Chemoprevention of colon and small intestinal tumorigenesis in APC(Min/+) mice by licofelone, a novel dual 5-LOX/COX inhibitor: potential implications for human colon cancer prevention

Abstract

Preclinical and clinical studies suggest that 5-lipoxygenase (5-LOX), such as COX-2, is a potential target for colon cancer inhibition and, in part, contributes to cardiovascular side effects associated with COX-2 inhibitors. Experiments were designed to assess the chemopreventive effects of a novel dual 5-LOX/COX inhibitor, licofelone {[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid}, in APC(Min/+) mouse intestinal tumorigenesis. Six-week-old male and female APC(Min/+) mice (n = 10 per group) were fed with control American Institute of Nutrition-76A diet or diets containing 150 or 300 ppm licofelone for 14 weeks (∼100 days), and intestinal tumors were evaluated for tumor multiplicity and size. Licofelone significantly inhibited total intestinal tumor multiplicity and size in a dose-dependent manner (P < 0.0001; mean tumors for 0, 150, and 300 ppm: 48.8, 17, and 8, respectively, in male mice; and 34.3, 8.8, and 5.5, respectively, in female mice). Licofelone at high dose showed more than 83% (P < 0.0001) tumor inhibition in both genders of mice. One hundred and fifty and 300 ppm licofelone resulted in 86% to 97% inhibition of polyps having size greater than 2 mm. One hundred and fifty and 300 ppm licofelone caused more than 72% and 100% inhibition of colonic tumors, respectively. Importantly, in mice fed with licofelone, tumors showed significantly reduced proliferating cell nuclear antigen expression (70%, P < 0.0001), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells (75%, P < 0.0001), and there was dose-dependent suppression of serum triglycerides (71%-83%, P < 0.0001), decreased inflammatory cytokines; and decreased COX and 5-LOX activities (57%-64%, P < 0.0001). Also, compared with 300 ppm celecoxib, 300 ppm licofelone provided better efficacy in suppressing tumor growth. These observations show that a novel dual 5-LOX/COX inhibitor dramatically suppresses small intestinal and colonic tumor formation in APC(Min/+) mice.

Figure 1

A. Chemical structure of licofelone,(2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl]acetic acid) B. Experimental design for the evaluation of the chemopreventive efficacy of licofelone in APC^min/+ mice. Groups (10 mince/group) of mice were fed diets containing 0, 150 or 300 ppm licofelone from 6 weeks of age to the end of the experiment. The study was terminated after 100 days of exposure to the experimental diets. (See Materials and Methods for more details). C & D. Changes in body weights over time for male (C) or female (D) mice fed control diets and/or experimental diets containing 150 or 300 ppm licofelone. Statistically significant differences in body weight gain were observed between licofelone-treated and control groups. Data are presented as Means ± SEM. Statistical Analysis was carried with ANOVA. Licofelone-treated animals were found to gain weight by the end of the study. E. MRI of the small intestine in APC^Min/+ mice. Small intestines of the treated and control animals were scanned for polyps by MRI. Comparing images from pre-and post-magnivest injection, polyps were identified as bright from accumulating magnivest. Left and right panels show polyp images in control and treated mice respectively.

Figure 2

(A). Inhibition of total small intestinal polyp formation in maleAPC^Min/+ mice by licofelone. Data values are means ± SEM of ten animals per treatment group. Control and treated groups are significantly different from one another (P < 0.001 or P < 0.0001). (B). Inhibition of total small intestinal polyp formation in female APC^Min/+ mice by licofelone. Data values are means ± SE of ten animals per treatment. Control and treated groups are significantly different from one another (P < 0.001 or P < 0.0001). (C). Average number of colon tumors per mouse in control and treated male APC^min/+ mice. A siginificant (P < 0.001) inhibition of colon tumors was observed with low dose licofelone and 100% inhibition was observed with high dose treatment. (D). Average number of colon tumors per mouse in control and treated APC^min/+ female mice. A siginificant (P < 0.001) inhibition of colon tumors was observed with low dose licofelone and 100% inhibition was observed with high dose treatment. (E). Photograph showing large polyps in control APC^min/+ mice compared with complete suppression of polyps in small intestine of licofelone treated mice. (F). Tumor sizes in the small intestine of APC^Min/+ mice. Intestines were divided into sections, examined under a stereomicroscope, and the size of polyps was determined. Data values are given as means ± SE of ten animals per treatment. Tumors >2-mm diameter were completely suppressed in high dose licofelone-treated mice. (G&H). Inverse correlation between tumor number and triglyceride levels was observed with control and licofelone-treated groups of male APC^min/+ mice. In licofelone-treated groups, the triglyceride levels were almost equal to the levels observed in wild-type mice. The decrease in triglyceride levels was significantly different from control mice versus treated male mice (P < 0.001 to P < 0.0001); albeit lesser in female mice (P < 0.04 to P < 0.001) (I&J). Packed blood cell volumes (PCV) of male or female APC^Min/+ mice fed either control or licofelone diets. A significant dose-dependent increase in PCV was observed in control mice vs treated mice

