H. pylori culture
The H. pylori cagA- and vacA-positive standard strain NCTC11637 was obtained from the Institute of Digestive Diseases, Renji Hospital, Shanghai Jiao Tong University, Shanghai, China. H. pylori was cultured on Columbia agar (Oxoid, Basingstoke Hampshire, UK) plates containing 5 % sheep blood and incubated at 37 °C under microaerophilic conditions for 48–72 h. Colonies were identified as H. pylori by Gram staining, morphology, and positive oxidase, catalase, and urease activities. Bacteria were suspended in phosphate-buffered saline (PBS) and the density was estimated by spectrophotometry (OD600 nm) and microscopic observation.
Immunohistochemical staining of COX-2, beta-catenin, VEGF, and CD34 in human gastric carcinoma tissues
A total of 106 different formalin-fixed, paraffin-embedded gastric cancer tissue samples and adjacent normal tissues were obtained from Shuguang Hospital, Shanghai University of Traditional Chinese Medicine. The use of all human tissue samples was approved by the Institutional Review Board of Shuguang Hospital, which is affiliated with Shanghai University of Traditional Chinese Medicine. Informed consent was obtained from every patient for the use of all human tissues used in this study. First, tissue samples were stained with Giemsa to determine the presence of H. pylori infection. Next, using standard methods, COX-2, beta-catenin, VEGF, and CD34 were detected immunohistochemically. Briefly, tissues were embedded in paraffin and 4-μm sections were cut, deparaffinized in xylene, and dehydrated through a graded alcohol series. Tissue sections were subjected to peroxidase clearance, antigen retrieval, and blocking of non-specific binding sites. Sections were first incubated with primary antibody (rabbit polyclonal antibodies against CD34, COX-2, beta-catenin, and VEGF (Abcam, Cambridge, MA, USA), followed by EnVision secondary antibody (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin. PBS served as a negative control for primary antibody. Staining intensity was assessed in each specimen on a scale of 0–3: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining.
Immunohistochemical analysis of the MVD
According to Weidner [24], areas of highest neovascularization were found by scanning tumor sections at low-power (×40) magnification. After the area of highest neovascularization was identified, individual microvessel counts were made in a single high-power (×200) magnification field. Three different visual fields were selected for microvessel counting, and the mean value was recorded. Brown-staining endothelial cells or endothelial cell clusters were considered as single, countable microvessels.
Cell culture and reagents
SGC7901 and MKN45 gastric cancer cells were obtained from the Institute of Digestive Diseases, Renji Hospital of Shanghai Jiao Tong University, Shanghai, China, and cultured in RPMI 1640(Gibco, Thermo Fisher Scientific Inc, Waltham, MA, USA) containing 10 % (v/v) fetal bovine serum (Gibco, Thermo Fisher Scientific Inc, Waltham, MA, USA) and 1 % penicillin and streptomycin (North China Pharmaceutical Company, Shijiazhuang, China). Cells were plated in 6-well plates and grown to confluency. FH535, a beta-catenin-specific inhibitor, was obtained from Cell Signaling (Beverly, MA, USA). All cells were grown in a humidified incubator containing 5 % CO2 at 37 °C.
Real-time fluorogenic quantitative polymerase chain reaction
RNA isolation
Total cellular RNA was prepared using RNAisol reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. RNAisol (1 ml) was added to each sample and incubated for 5 min at room temperature. Next, 200 μl chloroform was added and samples were shaken for 15 s and incubated at room temperature for 2-3 min and then centrifuged at 12,000 g for 15 min at 4 °C after formation of a biphasic solution. For RNA precipitation, the aqueous phase (top) was transferred to a new tube and 500 μl isopropanol was added. Samples were incubated at room temperature for 5-10 min and then centrifuged at 12,000 g for 15 min at 4 °C, after which a pellet was visible. After supernatant removal, 1000 μl of 75 % ethanol was added to wash the RNA pellet; this was vortexed and centrifuged at 8000 g for 5 min at 4 °C. After the ethanol was carefully removed by pipetting, the RNA pellet was air-dried for 5-10 min and then dissolved in diethylpyrocarbonate-treated water with vortexing. RNA quality was verified by agarose gel electrophoresis and visualization of 28S and 18S ribosomal RNA. RNA was quantified by spectrophotometry (OD260/280 nm). RNA was then immediately frozen at −70 °C.
