades: 1, weak; 2, moderate; and 3, strong. The proportional score of stained cells was also divided into three grades: 1, <10%; 2, 10-50%; and 3, >50%. A total score was calculated as the sum of the intensity and proportional scores. Two investigators evaluated the scores in a blind manner and the mean score was used. HCC tissue with a total score ≥4 was classified as having high expression and that with <4 was classified as having low expression. For P-STAT3, nuclear staining was considered to be a positive reaction. The P-STAT3 staining was classified as positive or negative (≥10% or <10% cells with nuclear staining, respectively). These expressions were evaluated manually under a light microscope (×100 magnification; Olympus Corporation). The Ki-67 index was calculated as the percentage of cells with positive nuclear Ki-67 immunostaining using e-Count cell counting software version 4.7 (e-Path Co, Ltd., Kanagawa, Japan) in those areas showing the highest nuclear labeling (so-called ‘hot spots’), which were determined using a light microscope (×400 magnification). The median number of tumor cells counted was 322 (range, 165-583 cells) per sample.

Immunofluorescence staining

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Following washing in PBS, the fixed cells were incubated in 50 mM glycine for 20 min at room temperature, and washed again. The cells were then permeabilized in 0.1% Triton X-100 for 30 min at room temperature. The cells were washed in PBS and blocking was performed using 10% goat serum (Wako Pure Chemical Industries, Ltd.) for 60 min at room temperature. The fixed cells were consecutively incubated with anti-PDIA3 mouse antibody (1:50) and anti-STAT3 rabbit antibody (cat. no. 12640S; 1:50; Cell Signaling Technology, Inc.), followed by Alexa 488-labeled anti-rabbit IgG antibody (cat. no. A11031) and Alexa 568-labeled anti-mouse IgG antibody (cat. no. A11034) (both 1:1,000; Thermo Fisher Scientific, Inc.). The slides were mounted in medium containing DAPI, and the images were captured using a Digital Eclipse C1 TE2000-E confocal microscope (×1,000 magnification; Nikon Corporation, Tokyo, Japan)

Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay

HCC tissues fixed as described above (4 µm thickness) were used for the TUNEL assay. The apoptotic cell death of HCC cells was determined by a TUNEL assay using an Apoptag® Peroxidase In Situ Apoptosis Detection kit (Merck KGaA). The peroxidase activity was visualized by 3,3′ diaminobenzidine. The sections were then counterstained with hematoxylin at room temperature for 1 min and dehydrated. 99% xylene with Malinol (Muto Pure Chemicals Co., Ltd.) was used as a water-insoluble mounting medium. Nuclear staining was considered a positive reaction. The images were captured under a light microscope (×400 magnification; Olympus). The TUNEL index was calculated by the same method as that used for the Ki-67 index. The median number of counted tumor cells was 337 (range, 179-649 cells) per sample.

Statistical analysis

The data are expressed as the mean ± standard error of the mean or standard deviation (SD). Statistical comparisons between and among the groups were conducted using one-way analysis of variance (ANOVA) with Dunnett's post-hoc test, two-way ANOVA with Tukey's post-hoc test or a Mann-Whitney U-test. The clinicopathological parameters were analyzed using the χ2 and Fisher's exact tests. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

PDIA3 expression is associated with cell proliferation and apoptosis in HCC tissues

To evaluate the effects of PDIA3 expression in HCC, cell proliferation, apoptosis and clinicopathological features were investigated in 53 HCC samples. The cell proliferation was evaluated using Ki-67 immunostaining, and apoptosis was measured by a TUNEL assay. The HCC tissues with high PDIA3 expression exhibited a significantly higher Ki-67 index than those with low expression (25.8 vs. 10.1%, respectively; P<0.001; Fig. 1A and B). By contrast, HCC tissues in which PDIA3 was highly expressed revealed significantly fewer apoptotic cells than those with low expression (2.4 vs. 8.9%, respectively; P<0.001; Fig. 1A and C). However, no associations were observed between the PDIA3 expression level and any clinicopathological features (Table I). These results indicate that HCC tissues with high PDIA3 expression are associated with increased cell proliferation and decreased apoptosis.

