Antibodies and reagents
The following antibodies were used for immunoblotting analysis using standard Western blotting procedures: superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), p22phox, and p-p40phox were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin, tublin, diphenyleneiodonium chloride (DPI), and capsaicin were purchased from Sigma-Aldrich, St. Louis, MS, USA.
Cell culture
The parental E6E7 cell line and K-ras–transformed cell line, which had been established by transfecting the immortalized human pancreatic duct epithelial E6E7 cell line with K-ras
G12V, were kindly provided by Dr. Paul Chiao from The University of Texas, MD Anderson Cancer Center and were cultured as reported previously [17]. Primary pancreatic cancer cell lines, including AsPC-1, Capan-1, and Panc-1, were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS).
Quantitative real-time Polymerase Chain Reaction (PCR) analysis
The sequences for the genes to be measured are as follows: 5′-GGAGTTTCAAGATGCGTGGAAACTA-3′ (sense) and 5′-GCCAGACTCAGAGTTGGAGATGCT-3′ (antisense) for NOX2, 5′-CAAGCCGTGACCAAGGACACCTG-3′ (sense) and 5′-CACACAGGACATCCACCGTGTC-3′ (antisense) for NOXA1. Real-time PCR analysis was performed by using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Otsu, Shiga, Japan) and Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA).
MTT assay
Cell growth was determined using MTT reagent in 96-well plates. After incubation, 20 μL MTT reagent was added to each well and incubated for an additional 4 hours and then the supernatant was removed. The cell pellets were dissolved in 200 μL DMSO. Absorbance was determined using a MultiSkan plate reader (Thermo, Helsinki, USA) at a wavelength of 570 nm.
Colony formation assay
Cells were seeded in six-well plates and cultured for about 2 weeks. Colonies were fixed with methanol for 10 minutes and stained with crystal violet solution (Beyotime, Jiangsu, China) for 30 minutes. All the experiment was repeated 3 times.
NOX activity
Cells were suspended in lysis buffer containing 20 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA,1 mmol/L EGTA, 100 mmol/L sucrose, and a cocktail of protease inhibitors. After homogenization, the samples were centrifuged at 800 g at 4°C for 5 minutes to pellet unbroken cells and nuclei. The supernatants were centrifuged at 100,000 g for 30 minutes to separate the membrane fraction (pellet) and the cytosolic fraction (supernatant). NOX activity was measured by lucigenin-derived chemiluminescence, with 100 μmol/L NADPH or NADH as substrate, 50 μmol/L lucigenin, and 25 μg of cell membrane proteins. Chemiluminescence was measured using a luminometer (Turner Designs, Sunnyvale, CA, USA) for 1 minute. The signal was normalized and expressed as arbitrary light units per microgram protein per minute.
Rac activity
The Rac activity assay was performed using the Rac-GEF (guanine-nucleotide exchange factors) Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, cells were washed in cold PBS, lysed in 1× Assay/Lysis Buffer, and centrifuged for 10 minutes at 14,000 g at 4°C. Aliquots from the supernatant were used for determining protein concentration. The supernatant was incubated with nucleotide-free Rac1 G15A agarose beads to pull down the active form of Rac-GEFs. The beads were washed 3 times with 1× Assay/Lysis Buffer, and the bound proteins were eluted. The active Rac proteins were detected by Western blotting using an anti-Rac-GEF antibody (Tiam1).
Invasion assay
Invasion assays were performed with BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). Pre-coated filter Matrigel inserts were re-hydrated with 0.5 mL of PBS for 2 hours in humidified tissue culture incubator at 37°C in 5% CO2 atmosphere. After rehydration, PBS was removed. Then, 1 × 105 parental or K-ras–transformed E6E7 cells and Capan-1, AsPC-1, Panc-1 cells in 0.5 mL of supplement-free medium with or without 10 μmol/L capsaicin were seeded onto the upper part of each chamber insert, and the 24-well plates were filled with 0.5 mL of their culture medium. Following incubation for 16 hours, non-invaded cells on the upper surface of the insert were wiped off with a cotton swab, and the cells that had migrated onto the lower surface of the filter, were fixed and stained with the Hema 3 Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA) containing a fixative and 2 stain solutions. The inserts were air dried and photographed. Invasiveness was determined by counting cells in 3 microscopic fields (×100) per well, and the extent of invasion was expressed as an average number of cells per microscopic field.
Measurement of ROS production and ATP generation
Cells were stained with 100 ng/mL hydroethidine (HET) (Invitrogen, Carlsbad, CA, USA) and 5 μmol/L DCF-DA (Invitrogen, Carlsbad, CA, USA) for 60 minutes before the measurement of superoxide and hydrogen peroxide using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cellular ATP generation was measured using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Wisconsin, USA) according to manufacturer’s recommendations.
Animal study
Four-week-old, BALB/c male nude mice were purchased from Medical Experimental Animal Center of Guangdong Province, China. A total of 2 × 106 AsPC-1 cells were inoculated into the right flanks of the mice by subcutaneous injection. When the volume of tumors reached 100 mm3, the mice were randomly divided into 2 groups of 10 mice each. The treatment group received 15 mg/kg capsaicin in 0.9% sodium chloride solution (intraperitoneal injection, 3 times per week). The control group received equal volume of 0.9% sodium chloride solution by intraperitoneal injection. Five weeks after inoculation, all mice were euthanized and the tumor weights were measured. Animal experiments were approved by Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center and performed under the guidelines of the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996).
Immunohistochemistry and TUNEL assay
Representative tumor tissues were sectioned and embedded in paraffin. The slides were then incubated with the primary antibody (mouse anti–8-oxoguanine monoclonal antibody, Abcam, Cambridge, UK) at 1:200 dilution overnight in a humidified chamber at 4°C. The slides were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Envision Detection Kit, Dako, Glostrup, Denmark) at 37°C for 30 minutes. Finally, the samples were stained with 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin and eosin (HE). Tumor cell death induced by capsaicin was detected by TUNEL assay with the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions.
Statistical analysis
Statistical significant differences were evaluated by using Student’s t test (Prism GraphPad, San Diego, CA, USA). The Kolmogorov-Smirnov test (Cell Quest Pro software, Becton-Dickinson, San Jose, CA, USA) was used to evaluate the significant difference between control and treatment groups in flow cytometry analysis. A P value of <0.05 was considered statistically significant.