D-Glucose and methylthiazoly-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemotherapy drug 5-fluorouracil (5-FU) was acquired from The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University (Xi’an, China). The primary antibodies used were as follows: Rabbit anti-human polyclonal anti-Nampt (AV42255; Sigma-Aldrich), rabbit anti-human polyclonal anti-p53 (sc-126; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)/p-glycoprotein (P-gp; sc-55510; Santa Cruz Biotechnology, Inc.), rabbit anti-human polyclonal anti-topoisomerase (topo)-IIα (20233-1-AP; Proteintech, Chicago, IL, USA) and rabbit anti-human monoclonal anti-Sirt1 (sc-15404; Santa Cruz Biotechnology, Inc.). The secondary antibodies and 3,3′-diaminobenzidine were purchased from ZSGB-BIO (Beijing, China).

A total of 68 patients with gastric cancer and 40 patients with gastric cancer with diabetes, who had undergone radical gastrectomy at The Department of General Surgery, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, between January 2000 and December 2004 were included in the present study. None of the patients had received preoperative neoadjuvant chemotherapy or radiotherapy. According to the tumor node metastasis classification of the International Union Against Cancer, those patients who were in stage I, II or III were matched based on gender and age. The control group was normal paired tissue samples collected from the patients from an area of tissue >5 cm away from the tumor. All studies were approved by the Ethics Committee of Xi’an Jiaotong University and every patient provided written informed consent prior to surgery. The baseline data of the study group are shown in Table I.

The human gastric cancer cell line SGC-7901 was provided by the Fourth Military Medical University (Xi’an, China). Cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (1,000 mg/l glucose; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and 100 IU/ml penicillin and 100 μg/ml streptomycin (Harbin Pharmaceutical Group Co., Ltd, Harbin, China) at 37°C in a humidified atmosphere containing 5% CO₂. Cells were subcultured with 0.25% Trypsin-EDTA (Amresco, LLC, Solon, OH, USA) once they reached >80% confluence.

For the effective detection of chemotherapy drugs, cells cultured in 1,000, 4,500 and 9,000 mg/l glucose were subjected to 5-FU at 3.75, 15, 30, 60 and 120 μg/ml. According to the results of daily cell counting, the growth curve with MTT was produced and the half maximal inhibitory concentration of each group was calculated.

The tissue samples were prepared cutting the paraffin-embedded tissue into serial sections 4 μm thick and evenly cut into three sections (one for hematoxylin staining and the other two for immunohistochemical staining). The tissue sections were preserved in 2.5% glutaraldehyde-polyoxymethylene solution, were dehydrated and embedded in paraffin following routine methods and were immersed in distilled water. Subsequently, the paraffin sections were washed (3 × 5 min) in phosphate-buffered saline with Tween-20 (PBS-T; 0.01 M; pH 7.4) and then blocked with 0.3% H₂O₂-methanol at room temperature for endogenous peroxidase ablation. The following steps were conducted in a moist chamber. The non-specific binding sites were blocked by 30-min incubation in 5% normal goat serum (cat. no. ZLI-9022; Beijing Zhongshan Golden Bridge Company, Beijing, China) at room temperature for 20 min. The primary antibody was then added dropwise, then the sections were incubated for 2 h or overnight at 4°C followed by washing again with in PBS-T (3 × 5 min). According to the PV-6000 direction to add the reagent, the sections were incubated for 30 min at room temperature, followed by washing with PBS-T(3 × 5min). The sections were then stained with 3,3-diaminobenzidine (cat. no. ZLI-9017; Beijing Zhongshan Golden Bridge Company), were kept at room temperature without light for 10 min and were immersed in distilled water for 10 min. The sections were then stained with hematoxylin for 2 min, completing the staining with the distilled water for 10 min. Dehydration of the sections was then conducted with sequential 1-min ethanol washes starting with 75%, followed by 80% and finishing with an 100% ethanol wash. The sections were mounted with neutral gum and then sealed and analyzed by optical microscopy (Nikon 80i; Nikon Corporation, Tokyo, Japan). The intensity of immunostaining was assessed by two different observers, with respect to the staining intensity into the following four grades: −, no staining (negative); +, faintly stained up to 25% (weak positive); ++, 25-50% cells moderately stained (moderate positive); +++, 50% or more cells markedly stained (strong positive) (16).