PDIA3 expression in HCC cells is higher than that in normal hepatocyte cells

The PDIA3 protein expression in HCC Huh-7 and HuH-1 cells was tested, and was demonstrated to be 1.7 and 2.0 times higher, respectively, than that in normal hepatocyte THLE-2 cells (P<0.001; Fig. 2A and B). This result indicates that Huh-7 and HuH-1 are appropriate cell lines for a model of HCC with increased PDIA3 levels.

Figure 2

Effects of PDIA3 knockdown on cell proliferation, apoptosis and cell cycle in HCC cell lines. (A) Representative western blots of PDIA3 protein expression in normal hepatocyte THLE-2 and HCC Huh-7 and HuH-1 cells. (B) Relative quantification of the PDIA3 protein expression. The data are expressed as the mean ± SEM. ***P<0.001 vs. THLE-2 (one-way ANOVA). (C) Representative western blots of the expression of PDIA3 in Huh-7 and HuH-1 cells lines transfected Ctrl si, PDIA3 si-1 and PDIA3 si-2. (D) Cell proliferation of the HCC cells transfected with Ctrl si, PDIA3 si-1 and PDIA3 si-2 was evaluated using the WST-8 cell counting reagent. At the indicated time points, the number of cells per well was estimated by measuring the absorbance (450 nm). The data are expressed as the mean ± SEM. ***P<0.001 vs. Ctrl si at each time point (two-way ANOVA). (E) Representative plots of the flow cytometry analysis of apoptosis. The apoptotic cells were counted by flow cytometry following double staining with annexin V and ethidium homodimer III. Q1 represents the necrotic cells; Q2 the late apoptotic cells; Q3 the live cells; and Q4 the early apoptotic cells. (F) The percentage of live, apoptotic and necrotic cells in the groups transfected with siRNAs. The data are expressed as the mean ± SEM. ***P<0.001 vs. Ctrl si (two-way ANOVA). (G) Representative histograms of flow cytometry analysis of the cell cycle, following propidium iodide staining. P4 represents the SubG1 (fragmented DNA); P5 the G0/G1 phase; P6 the S phase; and P7 the G2/M phase. (H) The percentage of cells in each cell cycle phase following siRNA treatment. ***P<0.001 vs. Ctrl si (two-way ANOVA). These results were from ≥3 independent experiments. PDIA3, protein disulfide-isomerase A3; HCC, hepatocellular carcinoma; PDIA si-1/PDIA si-2, PDIA3 siRNA; Ctrl si, control siRNA; ANOVA, analysis of variance; SEM, standard error of the mean.

PDIA3 knockdown inhibits cell proliferation of HCC cells

To investigate the role of PDIA3 in the progression of HCC, the proliferation of HCC cells was tested following PDIA3 knockdown. First, it was ascertained that PDIA3 si-1 and si-2 successfully suppressed PDIA3 expression in Huh-7 and HuH-1 cells (Fig. 2C). The proliferative ability of Huh-7 cells treated with PDIA3 si-1 and si-2 was 50.6 and 70.0% compared with those treated with Ctrl si at 96 h, respectively. The proliferation of HuH-1 cells treated with PDIA3 si-1 and si-2 was 48.0 and 71.4% compared with the control group at 96 h, respectively (P<0.001 at 72 and 96 h; Fig. 2D). Overall, the PDIA3 knockdown significantly inhibited cell proliferation in the two HCC cell lines.

PDIA3 knockdown induces apoptosis in HCC cells

To investigate the effect of PDIA3 silencing on HCC cell proliferation, the apoptosis and cell cycle progression of HCC cells were investigated following PDIA3 knockdown. The silencing of PDIA3 induced apoptosis in the HCC cells (P<0.001 compared with Ctrl si; Fig. 2E and F). Furthermore, the cell cycle analysis demonstrated that knockdown of PDIA3 significantly increased the SubG1 population and decreased the G0/G1 phase cells in the two HCC cell types (P<0.001 compared with Ctrl si in the SubG1 population; Fig. 2G and H). The increased SubG1 population signified the induction of apoptosis. These findings suggest that PDIA3 knockdown inhibits cell proliferation in HCC cell lines, likely through an apoptosis-dependent mechanism.

PDIA3 knockdown does not induce ER stress in HCC cells

As excessive ER stress causes apoptosis, the effect of the downregulation of PDIA3 expression on ER stress was investigated in the HCC cells. To evaluate the level of ER stress, the expression of ER stress marker GRP78 was tested following PDIA3 knockdown. No changes in GRP78 levels were observed, indicating that silencing of PDIA3 does not induce ER stress (Fig. 3).

