A total of 20 OLK patients with clinical and pathological diagnosis of OLK, with epithelial mild or mild-moderate dysplasia, at the Capital Medical University School of Stomatology (Beijing, China) were randomly selected for use in the present study. The OLK tissues of these patients were taken via biopsy, and 10 samples of normal oral mucosa were obtained from maxillofacial plastic surgery procedures for use as negative controls. Amongst the 20 OLK cases, 12 were female and 8 were male, aged 45–84 years (mean age, 64 years), including 13 cases of buccal mucosa, 1 case of lip mucosa and 6 cases of tongue mucosa. The present study was approved by the Human Research Ethics Committee of Capital Medical University School of Stomatology, and all patients signed an informed consent forms.

For quantification of messenger RNA (mRNA) expression, total RNA was extracted from human OLK and control tissues using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized by reverse transcribing 2 µg RNA with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Aliquots of cDNA (1 µl) were used as templates and SYBR Green Dye reagent (Applied Biosystems, Foster City, CA, USA) was used to quantify the products formed during the RT-qPCR reaction. For data analysis, the 2^−∆∆Ct method was used, and the raw data was normalized to the housekeeping gene GAPDH (13). The experiment was performed in triplicate. The sequences of primers were: GAPDH-forward (F), 5′-aggtcggtgtgaacggatttg-3′ and reverse (R), 5′-tgtagaccatgtagttgaggtca-3′; Prx1-F, 5′-gggtattcttcggcagatca-3′ and Prx1-R, 5′-tccccatgtttgtcagtgaa-3′; ASK1-F, 5′-aagtcccaacccatagaaattcct-3′ and ASK1-R, 5′-agccagtcggtaagttcagaatctt-3′; p38-F, 5′-gagctgaagattctggattttgg-3′ and p38-R, 5′-tagccacgtagccggtcatt-3′.

Human oral precancerous cell line DOK (presented by Professor Chen Xiaoxin, Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC, USA) were maintained in Dulbecco's modified Eagle's medium-nutrient mixture F-12, supplemented with 15% (v/v) fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA) containing 100 U/ml penicillin and 100 µg/ml streptomycin, in a 5% CO₂ atmosphere at 37°C.

The pEZ-M02-ASK1 and pEZ-M02-Prx1 plasmids were obtained from GeneCopoeia, Inc. (Rockville, MD, USA), while Prx1 short hairpin RNA (shRNA) plasmid and control shRNA Plasmid-A were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). DOK cells were plated in six-well plates at 1×10⁶ cells/well, then transfected with 2 µg plasmids using Lipofectamine® 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions.

Following transfection of Prx1 shRNA plasmid for 48 h, the cells were stimulated with 5 mM H₂O₂ for various time-periods (15, 30 and 45 min). Apoptotic cell death was measured by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) with Annexin V/fluorescein isothiocyanate and propidium iodide staining (R&D Systems, Inc., Minneapolis, MN, USA).

Cells and tissues were rinsed three times with ice-cold phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA) and lysed in immunoprecipitation assay buffer [50 mM Tris-Cl (pH 7.4), 1% NP40, 150 Mm NaCl, 1 mM EDTA, 1 M phenylmethylsulfonyl fluoride, 10 µg each of aprotinin and leupeptin, and 1 mM Na₃VO₄] (Invitrogen Life Technologies), to which the protease inhibitor mix Complete™ (Roche Diagnostics, Basel, Switzerland) was added. Following centrifugation at 12,000 × g for 30 min, the supernatant was collected, and the protein concentration was determined using the Lowry method (14). Equal quantities of protein were separated on 12% SDS-PAGE gels and blotted onto nitrocellulose membranes (Pierce Biotechnology, Inc., Appleton, WI, USA). The blots were subsequently incubated with rabbit polyclonal anti-human Prx1 antibody (1:1,000; cat. no. ab41906; Abcam, Cambridge, UK), rabbit polyclonal anti-human ASK1 (1:1,000; cat. no. 3762s; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-human p-ASK1 (1:1,000; cat. no. 3765s; Cell Signaling Technology, Inc.), rabbit monoclonal anti-human p38 (1:1,000; cat. no. ab7952; Abcam) and rabbit monoclonal anti-p-human p38 (1:1,000; cat. no. ab178867; Abcam) at 4°C overnight. Rabbit polyclonal anti-human β-actin antibody (1:1,000; cat. no. A2066; Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control. Immunoreactive bands were detected by 1-h incubation at room temperature with goat anti-rabbit (cat. no. ab136636) and goat anti-mouse (cat. no. ab979023) horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000; Abcam) and enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Chalfont, UK). Each experiment was repeated a minimum of three times.

To examine the association between Prx1 and ASK1, a co-immunoprecipitation assay was used to detect the interaction between Prx1 with ASK1 in vitro. Cell extract from H₂O₂-treated DOK cells was collected following centrifugation at 14,000 × g for 15 min at 4°C and boiled for 5 min with 2X loading buffer for examination by 4–12% SDS-PAGE. Whole cell extracts were incubated with the rabbit monoclonal anti-human Prx1 (1:1,000; cat. no. ab109506; Abcam) or rabbit polyclonal anti-human β-actin (1:1,000; cat. no. A2066; Sigma-Aldrich) antibodies at 4°C overnight and incubated with the HRP-conjugated secondary antibodies for 30 min at room temperature. For protein precipitation, protein A-Agarose beads (Invitrogen Life Technologies) in extract buffer were added prior to incubation with gentle mixing for 16 h at 4°C. Subsequently, the beads were pelleted by centrifugation at 14,000 × g for 15 min at 4°C and washed three times with extract buffer. The protein was eluted from the beads at 100°C using 1% SDS-PAGE sample buffer supplemented with 50 mM dithiothreitol and resolved with SDS-PAGE.

