Experimental design: Inflammation-promoted colorectal neoplasia was induced by administering 2% dextran sodium sulfate (DSS) to female Apc+/Min-FCCC mice in the drinking water as outlined in Figure 1A. At treatment week 0 (8 wk of age), mice were randomized to one of four experimental groups: DSS treatment alone (no plecanatide treatment (control)) or DSS plus 2, 10 or 20 ppm plecanatide in the diet (n = 23/group). All animals were administered DSS for 4 d beginning at treatment week 1 and received regular water for the remainder of the study (Figure 1A). At the end of the study, the entire small intestine and colon were excised, cut longitudinally and rinsed with saline. An equivalent strip of the small intestine and colon from each animal was snap frozen for molecular analysis of UG and GC-C transcript levels by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The remainder of the colon was fixed in 10% formalin overnight, cross-sectioned at 2 mm intervals and processed for histopathological review.
Figure 1 Inflammation-driven colorectal carcinogenesis in Apc+/Min-FCCC mice.
A: Outline depicting the experimental design of the animal study. Female Apc+/Min-FCCC mice (n = 23/group) were randomized into four treatment groups: DSS alone (vehicle control) or DSS plus diet supplemented with 2, 10 or 20 ppm plecanatide. One week later, all animals were administered 2% DSS in the drinking water for 4 d (shaded box with asterisk) and regular water for the remainder of the study. At the time of euthanasia, (7 wk of study), the entire colon were fixed in formalin for histopathological evaluation; B: Body weights of Apc+/Min-FCCC mice treated with either DSS alone or DSS plus a diet supplemented with indicated concentrations of plecanatide (n = 20-23/group). Body weights were obtained weekly, and DSS was administered to all animals on days 7-10 of study. DSS: Dextran sodium sulfate.
Histopathological analyses: Sections stained with H and E were histopathologically evaluated for neoplasia in a blinded manner, as described previously. All classifications were based on standardized morphology and nomenclature for the human pathology of inflammation-promoted colorectal neoplasia. A diagnosis of carcinoma was assigned when neoplastic glands had invaded into the muscularis mucosae or beyond. Any dysplasia or cancer exhibiting an elevated growth pattern was considered polypoid. Non-polypoid (flat) lesions were elevated less than 2-fold above the adjacent non-neoplastic colorectal mucosa. Lesions that could not be readily classified as either polypoid or non-polypoid were categorized as indeterminate.
Immunohistochemistry: Ki-67 was selected as a biomarker of cell proliferation. Antigen retrieval was performed prior to staining in a Ventana Benchmark XT automated stainer (Tucson, AZ). All buffers and washes were per standard XT protocol. For Ki-67 staining, sections were incubated with Ki-67 primary antibody (1:1500 dilution; Vector Laboratories, Inc., Burlingame, CA) for one hour at room temperature. Negative controls were processed with rabbit IgG at approximately the same protein concentration as the primary antibody. Staining was detected using a rabbit secondary antibody kit (Vector Laboratories, Inc.) according to the manufacturer’s instructions. All sections were counterstained with lite hematoxylin. Only cells with nuclear staining of Ki67 were considered positive. The number of Ki-67 positive cells in dysplasias (2 fields/dysplasia, 600 ×) and non-neoplastic colonic crypts (20 crypt columns/animal, 600 ×) were counted and recorded as a Ki-67 labeling index (number of positive cells/total number of cells evaluated). The rate of proliferation of each tumor was established and the mean rate of all tumors in the treatment group was calculated.
The cellular localization of β-catenin was determined using specific polyclonal antibodies (1:4000 dilution; Sigma, St Louis, MO). Negative controls were processed with rabbit IgG. Staining was detected using a goat anti-rabbit secondary antibody kit (Vector Laboratories, Inc.) as per the manufacturer’s instructions. Sections were counterstained with lite hematoxylin. The number of tumor cells with nuclear localization of β-catenin was counted and expressed as a percentage of the total number of tumor cells per field (400 ×).