PDIA3 interacts with STAT3 and regulates its phosphorylation in HCC cells

The interaction of PDIA3 with STAT3 in HCC cells was investigated. Double immunofluorescence staining revealed the colocalization (merge; yellow) of PDIA3 (red) and STAT3 (green) (Fig. 4A). PDIA3, an ER protein, was mainly observed in the cytoplasm where it colocalized with STAT3 (Fig. 4A; top panel; merged). Notably, PDIA3 also localized to the nucleus in ~5% of Huh-7 cells (Fig. 4A; bottom panel). In these cells, nuclear PDIA3 tended to colocalize with STAT3 (Fig. 4A; bottom panel; merged). Additionally, the co-immunoprecipitation experiments demonstrated that PDIA3 was bound to STAT3 in the HCC cells (Fig. 4B). These results suggest a physiological interaction between PDIA3 and STAT3 in HCC cells. Furthermore, the western blotting revealed that inhibition of PDIA3 expression reduced P-STAT3 levels (Fig. 4C). This result indicates that PDIA3 is associated with STAT3 activation via a certain interaction in HCC cells.

Downstream targets of STAT3 are suppressed by PDIA3 knockdown in HCC cells

The expression of downstream targets of STAT3 was investigated by immunoblotting in HCC cells following treatment with PDIA3 siRNAs (Fig. 5A and B). The knockdown of PDIA3 led to a decrease in the expression of XIAP and Bcl-XL in Huh-7 cells, and that of survivin, Mcl-1, XIAP and cyclin D1 in HuH-1 cells. The difference between the expression of Mcl-1 and cyclin D1 in the Huh-7 cells for si-1 and si-2 is possibly due to off-target effects of the siRNAs. These results indicate that PDIA3 knockdown affects apoptotic pathways by decreasing the levels of the downstream anti-apoptotic proteins of the STAT3 pathway in HCC cells.

PDIA3 knockdown with si-1 leads to a significant, but small, additional inhibitory effect on cell proliferation in HCC cells treated with AG490

The JAK/STAT3 signaling inhibitor AG490 suppressed the levels of P-STAT3 (Fig. 6A). Subsequently, to confirm that PDIA3 knockdown led to a decrease in cell proliferation mainly through STAT3 signaling, the cell proliferation of HCC cells treated with PDIA3 siRNAs was examined in the presence of AG490. AG490 markedly decreased cell proliferation in the two HCC cells (P<0.001 at 72 and 96 h; Ctrl si vs. Ctrl si + AG490; Fig. 6B). In the Huh-7 cells, PDIA3 si-1 or si-2 + AG490 decreased the cell proliferation only by a further 7.2 or 5.2%, respectively, compared with the effect of AG490 alone at 96 h, and only the additional effect of si-1 was statistically significant (PDIA3 si-1 + AG490 vs. Ctrl si + AG490, P<0.05; PDIA3 si-2 + AG490 vs. Ctrl si + AG490, P=0.22; Fig. 6B). In the HuH-1 cells, PDIA3 si-1 or si-2 + AG490 decreased the cell proliferation by 8.3 and 4.3% more than AG490 alone at 96 h, respectively, and only the effect of si-1 was statistically significant (P<0.001 PDIA3 si-1 + AG490 vs. Ctrl si + AG490, P<0.001; PDIA3 si-2 + AG490 vs. Ctrl si + AG490, P=0.086; Fig. 6B). Notably, these additional inhibitory effects of PDIA3 knockdown under AG490 treatment conditions were small compared with the inhibitory effect of AG490 alone (56.5 and 48.4% decrease in Huh-7 and HuH-1, respectively, compared with Ctrl si at 96 h). These results demonstrated that the inhibitory effect of PDIA3 knockdown on the proliferation of HCC cells with deactivated STAT3 was weak, suggesting that PDIA3 knockdown inhibits cell proliferation predominantly through STAT3 signaling.

