Patients and primary tissue samples. We enrolled 116 patients with stage II or III colon cancer who underwent curative resection at the University of Yamanashi Hospital between January 2007 and December 2014. We excluded patients who had undergone preoperative chemotherapy and those who had colitic cancer or synchronous cancers. Staging was performed according to the International Union Against Cancer/TNM classification (8th edition) (17). This study was approved by the ethics committee of the University of Yamanashi Hospital (approval number: 2294) and conducted in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the patients. The cecum, ascending colon, and transverse colon were defined as the right colon, and the descending colon and sigmoid colon were defined as the left colon, to indicate tumor localization.
IHC. IHC was performed as described previously (18). Briefly, formalin-fixed and paraffin-embedded cancer tissues were cut into 4-µm-thick slices. IHC staining for NOX2 was performed using the avidin-biotin complex method (VECTOR LABORATORIES INC, USA). NOX2 antibody (dilution 1:200; cat. no. ab80508; Abcam) at 4°C overnight. The NOX2 expression level was classified into three grades according to the strongest staining intensity of NOX2. Staining intensity was graded as 1 (weakly reactive), 2 (moderately reactive), or 3 (strongly reactive) (Fig. 1A-C). Samples with a grade of 1 were defined as negative for NOX2 expression, and those with a grade of 2 or 3 were positive for NOX2 expression.
Cell lines. The human colon cancer cell lines included HCT116, purchased from RIKEN BioResource Center; SW48, from Cell Resource Center for Biochemical Research (KAC Co, Ltd); and eight others (SW480, SW620, LOVO, RKO, T84, DLD-1, HT29, and Caco2), from the American Type Culture Collection. Cells were cultured according to the manufacturer’s instructions. SW48, SW480, and SW620 cells were incubated in a CO2-free environment at 37°C, while the other cells were incubated in an environment with 5% CO2 at 37°C.
Real-time quantitative PCR (RT-qPCR). RT-qPCR was performed as previously described (19). Briefly, total RNA was extracted from the cell lines using the R Neasy Mini Kit (QIAGEN). The NOX2 expression levels were measured (Hs00166163, Thermo Fisher Scientific, Inc.), and GAPDH (4310884E, Applied Biosystems™) was used as a housekeeping gene. RT-qPCR was performed using Applied Biosystems 7500 Real-Time PCR software (7500 Software v2.0.6, Applied Biosystems™). Each assay was performed in triplicate.
Western blotting. Western blotting was performed as previously described (19). Briefly, equal amounts of protein extracted from the cell lines were separated using SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were incubated with anti-NOX2 antibody (1:1000 dilution) and anti-β-actin antibody (1:2000 dilution) with shaking at 4°C for 24 h. After the membranes were probed with secondary antibodies at room temperature for 1 h, the proteins were visualized using Pierce™ ECL Plus Western blotting Substrate (Thermo Fisher Scientific, Inc.).
Small interfering RNA (siRNA) transfection. HCT116 and RKO cells were transfected with 10 nmol/L NOX2 siRNA (Cat. No10620318. Stealth, Invitrogen™). Lipofectamine was used as the transfection reagent. Controls were transfected with Stealth RNAi™ negative Control Med GC Duplex #2 (12935112, Invitrogen™). The medium with siRNA was replaced with fresh medium 24 and 48 h after transfection, total RNA and protein were extracted for RT-qPCR and western blotting, respectively.
Cell proliferation assay. HCT116 and RKO cells were seeded at a density of 0.5x105 cells/ml in 6-well plates. At 24 h after seeding, transfection was performed using siRNAs. The number of cells was counted 24, 48, and 72 h after transfection using a Countess Automated Cell Counter (Invitrogen™) to assess cell proliferation. Each assay was performed in triplicates.
Cell cycle analysis. HCT116 and RKO cells were lysed using TritonX-100 and treated with RNase. Nuclei of 1x106 cells were stained with propidium iodide and evaluated by fluorescence-activated cell sorting. Cell cycle measurements were performed 48 h after transfection. Each assay was performed in triplicate.
Cell migration and invasion assay. Migration and invasion assays were performed as described previously (19). Briefly, Falcon Cell Culture Inserts with 8-µm pore membranes were used in 24-well plates (Corning, Corning, NY, USA). The insert for the invasion assay was coated with Biocoat Matrigel (BD Biosciences, Franklin Lakes, New Jersey, USA). HCT116 and RKO cells were seeded at a density of 1x105 cells/mL in the upper chambers 24 h after transfection. After incubating for 48 h with 5% CO2 at 37°C, the migrated and invaded cells were stained with the Differential Quik III Stain Kit (Polysciences, Inc.) and counted in five independent fields of view. Each assay was performed in triplicate.
Statistical Analysis. The data were analyzed using JMP statistical software (version 16.1.0; SAS Institute, Cary, NC, USA). Fisher’s exact test was used to assess differences between proportions, and the Student’s t-test was used to assess continuous variables. Survival rates were estimated using the Kaplan–Meier method, and differences in survival were evaluated using the log-rank test. The Cox proportional hazards regression model was applied to determine independent prognostic factors, and differences were considered significant when the p-value was less than 0.05.