cDNA synthesis and real-time quantitative analysis
Reverse transcription was conducted using a PrimeScript RT-PCR Kit (TaKaRa Biotechnology, Dalian, China). Total RNA (1 μg) was used as a template for cDNA synthesis. Briefly, reverse transcription was carried out in a 20-μl solution including 4 μl 5× buffer, 1 μl oligo dT primer, 1 μl random 6-mers, 1 μl PrimeScript RT Enzyme Mix, and RNAse-free deionized H2O. Reverse transcription incubation conditions were 37 °C for 15 min and 85 °C for 5 s The resultant cDNA was stored at −20 °C until it was used for real-time quantitative polymerase chain reaction (PCR). Real-time PCR reactions were carried out using the ABI7300 Fast Real-Time PCR System (PE Biosystems, Foster City, CA, USA) using a PrimeScript RT-PCR Kit according to the manufacturer’s instructions. Primers and probes for human GAPDH, VEGF, and COX2 were designed and synthesized by Shanghai Shanjing Biotechnology (Shanghai, China) with FAM (6-carboxy-fluo-rescein-phosphoramidite)-labeled 5′ ends and TAMPA (carboxy-tetramethyl-rhodamine)-labeled 3′ ends. Primer and probe sequences were: human GAPDH-forward, 5′-CCACTCCTCCACCTTTGAC-3′; human GAPDH-reverse, 5′-ACCCTGTTGCTGTAGCCA-3′; GAPDH probe, 5′-TTGCCCTCAACGACCACTTTGTC-3′; human VEGF-forward, 5′-GGCCTCCGAAACCATGAACT-3′, human VEGF-reverse, 5′-ACCCTGTTGCTGTAGCCA-3′; and VEGF probe, 5′-TGTCTT GGGTGCATTGGAGC-3′. Briefly, each PCR was performed in a 20-μl reaction volume comprising 10 μl Premix EX Taq, 0.4 μl Rox reference dye, 0.4 μl each primer, 0.8 μl TaqMan probe, 6 μl deionized H2O and 2 μl cDNA. PCR cycling conditions were 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s (denaturation) and 60 °C for 31 s (annealing/extension). Each reaction was performed in triplicate, and data were analyzed by the 2−∆∆Ct method for comparing relative expression levels. GAPDH mRNA was used to normalize RNA levels from the various samples and mRNA expression was expressed as relative to the basal level without H. pylori stimulation.
Western blot analysis
Following treatment, cells were washed twice with ice-cold PBS and then protease inhibitors (Roch, Basel, Switzerland) were added. Cells were then scraped off the dish, and then cytoplasmic and nuclear fractions were prepared using a protein extraction kit (Fermentas, Waltham, MA, USA). Cell lysis buffer, nuclei washing buffer, and other reagent buffers were added to separate cytosolic proteins and nuclear proteins. The protein concentration in extracts was determined by bicinchoninic acid protein assay using a commercial kit (BCA Protein Assay Reagent; Merck, Whitehouse Station, NJ, USA). Protein samples were separated by 10 % SDS-PAGE and transferred to PVDF membrane. The membrane was incubated in blocking buffer (10 mmol/l Tris, pH 7.5, 100 mmol/l NaCl, 0.1 % Tween 20), containing 5 % nonfat powdered milk for 1 h. The membrane was then incubated with anti-phospho-beta-catenin or anti- beta-catenin polyclonal antibody (1:500; Cell Signaling Technology, USA). Following overnight incubation at 4 °C, blots were washed three times in TBS-Tween (0.05 %) solution and incubated with goat anti-rabbit antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature before visualizing using the Pierce ECL kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Results were analyzed by Image J software (NIH Image).
Enzyme-linked immunosorbent assay
Cell culture supernatant samples were collected and clarified at 3000 g for 5 min. ELISA was performed according to the manufacturer’s protocol. Briefly, microtiter plates were incubated with 100 μl samples at 37 °C for 120 min. After five washes in 10 mM PBS, plates were incubated with 100 μl anti-VEGF primary antibody labeled with biotin (from the ELISA kit) at 37 °C for 60 min. After five rinses with 10 mM PBS, 100 μl avidin-biotin-peroxidase complex was added to wells and incubated at 37 °C for 30 min. After extensive rinsing, 100 μl/well TMB Microwell Substrate and was added and plates were incubated in the dark at 37 °C for 15 min. The reaction was then stopped with 100 μl TMB stop solution and OD450 nm values were obtained within 30 min using a microplate reader. Finally, protein concentrations were determined from OD values using a calibration curve.
Statistical analysis
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 19.0). Statistical significance was determined by t tests and one-way ANOVA followed by Fisher’s least significant difference test and differences in rates were determined by the chi-squared test. Data are presented as means ± SE and a P value of <0.05 was considered statistically significant.