PDIA3 expression is associated with P-STAT3 levels in HCC tissues

Immunohistochemical staining of 35 HCC tissues revealed an association between PDIA3 and P-STAT3 levels. The HCC tissues that were positive for P-STAT3 exhibited significantly higher PDIA3 expression than those that were negative for P-STAT3 (mean PDIA3 expression score ± SD, 4.8±0.8 and 2.9±1.5, respectively; P<0.001; Fig. 7A and B). The HCC tissues that were P-STAT3 positive also demonstrated a tendency towards a higher Ki-67 index than those that were negative, although this was not statistically significant (mean Ki-67 index, 24.0 and 16.9%, respectively; P=0.069; Fig. 7C). By contrast, the P-STAT3-positive tissues demonstrated significantly fewer apoptotic cells than those that were negative (2.9 and 7.9% apoptotic cells, respectively; P<0.001; Fig. 7D). However, no association was observed between the P-STAT3 status and the clinicopathological features (Table I). Overall, PDIA3 expression was associated with a P-STAT3-positive status, which in turn was associated with decreased apoptosis in HCC tissues.

Discussion

Mutations within several genes can lead to alterations in the expression of various essential proteins and change the biological behavior of cancer cells. Certain of these proteins may be used as prognostic markers. In previous studies of patients with HCC, the upregulation of PDIA3 expression was associated with poor prognosis (20,23) and with increased cell proliferation and survival (20). To test the reproducibility of a previous study (20) on the collected samples in the present study and the contribution of PDIA3 to tumor progression, immunohistochemical staining of HCC tissues for PDIA3 was performed. PDIA3 expression in HCC was not associated with any clinicopathological features, including vascular invasion. However, high PDIA3 levels were associated with an increased Ki-67 labeling index, and a lower TUNEL index, suggesting a contribution of PDIA3 to HCC cell proliferation and survival. Therefore, an in vitro evaluation of cell proliferation and apoptosis was conducted.

Notably, the knockdown of PDIA3 significantly inhibited cell proliferation and induced apoptosis in HCC cells. These findings are reported for the first time in HCC. However, the question remains of how PDIA3 modulates cell proliferation and apoptosis in this cancer type.

ER chaperones protect cells from apoptosis induced by ER stress (24,25). As PDIA3 is an ER chaperone, the possibility was considered that its knockdown may cause ER stress-dependent apoptosis. Therefore, the expression of an ER stress marker, GRP78 (26,27), was examined following suppression of PDIA3. However, the GRP78 levels did not increase, indicating that ER stress is not involved in the induction of apoptosis by PDIA3 knockdown in HCC cells.

PDIA3 is associated with several proteins and signaling pathways, including STAT3 (16-18), mammalian target of rapamycin complex 1 (28,29), apurinic endonuclease/redox factor-1 (30), 1,25-dihydroxyvitamin D3 (31) and H2A histone family member X (32). Of these, STAT3 is particularly well known as an important pathway associated with cell proliferation and survival in HCC. In the present study, it was revealed that PDIA3 and STAT3 colocalized in the cytoplasm and nucleus of HCC cells and formed complexes. Furthermore, PDIA3 knockdown led to a decrease in the levels of P-STAT3. However, PDIA3 knockdown had little inhibitory effect on the cell proliferation in the presence of the JAK/STAT3 signaling inhibitor AG490. These findings may be the supportive evidence that PDIA3 functions mainly through the STAT3 signaling pathway. These results are consistent with those from several cancer cell lines (18,19), suggesting that PDIA3 is involved in the phosphorylation process and the DNA-binding activity of STAT3. Furthermore, an association was observed between PDIA3 and P-STAT3 expression in HCC tissues. To the best of our knowledge, these data are the first to demonstrate the association between PDIA3 and P-STAT3 in human HCC specimens. To better understand the mechanism of STAT3 signaling-activation mediated by PDIA3, further assessments into the transportation into the nucleus, binding to DNA fragments and protection of STAT3 from dephosphorylation are required.