Immunoblotting: One centimeter long colon tissues from 6 animals per group were pooled and homogenized in RIPA buffer (10 mmol/L Tris, pH 7.2; 150 mmol/L NaCl, 1% sodium deoxycholate, 1% triton × 100, 0.1% SDS, 0.1 mmol/L Na3VO4; 50 mmol/L NaF), containing a protease inhibitor cocktail (Boehringer Mannheim, GmbH, Germany). The homogenate was centrifuged at 12000 g for 15 min at 4 °C and the supernatant was used as crude tissue lysate. The crude lysates (approximately 50 g protein) were subjected to 10% SDS-PAGE under reducing conditions, followed by immunoblotting with antibodies specific for β-catenin (1:4000 dilution, Sigma), PKG-II (1:200, dilution Santa Cruz Biotechnology, Inc., CA), glyceraldehyde 3phosphate dehydrogenase (GAPDH; 1:10000 dilution, Ambion), GC-C (1:500, Santa Cruz Biotechnology Inc., CA), phospho-VASP and c-myc (1:1000, Cell Signaling, MA, United States), cyclin D1 (1:1000, Abcam, MA, United States), and β-actin (1:1000, Chemicon, CA). Blots were developed using the ECL plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, United Kingdom) or LiCor blotting system. The resulting images were analyzed using the FluorChem E system (Cell Biosciences, Santa Clara, CA).
Measurement of cyclic GMP in tissue lysates: Colon tissues (1 cm) from 6 animals per group were pooled to prepare crude tissue lysates. The levels of cGMP were determined using an ELISA kit (Cayman Chemical Co., Ann Arbor, MI). The protein concentration of the lysates was determined using the Pierce BCA protein assay (Thermo Fisher Scientific, Rockford, IL). Results were normalized per mg protein and expressed as mean pmol ± SEM.
Quantification of UG and GC-C transcript levels by RT-PCR: Representative areas of the proximal and distal small intestine and colon were randomly selected from DSS control and DSS + plecanatide-treated mice (n = 45/group) for analysis. Total RNA was extracted from 5-10 mg of tissue using the RNAqueous®-4PCR Kit (Ambion, Austin, TX) and quantified using a Nanodrop2000 (Thermo Fisher, Waltham, MA). Total RNA (1 μg) was reverse-transcribed to cDNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Carlsbad, CA), according to the manufacturer’s instructions. Quantitative RT-PCR amplification and analysis were carried out using a LightCycler480 (Roche, Basel, Switzerland), UG and GC-C specific TaqMan reagents (Integrated DNA Technologies, Coralville, IA) and RT-PCR Master Mix (Roche, Basel, Switzerland). All amplification reactions (20 L total volume) were performed in duplicate with 10 ng cDNA (based on input RNA) and subjected to 35 PCR cycles using parameters set by the manufacturer. GAPDH was used as an endogenous control. The data generated were analyzed and expressed as target gene expression relative to endogenous control, using the comparative Ct method and the 2ΔΔCt formula. Results are expressed as fold change in relative levels of UG or GC-C transcripts per segment of small intestine or colon of plecanatide-treated mice, as compared to those of control mice treated with only DSS.
DSS treatment to induce acute inflammation in Apc+/Min-FCCC mice: An independent study was designed to evaluate the effect of plecanatide on GC-C signaling and cytokine expression during the acute phase of colonic inflammation. Apc+/Min-FCCC mice (n = 12; 6-8 wk old) were administered 2% DSS in the drinking water for 4 d followed by 3 d of regular water. Starting on day 1, six animals received an oral gavage of plecanatide (2.5 mg/kg body weight) daily while the other six were administered vehicle (0.9% sodium chloride solution, Sigma, St. Louis, MO). Vehicle control animals (n = 6) received regular drinking water (no DSS) and were administered an oral gavage of saline daily. It should be noted that the amount of plecanatide delivered by a single oral gavage at this dose is similar to that ingested daily when animals were administered a diet supplemented with 10 ppm plecanatide in the main tumorigenesis study. Mice were euthanized on day 7, and the entire colon was excised. Part of the colon tissue from each animal was snap frozen for analysis of intracellular cGMP and determination of the expression of GC-C, PKG-II, p-VASP and β-actin by immunoblot. The remaining tissue was used immediately for explant cultures as described below.
Explant culture: Tissues were washed in PBS containing 100 units of penicillin, 0.1 mg streptomycin and 0.25 μg amphotericin B per milliliter (1 x antibiotic and anti-mycotic solution; Sigma, St. Louis, MO) and cut into 1 cm pieces. Tissue explants were cultured in a 24-well plate overnight in RPMI 1640 media (Mediatech, Manassas, VA) in the absence or presence of 1 μmol/L plecanatide at 37 °C in a CO2 incubator. After the incubation period, explants were snap frozen and stored for analysis of cyclin D1, c-myc and β-actin expression by immunoblot.
Cytokine analysis: Expression of select mouse cytokines was detected in the spent media of pooled explant cultures (n = 6/pool) using a mouse Proteome Profiler Panel A kit array panel (R and D Systems, Minneapolis, MN). The mean intensities of the dot blots were calculated using ImageJ software (NIH).