Zileuton, 5-Lipoxygenase Inhibitor, Acts as a Chemopreventive Agent in Intestinal Polyposis, by Modulating Polyp and Systemic Inflammation

<div><p>Purpose</p><p>Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APC^Δ468 mice.</p><p>Experimental Design</p><p>In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APC^Δ468 mouse model of polyposis and analyzed the effect of <i>in vivo</i> 5-LO inhibition on tumor-associated and systemic inflammation.</p><p>Results</p><p>Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice.</p><p>Conclusions</p><p>This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.</p></div

Frequency of lymphoid and myeloid cells isolated from APCΔ468Zileuton treated and APCΔ468 spleen and mesenteric lymph nodes (MLN).

<p>A) representative dot plots and the gating procedure of single cells suspencions isolated from APC^Δ468 and APC^Δ468Zileuton treated spleens. B) cummulative results of flow cytomentry of both spleen and MNL single cell suspencions. Black bars APC^Δ468, and open bars APC^Δ468Zileuton treated cellls. Flow cytometry analyzed with flow jo software, <i>One tailed unpaired</i> t test.</p

Zileuton treatment reduces the tumor infiltrating inflammatory cells in polyps.

<p>A) 1, 3, 5, 7: representative APC^Δ468/+ polyps; 2, 4, 6, 8: representative Zileuton treated APC^Δ468/+ polyps. B) calculation of positive cells as % frequency to the total cells; Black bars APC^Δ468/+, and open bars APC^Δ468/+ Zileuton treated. C) LTB4 ELISA of B6 (gray bars), APC^Δ468/+ (black bars), and APC^Δ468/+ Zileuton treated (open bars). D) ELISA human biopsy extracts. C) LTB4 ELISA of B6 (gray bars), APC^Δ468/+ (black bars), and APC^Δ468/+ Zileuton treated (open bars). D) ELISA human biopsy extracts. Black bars healthy tissue, Open bars tumor extracts. one tailed unpaired t test with Welch’s correction, * P<0.05, **P<0.005, one tail unpaired t test.</p

Zileuton treatment inhibits the proliferation rates of non epithelial cells in polyps, and increases the apoptosis rates in polyps.

<p>A) Polyp BrdU⁺ cells in small intestine and colon of Zileuton treated and control APC^Δ468 mice. Black bars total BrdU⁺ in APC^Δ468 polyps, open bars total BrdU⁺ in Zileuton APC^Δ468 polyps (17.4±0.91 BrdU⁺ cells per high power field in the control group vs 11.13±0.87 BrdU⁺ cells in Zileuton fed group; P = 0.0001). B) Polyp apoptotic cells in small intestine and colon of Zileuton treated APC^Δ468 polyps (open bars) and APC^Δ468 polyps (black bars).One tailed unpaired t test.</p

Zileuton is inhibiting the polyp formation in APCΔ468 colon and small intestine.

<p>A) Representative H&E stained 5μm Swiss rolls sections of small untreated intestine and colon of Zileuton treated and control APC^Δ468 mice, blue arrows indicate polyps appearing in this sections. B) Cumulative bar graphs of the polyp number appearing in APC^Δ468 mice small intestine (31.67±7.7 polyps in Zileuton group, n = 6, vs 83.88±6.5 polyps, n = 8, in the control group, p = 0.0002, closed bars) and colon (4.5±1.3 polyps in Zileuton group vs 8.9±0.8 polyps in control group, P = 0.0107, open bars, N = 6). C) Reflectance fluorescence of flayed opened colon, probe ProSense 680, representative whole mount of APC^Δ468 colon of APC^Δ468 Zileuton fed colon; red arrows polyps D) dot plot of the number of polyps; (3 closed dots) APC^Δ468 untreated colon polyps, (3 open dots) APC^Δ468 treated colon polyps. Statistics: unpaired one tailed t test.</p