Furthermore, PDIA3 knockdown was revealed to lead to a decrease in the levels of certain proteins associated with the STAT3 signaling pathway. First, the cell cycle protein cyclin D1 decreased in HuH-1 but not in Huh-7 cells following PDIA3-si2 treatment. Additionally, the cell cycle analysis demonstrated no G0/G1 arrest. Within the cell cycle, the effect of PDIA3 silencing remains unclear. Next, PDIA3 knockdown was previously revealed to increase the expression of p53 and induce apoptosis in tumor cells in a p53-dependent manner (19,29). However, in the present study, the expression of p53 was not increased following PDIA3 siRNA treatment in Huh-7 and HuH-1 cells, possibly because these cells have a homozygous loss-of-function mutation in p53 (33). Therefore, apoptosis was induced in Huh-7 and HuH-1 cells in a p53-independent manner. Finally, PDIA3 knockdown was revealed to decrease the levels of certain anti-apoptotic proteins, including survivin, XIAP, Mcl-1 and Bcl-XL. Therefore, it is hypothesized that PDIA3 expression in HCC promotes cell survival by modulating anti-apoptotic proteins (Fig. 8). Regarding the undetectable Bcl-2 expression in the Huh-7 cells, Wang et al (34) reported a similar result; however certain studies have detected it, but reported that it was lower in Huh-7 cells compared with other hepatoma cell lines (35,36). There is a possibility that the expression of Bcl-2 was not detected in the present study due to different experimental conditions. Differences in the anti-apoptotic protein levels following PDIA3 knockdown were observed between the Huh-7 and HuH-1 cells. This may be due to crosstalk between STAT3 signaling and other transcriptional mechanisms (37).

The association between PDIA3 and STAT3 signaling in HCC was demonstrated in the present study. However, it remained unclear whether PDIA3 specifically associates with P-STAT3 as well as non-phosphorylated STAT3. PDIA3 colocalized with STAT3 in the cytoplasm and nucleus of the HCC cells. In addition, it was observed in the nucleus of HCC cells in which the majority of STAT3 detection occurred. Therefore, PDIA3 and P-STAT3 may interact in nucleus. The present findings indicated that PDIA3 mainly associates with P-STAT3, however, the immunofluorescence staining was not sufficient to support that. To the best of our knowledge, this issue remains unclear and further assessments are required to improve our understanding.

In HCC therapy, sorafenib inhibits not only the downstream signaling of the receptor for the vascular endothelial growth factor, but also the phosphorylation level of STAT3 (38-40). However, previous studies have documented that P-STAT3 is upregulated in sorafenib-resistant patients with HCC and sorafenib-resistant HCC cell lines that were established following long-term exposure to sorafenib (41,42). In addition, dovitinib, a multi-targeted receptor tyrosine kinase inhibitor, is effective in sorafenib-resistant HCC cell lines due to inhibiting the STAT3 signaling pathway by a different mechanism (42). STAT3 serves an important role in sorafenib resistance and would therefore be an important therapeutic target. In addition, PDIA3 is known to contribute to chemoresistance (43) and radio-resistance (19) and, since it modulates the P-STAT3 levels, it is a potential therapeutic target in sorafenib-resistant HCC. The association of PDIA3 with chemoresistance in HCC requires further elucidation.

In conclusion, PDIA3 is upregulated in HCC tissues and cell lines, and is associated with cell survival factors. In the present study, it was revealed that the poor prognosis in HCC associated with a high expression of PDIA3 may be induced by a complex formation between PDIA3 and STAT3 in HCC cells, regulating the transcriptional potential of STAT3. These findings suggest that PDIA3 participates in the aggressive phenotype of HCC through its association with STAT3 signaling, hence PDIA3 is considered a potential therapeutic target for the treatment of HCC.

Funding

This study was supported by Grants-in-aid for the Clinical Rebiopsy Bank Project for Comprehensive Cancer Therapy Development from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant no. S1311022).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

RK, ZN and KI were involved in the conception and design of the study; RK, KI, YK, SK, WXP, MK, HT and NT performed the experiments; RK, KI, RW and HY analyzed the data; RK drafted the manuscript; HY and ZN reviewed and edited the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

This study was conducted according to the Declaration of Helsinki and the Japanese Society of Pathology, and was given official approval by the Ethics Committee of Nippon Medical School Hospital, Tokyo, Japan (approval no. 29-03-908). Written informed consent was obtained from all patients.

Patient consent for publication

Written informed consent was obtained from the patients.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

The authors thank Dr Eiji Uchida, Dr Hidemi Takahashi, Ms. Masumi Shimizu, Ms. Kiyoko Kawahara, Mr. Takenori Fuji, Mr. Kiyoshi Teduka, Ms. Yoko Kawamoto and Ms. Taeko Kitamura, Nippon Medical School, Tokyo, Japan, for their assistance.

