INTERNATIONAL JOURNAL OF ONCOLOGY 49: 639-645, 2016 Rifaximin, a non-absorbable antibiotic, inhibits the release of pro-angiogenic mediators in colon cancer cells through a pregnane X receptor-dependent pathway GIUSEPPE Esposito1*, Stefano GIGLI1*, LUISA SEGUELLA1, NICOLA NOBILE1, ALESSANDRA D'ALESSANDRO2, MARCEllA PESCE2, ELENA CAPOCCIA1, LUCA STEARDO1, CARLA CIRILLO3, ROSARIO CuOMO2 and Giovanni SARNELLI2 1Department of Physiology and Pharmacology, ‘Vittorio Erspamer’, La Sapienza University of Rome, I-00185 Rome; 2Department of Clinical Medicine and Surgery, ‘Federico II’ university of Naples, I-80131 Naples, Italy; 3Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, 3000 Leuven, Belgium Received February 22, 2016; Accepted March 3, 2016 DOI: 10.3892/ijo.2016.3550 Abstract. Activation of intestinal human pregnane X receptor VEGF secretion, NO release, VEGFR-2 expression, MMP-2 (PXR) has recently been proposed as a promising strategy for and MMP-9 expression vs. untreated cells. Rifaximin treat- the chemoprevention of inflammation-induced colon cancer. ment also resulted in a concentration-dependent decrease in The present study was aimed at evaluating the effect of rifax- the phosphorylation of Akt, mTOR, p38MAPK and inhibition imin, a non-absorbable antibiotic, in inhibiting angiogenesis of hypoxia-inducible factor 1-α (HIF-1α), p70S6K and NF-κB. in a model of human colorectal epithelium and investigating Ketoconazole (PXR antagonist) treatment inhibited these the role of PXR in its mechanism of action. Caco-2 cells were effects. These findings demonstrated that rifaximin causes treated with rifaximin (0.1, 1.0 and 10.0 µM) in the presence PXR-mediated inhibition of angiogenic factors in Caco-2 cell or absence of ketoconazole (10 µM) and assessed for cell line and may be a promising anticancer tool. proliferation, migration and expression of proliferating cell nuclear antigen (PCNA). The release of vascular endothelial Introduction growth factor (VEGF) and nitric oxide (NO), expression of Akt, mechanistic target of rapamycin (mTOR), p38 mitogen Colorectal cancer (CRC) represents one of the major causes activated protein kinases (MAPK), nuclear factor κB (NF-κB) of morbidity and mortality throughout the world and is the and metalloproteinase-2 and -9 (MMP-2 and -9) were also third most common form of human cancer worldwide (1,2). evaluated. Treatment with rifaximin 0.1, 1.0 and 10.0 µM Like other forms of cancer, CRC is characterized by angio- caused significant and concentration-dependent reduction of genesis, which is a crucial event in promoting cancer growth, cell proliferation, cell migration and PCNA expression in the progression and metastasis (3). Among the various signaling Caco-2 cells vs. untreated cells. Treatment downregulated molecules involved in the angiogenic process, vascular endothelial growth factor (VEGF) and nitric oxide (NO) are thought to be the key signaling molecules responsible for neo-vascularization (4-6). Once hypersecreted, VEGF binds to its type 2 receptor (VEGFR-2) and mediates the regulation Correspondence to: Dr Giovanni Sarnelli, Department of Clinical Medicine and Surgery, ‘Federico II’ university of Naples, Via of different pathways in the target cells, mainly the phosphati- Pansini 5, I-80131 Naples, Italy dylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian E-mail: [email protected] target of rapamycin (mTOR) pathway (7,8) and the phospho- p38 mitogen activated protein kinase (MAPK)-dependent Dr Giuseppe Esposito, Department of Physiology and Pharmacology activation of nuclear factor κB (NF-κB) (9,10). Activation of ‘Vittorio Erspamer’, La Sapienza University of Rome, Piazale Aldo the NF-κB and Akt/mTOR pathways increases the levels of Moro 5, I-00185 Rome, Italy the inducible nitric oxide synthase (iNOS) isoform, leading E-mail: [email protected] to the release and accumulation of nitric oxide (NO) that acts * as a pro-angiogenic stimulus on the blood vessels and favors Contributed equally neo-vascularization in solid tumors (5,11). Thus, targeting the angiogenic and mitogenic pathways is a rational and poten- Key words: angiogenesis, Caco-2 cell line, nitric oxide, pregnane X receptor, rifaximin, vascular endothelial growth factor tially effective strategy in the treatment of CRC (12). Rifaximin is a semi-synthetic antibiotic largely used for the treatment of travelers' diarrhea and hepatic encephalopathy (13,14). It is poorly absorbed on oral administration (15) 640 Esposito et al: Rifaximin downregulates ThE release of pro-angiogenic mediators in Caco-2 cells and as such has an optimum safety profile. Apart from its depending upon the experiments. Rifaximin and ketoconazole antibiotic potential, rifaximin has also been studied for its concentrations were selected based on the data from the avail- anti-inflammatory effects; several studies have highlighted able literature (16,18) and the pilot experiments (data not shown) the anti-inflammatory potential of rifaximin, which is mainly