Cells and culture conditions
A431 cells were purchased from ATCC (Rockville, MD) and authenticated using a panel of microsatellite markers (Istituto Nazionale Tumori, Milano, Italy). Cells were cultured in RPMI 1640 medium (Euroclone, Pero, Italy) supplemented with 10% FBS (Euroclone), 1% glutamine (Euroclone) and 1% penicillin/streptomycin solutions (Euroclone). A431 cells in log phase were digested and cell suspensions were inoculated subcutaneously under sterile conditions into mice.
The synthesis and a preliminary profiling of BLF501 have been described . BLF501 resembles the structure (isosteric) of the natural O-glycoside, but cannot be metabolized (C-glycosides are unable to undergo glycolysis). BLF501 is water-soluble (5 mM) and stable for several days at 25°C at pH 1-12 (stored in the dark).
Mice and in vivo treatments
Eight-week-old female BALB/c and nude SKH-1 mice were purchased from Charles River Italy (Calco, Italy). SGLT-1-/- mice on a C57BL/6 background have recently been described . Mice were housed in specific aseptic conditions at constant temperature and humidity, with food and water given ad libitum. Mice were fed a specific normocaloric chow which does not contain glucose and galactose (Altromin C1000, Rieper, BZ, Italy). Experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Istituto Nazionale Tumori (Milano, Italy), and conducted according to the guidelines of the United Kingdom Coordinating Committee on Cancer Research for animal welfare in experimental neoplasia (1998).
The effect of BLF501 on intestinal injury induced by a single injection of DXR (Pfizer; NY, USA) was evaluated in four groups of BALB/C mice: controls (n = 7); DXR (n = 14); DXR plus BLF501 (n = 14); and BLF501 (n = 14). DXR (20 mg/kg in saline, total volume 300 μl) was administered by i.p. injection. BLF501 (25 μg/kg in saline, total volume 100 μl) was administered at the same time by gavage using a gastric tube. Control mice were treated i.p. with saline. One hour before sacrifice, bromodeoxyuridine (BrdU, Novocastra, Newcastle, UK) was injected i.p to determine cell proliferation. Control mice were sacrificed after 72 h. Mice from the other groups were sacrificed at 48 (n = 7) and at 72 h (n = 7) after treatment (Table 1) . SGLT-1-/- mice were randomly divided into three groups: UNTR; DXR; and DXR + BLF501 25 μg/kg. (n = 7/group) (Table 1) and sacrificed after 72 h.
The effect of BLF501 on intestinal injury induced by repeated injections of DXR and 5-FU (TEVA; Petah Tikva, Israel) was evaluated in six groups (n = 7/group): controls; DXR/5 + FU; DXR/5 + FU + BLF501 (0.25 μg/kg); DXR + 5-FU + BLF501 (2.5 μg/kg); DXR + 5-FU + BLF501 (25 μg/kg); BLF501 (25 μg/kg). DXR (7 mg/kg) and 5-FU (100 ng/kg), dissolved in saline solution in a final volume of 300 μl, were administered i.p. once per week for 3 weeks. BLF501 (in saline, 100 μl final volume) was administered at the same time by gavage using a gastric tube. Mice were sacrificed on day 19 after starting treatment (Table 1).
The effect of BLF501 on the antitumor activity of DXR was tested in four groups (n = 8/group) of nude SKH-1 mice bearing SGLT-1-positive A431 mammary tumors that had reached a mean weight of 240 mg: controls; DXR (6 mg/kg in 200 μl saline) + BLF501 (25 μg/kg in 100 μl saline); DXR alone; BLF501 alone. DXR was administered intravenously (i.v.) once per week for 3 weeks. BLF501 was administered at the same time by gavage using a gastric tube. Mice were sacrificed on day 26 of treatment (Table 1). Statistical significance was assessed using χ2 analysis.
Processing of samples and histological evaluation
Mice were sacrificed and jejunum samples were collected and fixed in 10% formalin with 2% sucrose in phosphate buffer for 4 h at 4°C and processed for paraffin embedding. Other jejunum samples were collected and preserved in liquid nitrogen for mRNA and protein extraction.
For histological examination, slides were stained with hematoxylin and eosin. Histological images were captured and digitized. Villus height was measured using Image Pro Plus 4 image analysis software (Media Cybernetics, Baltimore, MD). The degree of intestinal tissue injury was evaluated on a grading scale of : +++ = severe; ++ = mild; + = light; 0 = absent.