References

1 

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI

2 

Fitzmorris P, Shoreibah M, Anand BS and Singal AK: Management of hepatocellular carcinoma. J Cancer Res Clin Oncol. 141:861–876. 2015. View Article : Google Scholar

3 

Imamura H, Matsuyama Y, Tanaka E, Ohkubo T, Hasegawa K, Miyagawa S, Sugawara Y, Minagawa M, Takayama T, Kawasaki S, et al: Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol. 38:200–207. 2003. View Article : Google Scholar : PubMed/NCBI

4 

Bellavance EC, Lumpkins KM, Mentha G, Marques HP, Capussotti L, Pulitano C, Majno P, Mira P, Rubbia-Brandt L, Ferrero A, et al: Surgical management of early-stage hepato-cellular carcinoma: Resection or transplantation? J Gastrointest Surg. 12:1699–1708. 2008. View Article : Google Scholar : PubMed/NCBI

5 

Nakajima Y, Ko S, Kanamura T, Nagao M, Kanehiro H, Hisanaga M, Aomatsu Y, Ikeda N and Nakano H: Repeat liver resection for hepatocellular carcinoma. J Am Coll Surg. 192:339–344. 2001. View Article : Google Scholar : PubMed/NCBI

6 

Ge S and Huang D: Systemic therapies for hepatocellular carcinoma. Drug Discov Ther. 9:352–362. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Daher S, Massarwa M, Benson AA and Khoury T: Current and Future Treatment of Hepatocellular Carcinoma: An Updated Comprehensive Review. J Clin Transl Hepatol. 6:69–78. 2018. View Article : Google Scholar : PubMed/NCBI

8 

Swamy SG, Kameshwar VH, Shubha PB, Looi CY, Shanmugam MK, Arfuso F, Dharmarajan A, Sethi G, Shivananju NS and Bishayee A: Targeting multiple oncogenic pathways for the treatment of hepatocellular carcinoma. Target Oncol. 12:1–10. 2017. View Article : Google Scholar

9 

Yu H, Pardoll D and Jove R: STATs in cancer inflammation and immunity: A leading role for STAT3. Nat Rev Cancer. 9:798–809. 2009. View Article : Google Scholar : PubMed/NCBI

10 

Nikolaou K, Sarris M and Talianidis I: Molecular pathways: The complex roles of inflammation pathways in the development and treatment of liver cancer. Clin Cancer Res. 19:2810–2816. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Subramaniam A, Shanmugam MK, Perumal E, Li F, Nachiyappan A, Dai X, Swamy SN, Ahn KS, Kumar, Tan BK, et al: Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma. Biochim Biophys Acta. 1835:46–60. 2013.

12 

Wu WY, Li J, Wu ZS, Zhang CL, Meng XL and Lobie PE: Prognostic significance of phosphorylated signal transducer and activator of transcription 3 and suppressor of cytokine signaling 3 expression in hepatocellular carcinoma. Exp Ther Med. 2:647–653. 2011. View Article : Google Scholar : PubMed/NCBI

13 

Zhang CH, Xu GL, Jia WD, Li JS, Ma JL, Ren WH, Ge YS, Yu JH, Liu WB and Wang W: Activation of STAT3 signal pathway correlates with twist and E-cadherin expression in hepatocellular carcinoma and their clinical significance. J Surg Res. 174:120–129. 2012. View Article : Google Scholar

14 

Mano Y, Aishima S, Fujita N, Tanaka Y, Kubo Y, Motomura T, Taketomi A, Shirabe K, Maehara Y and Oda Y: Tumor-associated macrophage promotes tumor progression via STAT3 signaling in hepatocellular carcinoma. Pathobiology. 80:146–154. 2013. View Article : Google Scholar : PubMed/NCBI

15 

Hettinghouse A, Liu R and Liu CJ: Multifunctional molecule ERp57: From cancer to neurodegenerative diseases. Pharmacol Ther. 181:34–48. 2018. View Article : Google Scholar

16 

Ndubuisi MI, Guo GG, Fried VA, Etlinger JD and Sehgal PB: Cellular physiology of STAT3: Where's the cytoplasmic monomer? J Biol Chem. 274:25499–25509. 1999. View Article : Google Scholar : PubMed/NCBI

17 

Eufemi M, Coppari S, Altieri F, Grillo C, Ferraro A and Turano C: ERp57 is present in STAT3-DNA complexes. Biochem Biophys Res Commun. 323:1306–1312. 2004. View Article : Google Scholar : PubMed/NCBI