To further investigate whether Prx1 protein interacts with ASK1 protein directly in vitro, a GST pull-down assay kit (21516; Thermo Fisher Scientific, Waltham, MA, USA) was used. Cell lysates were prepared and centrifuged at 15,000 × g for 15 min and the supernatants were collected. Histidine (His)-Prx1 (Prx1 protein with a 6-His tag, prepared in our laboratory) were incubated with GST or GST-fused ASK1 conjugated to sepharose beads in reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 5 mM NaF, 1% Triton X-100 and protease inhibitor mixture Complete; Roche Diagnostics) at 4°C for 12 h. Following centrifugation at 500 × g for 5 min at 4°C, the proteins bound to sepharose beads were washed with ice-cold PBS, mixed with 2X SDS sample buffer and eluted by the addition of 10 µl glutathione elution buffer (G-Biosciences, St. Louis, MO, USA). The sepharose beads were suspended and incubated at room temperature for 5 min. They were then centrifuged for at 500 × g for 5 min at 4°C to sediment the sepharose beads, and the supernatants were transferred to fresh tubes for SDS-PAGE. Western blot analysis was then performed to examine Prx1 binding to ASK1 using rabbit monoclonal anti-human Prx1 antibody (1:1,000; cat. no. ab109506; Abcam).

Data were expressed as the mean ± standard deviation. Comparisons were performed by two independent Student's t-test (independent 2-sample t-test) using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Prx1 mRNA expression was significantly higher in 20 OLK tissues than that of normal oral mucosa samples, with approximately 2-fold greater expression than that of the normal group (P=0.047; Fig. 1A). Closely correlated with the mRNA levels, the protein expression of Prx1 was also enhanced in 20 cases of OLK, compared with that of the normal group, and the mean relative Prx1 content was 1.11 in the OLK group (Fig. 1B).

The mRNA expression levels of ASK1 and p38 were both significantly higher in OLK tissues than that in the normal oral mucosa, with ~3- (P=0.024) (Fig. 2A) and 4-fold (P=0.022) (Fig. 2B) increase compared with the normal group, respectively. The protein expression of ASK1 and p-ASK1 were slightly increased in the OLK tissues, and the mean relative content of ASK1 and p-ASK1 was 1.06 and 1.11, respectively, in the OLK group (Fig. 2C). The protein expression of p38 and p-p38 in OLK group were also both increased slightly, and the mean relative p38 and p-p38 content was 1.09 and 1.11, respectively, in the OLK group (Fig. 2D) The results of the western blot were not statistically analyzed.

Western blot analysis with anti-Prx1 antibody was performed in DOK cells treated with 5 mM H₂O₂ for the indicated time-periods (Fig. 3A). The expression of Prx1 was not markedly altered until 45 min. To investigate whether endogenous Prx1 affected ASK1 expression and ASK1-induced apoptosis, the suppression of endogenous Prx1 expression was induced by transfecting Prx1 shRNAs into DOK cells (Fig. 3B). The expression of phosphorylated ASK1 and p38 were subsequently evaluated by immunoblotting assay. As shown in Fig. 3C, upon stimulation with H₂O₂, the levels of p-ASK1 rose rapidly and strongly from 15 min. Following Prx1 knockdown, higher levels of p-ASK1 and p-p38 were observed, compared with that of control (mock-transfected) DOK cells. In Prx1-knockdown DOK cells, the expression of phosphorylated ASK1 and p38 gradually increased in a time-dependent manner, and then decreased at 45 min.

Subsequently, the functional roles of Prx1 in H₂O₂-induced apoptosis were examined. DOK cells were transiently transfected with Prx1 shRNA. Following transfection for 48 h, the cells were treated with 5 mM H₂O₂ (0, 15, 30 and 45 min)and apoptotic cell death was measured by flow cytometry. Notably, H₂O₂-induced apoptosis was significantly enhanced in DOK cells transfected with Prx1 shRNA compared with that of the mock-transfected cells (Fig. 4), particularly following treatment with 5 mM H₂O₂ for 30 min (56.0±2.8 vs. 20.3±3.3%, respectively; P=0.008).

To investigate the association between Prx1 and ASK1, whether Prx1 interacts with ASK1 in vitro was examined (Fig. 5A). DOK cells were transiently transfected with expression plasmids encoding ASK1 and Prx1. Following co-transfection for 48 h, the cells were stimulated with 5 mM of H₂O₂ for 30 min, extracted and immunoprecipitated with anti-Prx1 antibody. No ASK1 was detected in immunoprecipitates of endogenous Prx1 under these conditions.

Whether Prx1 directly interacts with ASK1 in vitro was further examined by GST pull-down assay. A GST pull-down assay kit was used, and the results indicated that ASK1 did not markedly bind to Prx1 (Fig. 5B). These results suggested that Prx1 and ASK1 were not able to directly interact in vitro under these conditions.