Briefly, sample sections on slides were deparaffinized and hydrated for 1 h through a descending scale of alcohols. After a quick rinse with 0.1 M Tris-HCl, pH 7.4, sections were incubated with proteinase K (20 g/ml) Tris-EDTA buffer, pH 8, for 15 min at 37°C, washed with Tris-HCl and permeabilized with 1% Triton X-100 in Tris-HCl for 5 min. Sections were treated with blocking solution for 1 h at room temperature and incubated overnight at 4°C with 20 μg/ml primary anti-mouse ZO-1 rabbit antibody (α-ZO-1, Invitrogen, Camarillo, CA). After washing, samples were incubated with secondary goat anti-rabbit antibody conjugated to tetramethyl-rhodamine isothiocyanate (TRITC; DyLight Jackson, West Baltimore Pike West Grove, PA, US) diluted 1:1000 in Tris-HCl for 45 min at room temperature. Sections were incubated with DAPI (1:10000 in Tris-HCl) for 5 min at room temperature and washed 3 times with Tris-HCl and 0.01% Triton X-100. Slides were mounted with Mowiol.
Sample sections on slides were deparaffinized and hydrated for 1 h through a descending scale of alcohols. Antigen retrieval was performed using two antigen unmasking steps of 5 min in a microwave oven with citrate buffer, pH 6 (0.005 M). Sections were cooled and then washed with 0.1 M Tris-HCl, pH 7.4, + 0.025% Triton X-100. Samples were treated with a peroxidase inhibition solution of 3% H2O2 in 0.1 M Tris-HCl, pH 7.4, for 20 min and nonspecific sites were blocked with HHG solution (1 mM Hepes, 2% goat serum, 1X HBSS, 0.5% Triton X-100) in Tris-HCl for 1 h at room temperature. Sections were then incubated for 2 h at room temperature with a primary anti-beta-catenin rabbit antibody diluted 1:500 (Abcam, Cambridge, UK), washed, and incubated for 45 min at room temperature with a biotinylated secondary goat anti-rabbit antibody diluted 1:1000. Finally, sections were incubated with ABC-kit and DAB (Vector, Burlingame, VT, US), counterstained with hematoxylin, dehydrated through an ascending scale of alcohols and xylene, and mounted with coverslips using Entellan (Merck, Darmstadt, Germany).
Sample sections on slides were deparaffinized and hydrated for 1 h through a descending scale of alcohols, rinsed in PBS and incubated at room temperature with 2 N HCl for 30 min and with Na2B4O7 for 10 min. Sections were incubated with PBS/3% BSA for 20 min at room temperature and with proteinase K (20 g/ml) Tris-EDTA buffer, pH 8, for 15 min at 37°C. After washing, slides were incubated for 1 h at room temperature with a primary anti-BrdU mouse antibody (Novocastra) diluted 1:200 in PBS/3% BSA, washed, and incubated with secondary goat anti-mouse DyLight 488 antibody (Jackson) diluted 1:500 in PBS/BSA 3%. Sections were washed, incubated for 5 min at room temperature with DAPI diluted 1:2500 in PBS, and mounted with Mowiol.
All samples were observed and photographed with a microscope Nikon Eclipse 80 with a digital camera Nikon DS-L1.
Real-time polymerase-chain reaction (PCR)
RNA was extracted from frozen jejunum samples using TRI-reagent (Sigma-Aldrich, St. Louis, MO, US) and transcribed with a reverse transcription kit (Applied Biosystems, Foster City, CA, US). Real-time PCR experiments were performed according to the manufacturer’s instructions using a 7900HT Fast Real-Time PCR System (Applied Biosystems). Primers for SI, TFF3, DLL1, beta-actin and the housekeeping gene 18S were purchased from Applied Biosystems.
Western blot analysis
Expression of caspase-3 and ERM complex was assessed on total phosphorylated protein extracted from small intestine samples of mice using our TNTG buffer (40 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 1% Triton X-100, proteases inhibitors). Protein extracts were quantified using the BCA method (BCA Protein Assay Kit, Pierce, Rockford, IL, US). Proteins (30 μg) were fractionated on a polyacrylamide gel (BIO-RAD Labs, Hercules, CA) and electroblotted onto nitrocellulose filters (American Biosciences, Buckinghamshire, UK). Filters were incubated for 1 h in TBS containing 5% milk powder to block nonspecific binding sites, followed by anti-caspase-3 antibody (1 μg/ml; Abcam), anti-GAPDH antibody (1:1000; Abcam) and anti-ERM antibody (1:1000; Cell Signaling Technologies, Danvers, MA, US), and finally with appropriate anti-goat and anti-rabbit secondary anti-peroxide antibody (Vector Laboratories). Bands were visualized using ECL™ Western Blotting Detection Reagents and plates by autoradiography (American Biosciences).
Student’s t-test (paired two-tailed), χ2 analysis and GraphPad Prism software (GraphPad Prism Software Inc., San Diego, CA) were used for comparisons between groups. Differences were considered significant at p < 0.05.