18 

Chichiarelli S, Gaucci E, Ferraro A, Grillo C, Altieri F, Cocchiola R, Arcangeli V, Turano C and Eufemi M: Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line. Arch Biochem Biophys. 494:178–183. 2010. View Article : Google Scholar

19 

Choe MH, Min JW, Jeon HB, Cho DH, Oh JS, Lee HG, Hwang SG, An S, Han YH and Kim JS: ERp57 modulates STAT3 activity in radioresistant laryngeal cancer cells and serves as a prognostic marker for laryngeal cancer. Oncotarget. 6:2654–2666. 2015. View Article : Google Scholar : PubMed/NCBI

20 

Takata H, Kudo M, Yamamoto T, Ueda J, Ishino K, Peng WX, Wada R, Taniai N, Yoshida H, Uchida E, et al: Increased expression of PDIA3 and its association with cancer cell proliferation and poor prognosis in hepatocellular carcinoma. Oncol Lett. 12:4896–4904. 2016. View Article : Google Scholar

21 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Δ Δ C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar

22 

Ishino K, Kudo M, Peng WX, Kure S, Kawahara K, Teduka K, Kawamoto Y, Kitamura T, Fujii T, Yamamoto T, et al: 2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells. Biochem Biophys Res Commun. 501:668–673. 2018. View Article : Google Scholar : PubMed/NCBI

23 

Liu M, Du L, He Z, Yan L, Shi Y, Shang J and Tang H: Increased ERp57 Expression in HBV-Related Hepatocellular Carcinoma: Possible Correlation and Prognosis. Biomed Res Int. 2017:12526472017.PubMed/NCBI

24 

Ni M and Lee AS: ER chaperones in mammalian development and human diseases. FEBS Lett. 581:3641–3651. 2007. View Article : Google Scholar : PubMed/NCBI

25 

Corazzari M, Gagliardi M, Fimia GM and Piacentini M: Endoplasmic reticulum stress, unfolded protein response, and cancer cell fate. Front Oncol. 7:782017. View Article : Google Scholar : PubMed/NCBI

26 

Bánhegyi G, Baumeister P, Benedetti A, Dong D, Fu Y, Lee AS, Li J, Mao C, Margittai E, Ni M, et al: Endoplasmic reticulum stress. Ann N Y Acad Sci. 1113:58–71. 2007. View Article : Google Scholar : PubMed/NCBI

27 

Luo B and Lee AS: The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies. Oncogene. 32:805–818. 2013. View Article : Google Scholar

28 

Ramírez-Rangel I, Bracho-Valdés I, Vázquez-Macías A, Carretero-Ortega J, Reyes-Cruz G and Vázquez-Prado J: Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57. Mol Cell Biol. 31:1657–1671. 2011. View Article : Google Scholar : PubMed/NCBI

29 

Hussmann M, Janke K, Kranz P, Neumann F, Mersch E, Baumann M, Goepelt K, Brockmeier U and Metzen E: Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics. Oncotarget. 6:39247–39261. 2015. View Article : Google Scholar : PubMed/NCBI

30 

Grillo C, D'Ambrosio C, Scaloni A, Maceroni M, Merluzzi S, Turano C and Altieri F: Cooperative activity of Ref-1/APE and ERp57 in reductive activation of transcription factors. Free Radic Biol Med. 41:1113–1123. 2006. View Article : Google Scholar : PubMed/NCBI

31 

Sequeira VB, Rybchyn MS, Tongkao-On W, Gordon-Thomson C, Malloy PJ, Nemere I, Norman AW, Reeve VE, Halliday GM, Feldman D, et al: The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3. Mol Endocrinol. 26:574–582. 2012. View Article : Google Scholar : PubMed/NCBI

32 

Krynetskaia NF, Phadke MS, Jadhav SH and Krynetskiy EY: Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage. Mol Cancer Ther. 8:864–872. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Forbes SA, Beare D, Boutselakis H, Bamford S, Bindal N, Tate J, Cole CG, Ward S, Dawson E, Ponting L, et al: COSMIC: Somatic cancer genetics at high-resolution. Nucleic Acids Res. 45(D1): D777–D783. 2017. View Article : Google Scholar :