that helped in identifying the lowest effective concentrations. attributed to the inhibition of the NF-κB signaling and NO release via activation of intestinal human pregnane X (PXR) Western blot analysis. Protein expression in the Caco-2 cells receptors (16,17). was evaluated by western blot analysis. After the different The aim of the present study was to explore the anti-prolif- treatments, cells (1x106) were harvested, washed twice with erative and anti-migration effects of rifaximin, and to evaluate ice-cold PBS and centrifuged at 180 x g for 10 min at 4˚C. the possible control of the angiogenic mediator release by The cell pellet obtained after centrifugation was re-suspended rifaximin using a human intestinal epithelial cell line to model in 100 µl ice-cold hypotonic lysis buffer (10 mM hEPES, the intestinal barrier. The effect of rifaximin on VEGF and NO 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulpho- signaling and the mechanisms involved were also investigated. nylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 0.1 mM benzamidine and Materials and methods 0.5 mM DTT). To lyse the cells, the suspension was rapidly passed through a syringe needle five to six times and then Caco-2 cells were purchased from the European Collection of centrifuged for 15 min at 13,000 x g to obtain the cytoplasmic Cell Cultures (ECACC, Public Health England Porton Down, fraction. The cytoplasmic fraction proteins were mixed with Salisbury, uK). Cell medium, drugs and reagents for cell non-reducing gel loading buffer [50 mM Tris, 10% sodium culture were purchased from Sigma-Aldrich (St. louis, MO, dodecyl sulphate (SDS), 10% glycerol, 2 mg bromophenol/ml] uSA), unless otherwise specified. Instruments, reagents and at a 1:1 ratio, boiled for 3 min and centrifuged at 10,000 x g for materials for western blot analysis were obtained from Bio-Rad 10 min. The protein concentration was determined using the laboratories (Milan, Italy). Rifaximin and ketoconazole were Bradford assay and 50 µg of each sample was electrophoresed purchased from Tocris Cookson, Inc. (Ballwin, MO, uSA). on a 12% discontinuous polyacrylamide mini-gel. Proteins Mouse anti-total Akt, rabbit monoclonal anti-phospho-Akt were then transferred onto nitrocellulose membranes that had (Ser473), rabbit polyclonal anti-phospho-mTOR (pSer2448), been saturated by incubation with 10% non-fat dry milk in rabbit polyclonal anti-total p70S6K, rabbit polyclonal 1X PBS overnight at 4˚C with the following antibodies: total anti-phospho-p70S6K (Thr421/Ser424, Thr389) and rabbit Akt (1:1,000), phospho-Akt (1:2,000), total mTOR (1:1,000), monoclonal anti-VEGF receptor were purchased from Cell phospho-mTOR (1:1,000), total p70S6K (1:1,000), phospho- Signaling Technology (Euroclone, Pero, Milan, Italy). Rabbit p70S6K (1:1,000), anti-hIF-1α (1:500), anti-iNOS (1:1,000), polyclonal anti-total mTOR was purchased from Abcam anti-VEGFR-2 (1:1,000), anti-MMP-2 (1:1,000), anti-MMP-9 (Cambridge, uK); mouse monoclonal anti-hypoxia-inducible (1:1,000), anti-phospho-p38 (1:1,000), and anti-β-actin protein factor 1-α (HIF1-α) was purchased from Sigma-Aldrich (Milan, expression was performed on total protein fractions of homog- Italy); rabbit polyclonal anti-matrix metalloprotease-2 and 9 enates. Membranes were then incubated with the specific (MMP-2 and MMP-9) and mouse anti-β-actin were purchased secondary antibodies conjugated to hRP. Immune complexes from Santa Cruz Biotechnology (Santa Cruz, CA, uSA). were identified by enhanced chemiluminescence detection Polyclonal rabbit anti-mouse immunoglobulin G was procured reagents and blots were analyzed by scanning densitometry from Dako (Glostrup, Denmark), 32P-γ-ATP fromAmersham (GS-700 imaging densitometer; Bio-Rad laboratories). Results Biosciences (Milan, Italy), poly(deoxyinosinic-deoxycytidylic) were expressed as optical density (OD) (arbitrary units; mm2) acid (poly(dI-dC)), from Boehringer Mannheim (Milan, and normalized against the expression of the housekeeping Italy) and horseradish peroxidase (hRP) from Dako (Milan, protein β-actin. Italy). Chemiluminescence detection reagents were purchased from Amersham Biosciences and custom oligonucleotides Electrophoretic mobility shift assay. Electrophoretic mobility were synthesized by TIB Molbiol (Boehringer-Mannheim, shift assay (EMSA) was performed to detect NF-κB activation Mannheim, Germany). in Caco-2 cells. Briefly, 10 mg of the cell extracts were incu- bated in a binding buffer (8 mM hEPES, ph 7.0, 10% glycerol, Cell culture. Caco-2 cells were cultured in 6-well plates in 20 mM KCl, 4 mM MgCl2, 1 mM sodium pyrophosphate) Dulbecco's modified Eagle's medium (DMEM) containing containing 1.0 mg of poly(dI-dC) and 32P-γ end-labeled probe 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, with the following sequence: i) 5' AAC TCC GGG AAT TTC 2 mM l-glutamate and 1% non-essential amino acids. A CCT GGC CC 3' and ii) 5' GGG CCA GGG AAA TTC CCG total of 1x106 cells/well were plated and incubated for 24 h.