Rat colon tumors
Colon tumors and adjacent normal-looking mucosa were from a study in which male F344 rats received intermittent exposure to PhIP alternating with a high-fat diet, as reported elsewhere.20
An interim sacrifice was included to assess early molecular changes, before the onset of tumors. Specifically, 24 h after the last dose of PhIP, rats in each group (n
= 5) were euthanized, the colon was removed and opened longitu-dinally, and the mucosa was scraped and frozen in liquid nitrogen before storing at −80°C. The remaining animals in each group (n
= 36) were euthanized at 52 weeks (Supporting Information Fig. 1
). A complete necropsy examination was performed on each animal.20
This work received prior approval from the Institutional Animal Care and Use Committee.
Human primary colon cancers
Ten pairs of primary human colon cancers and their matched adjacent normal-looking tissues were kindly provided by Steven F. Moss, M.D. and Lelia Simao (Rhode Island Hospital, Providence, RI). The patients (6 female, 4 male, 53–93 years of age) had been diagnosed with adenocarcinoma of the colon.
Quantitative real-time RT-PCR (qPCR)
Frozen colon tumor samples and their matched controls were thawed, and mRNA was extracted using the RNeasy kit (Qiagen, Valencia, CA). RNA (2 μg) was reverse-transcribed in 20 μl of 1× RT buffer, containing 10 U RNase inhibitor (Invitrogen, Carlsbad, CA), 0.5 mM each dNTP, 4 U Omni-script Reverse Transcriptase (Qiagen) and 50 ng random hexamers (Invitrogen). Primers were as listed in Supporting Information Table I
. Forty cycles of PCR were run on an Opticon Monitor 2 system (Finnzymes, Finland), in 20 μl total reaction volume containing cDNAs, SYBR Green I dye (DyNAmo master solution, Finnzymes) and primer set. The PCR conditions were 95°C/10s, 58°C/20s and 72°C/20s, except for rat Nox1
and rat Nox4
, in which the annealing temperature was 60°C. The amount of specific mRNA was quantified by determining the point at which the fluorescence accumulation entered the exponential phase (Ct
), and the Ct
ratio of the target gene to glyceraldehyde-3-phosphate dehydrogenase
) was calculated for each sample. At least two separate experiments were performed for each sample. In some experiments, malignant regions were microdissected from PhIP-induced colon tumors using a Zeiss PALM MicroBeam IV Laser Capture System (Carl Zeiss, Thorn-wood, NY), and compared with microdissected normal colon. Briefly, mRNA from captured cells was extracted using an RNeasy Micro Kit (Qiagen) and cDNA was synthesized using the Superscript III kit (Invitrogen). Forty-five cycles of qPCR (95°C/10s, 60°C/10s, 72°C/10s) were run on a Roche Light-Cyler 480 II (Roche, Indianapolis, IN), in a 10 μl reaction containing primer set, cDNA and SYBR Green I dye (Roche).
ProteoExtract Native membrane protein extraction kit (EMD Biosciences, San Diego, CA) was used for enrichment of membrane proteins, whereas NE-PER reagents (Pierce Biotechnology, Rockford, IL) were used to separate cytoplasmic and nuclear fractions. Membrane-enriched fractions were subjected to SDS-PAGE and immunoblotted using anti-Nox1 (H-75, sc-25545, 1:400 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Nox4 (rat, 1:100 dilution, abcam, Cambridge, MA, ab41886; human, 1:500 dilution, Novus, Biologicals, NB110-58851, Littleton, CA). The correct molecular weights were confirmed by reference to the marker ladder on each blot (Nox1 65-kD, Nox4 67-kD), and antibody specificity was corroborated via the use of blocking peptides (sc-5821p against sc-5821 Nox1) (Santa Cruz), 1:200 dilution; Nox4 peptide (ND110-58851 PEP Novus Biologicals), 1:250 dilution). Whole cell lysates were immunoblotted with rabbit polyclonal antibody to IKKα and IKKβ (1:1000 dilution, Cell Signaling, Nos. 2682 and 2684) or phospho-IKKα/β (1:500 dilution, Cell Signaling, no. 2681). Cytoplasmic extracts were probed with antibodies to cleaved caspase 3 (1:1000 dilution, Cell Signaling, no. 9661), cleaved caspase 6 (1:1000 dilution, Cell Signaling, no. 9761), and cleaved caspase 7 (1:1000 dilution, Cell Signaling, no. 9492). Nuclear fractions were immunoblotted with polyclonal rabbit antibody to NFκB p50 (1:600 dilution, Santa Cruz, sc-7178), NFκB p65 (1:600 dilution, Santa Cruz, sc-109), and poly-(ADP-ribose)polymerase (PARP, 1:1000 dilution, Cell Signaling, no. 9532). Amido black staining was used to ensure equal protein loading, followed by β-actin. For nuclear extracts, histone H1 also was used as a loading control (mouse monoclonal antibody sc-8030, Santa Cruz, 1:500 dilution). Proteins were visualized by Western Lightning Chemiluminescence Reagent Plus (Perkin–Elmer Life Sciences, Boston MA), with quantification via an AlphaInnotech photodocumentation system and associated software (AlphaInnotech, San Leandro, CA).