Figure 3

(A). Effects of a diet with 150 ppm licofelone on COX activity in intestinal polyps from APC^min/+ mice as assessed with the Radio-HPLC method. Values are means ± SEM, N=6/treatment group. A significant (P < 0.001) inhibition of arachidonic acid metabolites (PGs and TXB₂) was observed in polyps from licofelone-treated mice compared with polyps from control mice. (B). Effects of a diet with 150 ppm licofelone on 5-LOX activity in intestinal polyps from APC^Min/+ mice as assessed with the Radio-HPLC method. Values are means ± SEM, N=6/treatment group. A significant (P < 0.0001) inhibition of the 5-LOX metabolite 5-HETE was observed in polyps l from icofelone-treated mice compared with polyps from control mice. (C). Effects of 150 ppm licofelone diet on PGE₂ generation in intestinal polyps from APC_min/+ mice as assessed with the Radio-HPLC method. Values are means ± SEM, N=6/treatment group. A significant (P < 0.001) decrease in the arachidonic acid metabolite PGE₂ was observed in polyps from treated mice compared with those from control mice. (D). Effects of 150 ppm licofelone diet on PGE₂ levels in intestinal polyps from APC^min/+ mice as determined with ELISA. Values are means ± SEM, N=6/treatment group. A significant (P < 0.001) decrease of the arachidonic acid metabolite PGE₂ was observed in polyps from treated mice compared with those from control mice.

Figure 4

A. Immunohistochemical staining for PCNA in intestinal tumors from APC^Min/+ mice fed control diet or treated with licofelone. B. Significant difference was observed in proliferative index between licofelone-treated and control group polyps. C. TUNEL assay was performed for apoptotic cells in intestinal tumors from APC^Min/+ mice fed control diet or treated with licofelone. A significant induction of apoptosis was observed in hyperplastic regions and polyps of treated mice compared with untreated mice. D. A significant difference was observed in apoptotic index between licofelone-treated and control groups. Polyps from treated showed ~75% induction of apoptosis compared with polyps from control mice.

Figure 5

A. Immunohistochemical staining for COX-2 expression in intestinal tumors from APC^Min/+ mice fed control diet or treated with licofelone. B. Modulatory effects of licofelone on COX-2 and PGES-2 mRNA expression in intestinal polyps of treated and untreated male APC^Min/+ mice. A significant dose-dependent suppression of COX-2 mRNA was observed upon licofelone treatment in APC^Min/+ mice. C. Modulatory effects of licofelone on COX-2 and PGES-2 mRNA expression in intestinal polyps of treated and untreated female APC^Min/+ mice. A significant dose-dependent suppression of COX-2 mRNA was observed in licofelone treated APC ^min/+ mice. D. Immunohistochemical staining for 5-LOX expression in intestinal tumors from APC^Min/+ mice fed control diet or treated with licofelone. E. Modulatory effects of licofelone on 5-LOX and FLAP mRNA expression in intestinal polyps of treated and untreated male APC^Min/+ mice. A significant dose dependent suppression of 5-LOX and FLAP mRNA was observed in licofelone treated APC^Min/+ mice. F. Modulatory effects of licofelone on 5-LOX and FLAP mRNA expression in intestinal polyps of treated and untreated female APC^Min/+ mice. A significant dose-dependent suppression of 5-LOX and FLAP mRNA was observed in licofelone treated APC^Min/+ mice. G. Modulatory effects of licofelone on LTB4 receptor mRNA expression in intestinal polyps of treated and untreated APC^Min/+ mice. A significant dose-dependent suppression of LTB₄ receptor mRNA was observed in licofelone treated polyps. GAPDH was used as loading control. H. Real time PCR analysis for COX-2 mRNA expression in intestinal polyps of treated and untreated male APC^Min/+ mice. A significant dose-dependent suppression of COX-2 mRNA was observed upon licofelone treatment in APC^Min/+ mice. I. Real time PCR analysis for PGES-2 mRNA expression in intestinal polyps of treated and untreated male APC^Min/+ mice. A significant dose-dependent suppression of PGES-2 mRNA was observed upon licofelone treatment in APC^Min/+ mice. J. Real time PCR analysis for 5-LOX mRNA expression in intestinal polyps of treated and untreated male APC^Min/+ mice. A significant dose-dependent suppression of 5-LOX mRNA was observed upon licofelone treatment in APC^Min/+ mice.

Figure 6

Effect of licofelone on Inflammatory cytokines in serum samples from treated and untreated APC^Min/+ mice as analyzed by ELISA.