34 

Wang B, Ni Z, Dai X, Qin L, Li X, Xu L, Lian J and He F: The Bcl-2/xL inhibitor ABT-263 increases the stability of Mcl-1 mRNA and protein in hepatocellular carcinoma cells. Mol Cancer. 13:982014. View Article : Google Scholar : PubMed/NCBI

35 

Chen P, Hu T, Liang Y, Jiang Y, Pan Y, Li C, Zhang P, Wei D, Li P, Jeong LS, et al: Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer. Oncotarget. 6:9002–9017. 2015.PubMed/NCBI

36 

Zhou M, Zhang Q, Zhao J, Liao M, Wen S and Yang M: Phosphorylation of Bcl-2 plays an important role in glycoche-nodeoxycholate-induced survival and chemoresistance in HCC. Oncol Rep. 38:1742–1750. 2017. View Article : Google Scholar : PubMed/NCBI

37 

Zheng HC: The molecular mechanisms of chemoresistance in cancers. Oncotarget. 8:59950–59964. 2017.PubMed/NCBI

38 

Tai WT, Cheng AL, Shiau CW, Huang HP, Huang JW, Chen PJ and Chen KF: Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma. J Hepatol. 55:1041–1048. 2011. View Article : Google Scholar : PubMed/NCBI

39 

Gu F-M, Li QL, Gao Q, Jiang JH, Huang XY, Pan JF, Fan J and Zhou J: Sorafenib inhibits growth and metastasis of hepato-cellular carcinoma by blocking STAT3. World J Gastroenterol. 17:3922–3932. 2011. View Article : Google Scholar : PubMed/NCBI

40 

Huang CY, Lin CS, Tai WT, Hsieh CY, Shiau CW, Cheng AL and Chen KF: Sorafenib enhances radiation-induced apoptosis in hepatocellular carcinoma by inhibiting STAT3. Int J Radiat Oncol Biol Phys. 86:456–462. 2013. View Article : Google Scholar : PubMed/NCBI

41 

van Malenstein H, Dekervel J, Verslype C, Van Cutsem E, Windmolders P, Nevens F and van Pelt J: Long-term exposure to sorafenib of liver cancer cells induces resistance with epithelial-to-mesenchymal transition, increased invasion and risk of rebound growth. Cancer Lett. 329:74–83. 2013. View Article : Google Scholar

42 

Tai WT, Cheng AL, Shiau CW, Liu CY, Ko CH, Lin MW, Chen PJ and Chen KF: Dovitinib induces apoptosis and overcomes sorafenib resistance in hepatocellular carcinoma through SHP-1-mediated inhibition of STAT3. Mol Cancer Ther. 11:452–463. 2012. View Article : Google Scholar

43 

Cicchillitti L, Della Corte A, Di Michele M, Donati MB, Rotilio D and Scambia G: Characterisation of a multimeric protein complex associated with ERp57 within the nucleus in paclitaxel-sensitive and -resistant epithelial ovarian cancer cells: The involvement of specific conformational states of β-actin. Int J Oncol. 37:445–454. 2010. View Article : Google Scholar : PubMed/NCBI

44 

Brierley JD, Gospodarowicz MK and Wittekind C: TNM Classification of Malignant Tumours. 8th edition. Wiley-Blackwell; New York, NY: 2016

Related Articles

Journal Cover

April-2019
Volume 54 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Kondo R, Ishino K, Wada R, Takata H, Peng WX, Kudo M, Kure S, Kaneya Y, Taniai N, Yoshida H, Yoshida H, et al: Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma. Int J Oncol 54: 1409-1421, 2019.
APA
Kondo, R., Ishino, K., Wada, R., Takata, H., Peng, W., Kudo, M. ... Naito, Z. (2019). Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma. International Journal of Oncology, 54, 1409-1421. https://doi.org/10.3892/ijo.2019.4710
MLA
Kondo, R., Ishino, K., Wada, R., Takata, H., Peng, W., Kudo, M., Kure, S., Kaneya, Y., Taniai, N., Yoshida, H., Naito, Z."Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma". International Journal of Oncology 54.4 (2019): 1409-1421.
Chicago
Kondo, R., Ishino, K., Wada, R., Takata, H., Peng, W., Kudo, M., Kure, S., Kaneya, Y., Taniai, N., Yoshida, H., Naito, Z."Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma". International Journal of Oncology 54, no. 4 (2019): 1409-1421. https://doi.org/10.3892/ijo.2019.4710