Rat colon tumors were processed to paraffin, sectioned at 4 lm, and placed on charged slides. Sections were rehydrated through xylene, 100% ethanol, 95% ethanol, 80% ethanol and water. Antigen retrieval was carried out in a microwave pressure cooker for 10 min, followed by 20 min at room temperature. Antigen retrieval solution was Dako Target Retrieval Solution pH 6.0 (Dako, Carpentaria, CA). Slides were washed in water and loaded into a Dako autoimmunostainer. Endogenous peroxides were blocked with 3% H2O2 in TBST (Dako Tris-buffered saline with Tween 20) for 10 min, and slides were then washed in TBST. Dako serum-free protein block was applied for 10 min, followed by a burst of air to blot the slides. Incubation with the primary antibody was for 30 min (Nox4, 1:250 dilution, Novus Biologicals, NB110-58851, Littleton, CA). As immunohistochemical controls, the corresponding blocking peptide (see above) was used to confirm antibody specificity, and Dako Universal Negative Rabbit control was used in place of the primary antibody. After washing in TBST, Dako Envision+ anti-rabbit HRP was applied for 30 min, followed by Nova Red (Vector Laboratories, Burlingame, CA) and hematoxylin (Dako) counter staining.
The same protocol was used to immunostain Nox4 in human tissues. Tissue microarrays (TMAs) were constructed using a Beecher Instruments MTA-1 tissue arrayer (Beecher Instruments, Sun Prairie, WI). At least duplicate tumor samples were taken from donor tissue blocks, and a retrospective analysis for outcome assessment was based on detailed clini-copathological information linked to the TMA specimens. For further details, see Ashktorab et al
Cell culture, siRNA transfection and LPS treatment
Human colorectal cancer lines HT29 and Caco2 (American Type Culture Collection, Manassas, VA) were maintained in McCoy's 5A medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. SmartPool siRNA against human NOX1 and non-specific control siRNAs were purchased from Dhar-macon and transfected according to the manufacturer's instructions. Briefly, cells (1 × 105) were seeded in 6-well plates overnight. The media was aspirated and replaced with fresh antibiotic free-transfection media containing Dharma-FECT (4 μl for Caco2 cells, 8 μl for HT29 cells), 100 nM NOX1 siRNA, or 100 nM non-specific siRNAs (non-target controls), or DharmaFECT only (mock controls). Untreated controls received antibiotic-free media only. At 48 and 72 h after transfection, cells were harvested for flow cytometry, morphological assessment of apoptosis using Acridine Orange/Ethidium Bromide staining, and protein and mRNA analyses. In additional experiments, 72 h after transfection of NOX1 siRNAs the media was aspirated and replaced with fresh media containing 1.2 μg/μl of LPS (Sigma), or media alone. Thirty minutes later the cells were washed twice with PBS and lysed in IP buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail I and II (Sigma). Insoluble debris was removed by centrifugation at 10,000g for 5 min at 4°C. Cell lysates were subjected to SDS-PAGE, as described above, and immunoblotted for phospho-IKKα/β (p-IKK, Ser176/180, 1:500, Cell Signaling, no. 2697).
Cells (5 × 103) in 100 μl media were seeded in 96-well plates overnight and transfected with siRNA, as described above. At 24, 48, 72 and 96 h after transfection 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and incubated for 3 h, followed by 100 μl of 10% SDS in 0.01 N HCl. Formation of colored formazan dye was assessed colorimetrically at 550 nm.
Cell cycle distribution
Cells treated with NOX1 siRNA for 72 h (see above) were harvested in cold PBS, fixed in 70% ethanol, and stored at −20°C for 48 h. Fixed cells were washed with PBS and resuspended in propidium iodide/Triton X-100 staining solution containing RNase A. Samples were incubated in the dark for 30 min before determining the DNA content on an EPICS XL Beckman Coulter flow cytometer. Cell cycle distribution was assessed using Multicycle Software (Phoenix Flow Systems, San Diego, CA).
Cell suspensions (25 μl) were incubated with 1 μl acridine orange/ethidium bromide solution (50 μg/ml of each reagent in PBS), mixed gently, and placed onto a microscope slide. Cell morphology was examined under a fluorescence microscope for signs of chromatin condensation, fragmented nuclei and/or membrane blebbing, indicative of apoptosis. At least 500 cells were counted per treatment, and three independent experiments were performed.
DNA binding activity of NFκB-p50
Nuclear extracts were examined via enzyme-linked immunosorbent assay (NFκB-p50 transcription factor assay kit, Cat no. 10006912, Cayman Chemical, Michigan), according to the manufacturer's instructions.
Unless stated otherwise, results shown in each figure were from a single experiment, and are representative of the findings from three or more independent experiments. Data were expressed as mean ± standard error (mean ± SE), and comparisons between control and treatment groups were made using the paired t-test (SigmaPlot 8.0). In the figures, significant results were indicated as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.