Total RNA was isolated using the RNA Fast 200 purification kit from Fastagen Biotech (Shanghai, China) according to the manufacturer’s instructions. Reverse transcription of RNA was performed with ExScriptTM RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The cDNA was then subjected to RT-PCR. All primer pairs shown in Table II were designed by Oligo 6.0 primer analysis software (Oswel Research Products, Beijing, China). qPCR was performed in duplicate reactions of 12.5 μl volume SYBR II (Takara Biotechnology Co., Ltd.), 1 μl each of specific forward and reverse primers and 9.5 μl RNase Free H₂O up to 24 μl. Samples were heated for 85°C followed by 40 cycles of amplification for 15 sec at 95°C and 1 min at 60°C. The mRNA level of β-actin was measured as an internal reference.

Cells were treated with lysis buffer. Protein samples (30 μg) were separated by 10% gels and then transferred onto a polyvinylidene difluoride membrane (Sterlitech Corp., Kent, WA, USA). Blots were probed with antibodies against Nampt (1:1,000), Sirt1 (1:500), p53 (1:500), P-gp (1:200) and Top-IIα (1:200). Following primary antibody incubation, membranes were incubated with horseradish peroxidase-conjugated secondary immunoglobulin G (1:2,000). The immunoreaction was visualized using ECL-Plus reagent (Pierce Biotechnology, Inc., Rockford, IL, USA) and analyzed using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD, USA).

The data are presented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed using a χ² test. P<0.05 was considered to indicate a statistically significant difference. Correlations were analyzed using Spearman’s rank correlation coefficient test.

At present, P-gp and p53 are recognized as typical chemoresistance protein markers (17,18). The present study demonstrated increased levels of P-gp and p53 in patients with gastric cancer with diabetes compared with gastric cancer alone (Fig. 1 and Table III). It is well established that Topo-IIα is a target for anti-cancer drugs (19). Notably, the level of Topo-IIα in gastric cancer with diabetes tissues was lower than that in gastric cancer alone (Fig. 1 and Table III). p53 and Topo-IIα were found primarily in the nuclei, while P-gp was identified in the cytomembrane, which is consistent with previous studies (20,21). The clinical pathological features are summarized in Table III.

To further investigate the mechanism underlying chemoresistance, the present study examined whether Nampt and Sirt1 were involved in the process. Levels of Nampt and Sirt1 were observed to be higher in gastric cancer with diabetes than gastric cancer tissues (Fig. 2 and Table III). To further investigate the impact on survival time, the survival curve was also examined. Increased Nampt was associated with reduced overall survival rate of gastric cancer with diabetes by univariate analysis (Fig. 3). However, Sirt1 had little effect on the overall survival rate (Fig. 3).

PCR analysis indicated that cells treated with glucose at 4,500 and 9,000 mg/l exhibited increased mRNA expression of Nampt, Sirt1, p53 and P-gp compared with 1,000 mg/l. In addition, the levels of Topo-IIα were reduced at 4,500 and 9,000 mg/l glucose (Fig. 4). Western blot analysis confirmed these effects at the protein level (Fig. 5). These results suggested that high glucose induced chemoresistance protein markers at the transcriptional and translational levels.

Subsequently, it was examined whether high glucose levels induced chemoresistance to 5-FU in SGC-7901 cells. The number of live cells was measured using an MTT assay. When exposed to 5-FU, SGC7901 cells exhibited a higher cell proliferation rate in the presence of high glucose (9,000 mg/l) compared with lower glucose concentrations (1,000 mg/l) over 48 h, suggesting that high glucose attenuated the growth inhibitory effect of 5-FU (Fig. 6).