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. 2010 Jun 1;70(11):4433-42.
doi: 10.1158/0008-5472.CAN-09-4289. Epub 2010 May 18.

Persistent cyclooxygenase-2 Inhibition Downregulates NF-{kappa}B, Resulting in Chronic Intestinal Inflammation in the Min/+ Mouse Model of Colon Tumorigenesis

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Persistent cyclooxygenase-2 Inhibition Downregulates NF-{kappa}B, Resulting in Chronic Intestinal Inflammation in the Min/+ Mouse Model of Colon Tumorigenesis

Adelaide M Carothers et al. Cancer Res. .
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Abstract

Cyclooxygenase-2 (COX-2) inhibition prevents adenoma formation in humans and mouse models of colon cancer. The selective COX-2 inhibitor celecoxib reduces COX-2 and prostaglandin E(2) (PGE(2)) expression and adenomas in the intestine of Min/+ mice after treatment for several weeks, but prolonged treatment increases PGE(2) production, resulting in drug-resistant tumor formation and transforming growth factor beta (TGFbeta)-dependent intestinal fibrosis. In this study, we examined pathways that regulate COX-2 expression and suppress chronic intestinal inflammation. We show that NF-kappaB signaling was inhibited in the ileum of Min/+ mice receiving long-term treatment with celecoxib. This effect was associated with inhibition of TGFbeta-associated kinase-1 and IkappaB kinase alpha/beta activities and reduced expression of the Toll-like receptor (TLR) 2 and TLR4 that enhance colonic barrier function. Additionally, we observed reduced activities of protein kinases c-Jun NH(2)-terminal kinase 1 and protein kinase A and transcription factor cyclic AMP-responsive element binding protein, regulators of COX-2 expression, which cross-talk with NF-kappaB. In ileum subjected to long-term celecoxib treatment, we noted relatively higher expression of COX-2, vascular endothelial growth factor, and interleukin-1beta in Paneth cells, whereas NF-kappaB and COX-2 were more strongly expressed by an expanded population of stromal myofibroblasts. Our findings argue that celecoxib resistance is an acquired adaptation to changes in the crypt microenvironment that is associated with chronic intestinal inflammation and impaired acute wound-healing responsiveness.

Figures

Figure 1
Figure 1. Long term celecoxib treatment suppressed NF-κB activity in enterocytes, a condition associated with chronic intestinal inflammation
IBs used lysates prepared as detailed (37) from Min/+ mice that were untreated or treated with celecoxib for 4 days, 3 weeks, or 5 months (A). The antibodies used in this study are listed in Supplementary Methods Table 1. In this and subsequent IBs, the protein concentration of each sample was the same except where indicated, β-actin served as a control for the uniformity of sample loading. IBs of total vs. phosphorylated proteins were performed in parallel. The fold difference at each treatment time for NF-κB p65 and p65-p-Ser536 is shown (Right); filled bars denote total NF-κB; hatched bars denote active phospho-NF-κB. In this and subsequent figures, error bars represent standard error of the mean (SEM). P values for NF-κB p65: untreated vs. 4 day: P=0.0194, 4 day vs. 3 week: P=0.0002, 3 week vs. 5 month: P=.0030, and untreated vs. 5 months: P=0.06. P values for NF-κB p65-p-Ser356: untreated vs. 4 day: P=0.0207, 4 day vs. 3 week: P=0.0001, 3 week vs. 5 month: P=0.0363, and untreated vs. 5 months: P=0.0006. IBs of NF-κB and STAT3 downstream signaling targets are shown (B). Representative photomicrographs of IHC for MPO using sectioned ileum from Min/+ mice untreated or treated with celecoxib for 4 days is shown (C). The number of myeloid cells (MPO+ or F4/80+) were significantly reduced at subsequent treatment times (data not shown & 4). Representative photomicrographs of serial sections of ileum from Min/+ mice untreated or treated with celecoxib for indicated times were immunostained for Vimentin and p65 to show the expression and location of NF-κB+ in myofibroblasts (D).
Figure 2
Figure 2. NF-κB inhibition upon long term celecoxib exposure was associated with reduced TLR2 and TLR4 expression, and TAK-1 activity
IBs examined treatment time-dependent changes in expression of TAK-1 vs. its active isoforms (A). IB analysis is shown of treatment time-dependent changes in TLR2, and TLR4 but not in NOD-2 expression (B, Left). Representative photomicrographs of serial sections of ileum from Min/+ mice treated with celecoxib for 4 days are shown that were immunostained separately for TLR4 and TLR2 (B, Right).
Figure 3
Figure 3. Long term celecoxib treatment increased microvessel density, the number of CD34+ vessels in the submucosa, and VEGF expression in Paneth cells
Representative photomicrographs of sectioned ileum from Min/+ mice untreated and treated for indicated times are shown that were immunostained for vWF (Left). A graph of the mean number of vWF+ cells per high power magnification (40X) field in crypt-villus units is shown (Right) (A). P values: untreated vs. 3 week: 0.0432; untreated vs. 5 months: 0.0009; 3 weeks vs. 5 months: P<0.0001. Representative photomicrographs of sectioned ileum from Min/+ mice treated with celecoxib for indicated times are shown that were immunostained for CD34 at original magnifications of 10X (Top) and 40X (Bottom) (B). Green arrows point to blood vessels in the submucosa. Representative photomicrographs of sectioned ileum from Min/+ mice untreated and treated for indicated show crypt-villus units that were immunostained for VEGF (Top). Images of ileum cross-sectioned at the base of crypts at 40X show positive staining for VEGF in Paneth cells of Min/+ mice that were untreated or treated with celecoxib for 5 months (Bottom) (C).
Figure 4
Figure 4. Paneth cell number and IL-1β and COX-2 expression were modulated by the duration of celecoxib treatment
Representative photomicrographs of sectioned ileum from Min/+ mice untreated or treated for indicated times are shown that were immunostained for Lysozyme (Right). A graph of the mean number of Paneth cells/10 crypt length of ileum vs. the duration of treatment time is shown (Left) (A). Representative photomicrographs of ileum from Min/+ mice untreated or treated for indicated times are shown that were immunostained for IL- 1β (Top, Left). Below is a 40X magnification image of ileum from Min/+ treated with celecoxib for 5 months; an arrow points to IL-1β in the secretory granules of Paneth cells (Bottom, Right). IB analysis of IL-1β expression in lysates of ileum from Min/+ mice untreated or treated for indicated times is shown (Left) (B). Representative photomicrographs of ileum from Min/+ mice untreated or treated for indicated times are shown that were immunostained for COX-2 is shown; arrows indicate COX-2 expression, or the lack of its expression, in Paneth cells (C).
Figure 5
Figure 5. Long term celecoxib treatment was associated with decreased PKA-CREB and MKK4-JNK1 activities, pathways that crosstalk with NF-κB
IB is shown of the relative expression of total MKK4 vs. its active phosphorylated isoform and of total JNK1 vs. its active 42 kDa phosphorylated isoform (Left) (A). IB is shown of the relative expression of total CREB vs. its active phosphorylated isoform and of total PKA β catalytic subunit vs. its active phosphorylated isoform (Right) (B).

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