Colorectal cancer is a common cancer worldwide. Carbonic anhydrase (CA) catalyzes the reversible conversion of carbon dioxide to bicarbonate ion and a proton, and its inhibitor is reported to reduce cancer cell proliferation and induce apoptosis. Therefore, we asked whether acetazolamide, a CA inhibitor, could inhibit intestinal carcinogenesis. Five-week-old male Apc-mutant mice, Min mice, were fed a AIN-76A diet containing 200 or 400 ppm acetazolamide. As a result, acetazolamide treatment reduced the total number of intestinal polyps by up to 50% compared to the control group. In addition, the acetazolamide-treated group had low cell proliferation and a high apoptosis ratio in the intestinal polyp epithelial cells. Moreover, the mRNA expression level of proinflammatory cytokines, such as IL-6, involved in the cell proliferation was decreased in the polyp part of the acetazolamide-treated group. Next, we examined the effects of acetazolamide on the activation of several transcriptional factors (AP-1, HIF, HSF, NF-κB, NRF2, p53, and STAT3) using a reporter gene assay in human colon cancer cells, Caco-2 cells. Among the examined transcriptional factors, NRF2 transcriptional activation was strongly induced. NRF2-targeting genes, γGCS, GPx1, HO-1, and NQO-1, were also elevated in the intestinal polyps of acetazolamide-treated Min mice. Our results suggested that CA is involved in intestinal carcinogenesis. Acetazolamide could inhibit polyp formation through suppressing local/general cytokine levels, i.e., IL-6, via NRF2 activation.

Mieap, a novel p53-inducible protein, plays a key role in maintaining healthy mitochondria in various pathophysiological states. Here, we show that Mieap deficiency in Apc(Min/+) mice is strikingly associated with the malignant progression of murine intestinal tumors. To understand the role that Mieap plays in in vivo tumorigenesis, we generated Mieap heterozygous (Apc(Min/+) Mieap(+/-)) and homozygous (Apc(Min/+) Mieap(-/-)) Apc(Min/+) mice. Interestingly, the Apc(Min/+) mice with the Mieap(+/-) and Mieap(-/-) genetic background revealed remarkable shortening of the lifetime compared to Apc(Min/+) mice because of severe anemia. A substantial increase in the number and size of intestinal polyps was associated with Mieap gene deficiency. Histopathologically, intestinal tumors in the Mieap-deficient Apc(Min/+) mice clearly demonstrated advanced grades of adenomas and adenocarcinomas. We demonstrated that the significant increase in morphologically unhealthy mitochondria and trace accumulations of reactive oxygen species may be mechanisms underlying the increased malignant progression of the intestinal tumors of Mieap-deficient Apc(Min/+) mice. These findings suggest that the Mieap-regulated mitochondrial quality control plays a critical role in preventing mouse intestinal tumorigenesis.

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.

Colorectal cancer is the fourth leading cause of cancer death worldwide, and it is important to establish effective methods for preventing colorectal cancer. One effective prevention strategy could be the use of antioxidants. However, the role of the direct antioxidative function of antioxidants against carcinogenesis has not been clarified. Thus, we aimed to determine whether the direct removal of reactive oxygen species by a hydroxyl radical scavenger, NZ-419, could inhibit colorectal carcinogenesis. NZ-419 is a creatinine metabolite that has been shown to be safe and to inhibit the progression of chronic kidney disease in rats, and it is now under clinical development. In the present study, we demonstrated that NZ-419 eliminated reactive oxygen species production in HCT116 cells after H2O2 stimulation and suppressed H2O2-induced Nrf2 promoter transcriptional activity. The administration of 500 ppm NZ-419 to Apc-mutant Min mice for 8 weeks resulted in a decrease in the number of polyps in the middle segment of the small intestine to 62.4% of the value in the untreated control (p < 0.05 vs. control group). As expected, NZ-419 treatment affected the levels of reactive carbonyl species, which are oxidative stress markers in the serum of Min mice. Suppression of the mRNA levels of the proliferation-associated factor c-Myc was observed in intestinal polyps of Min mice after NZ-419 treatment, with a weak suppression of epithelial cell proliferation assessed by proliferation cell nuclear antigen (PCNA) staining in the intestinal polyps. This study demonstrated that NZ-419 suppress the development of intestinal polyps in Min mice, suggesting the utility of radical scavenger/antioxidants as a cancer chemopreventive agent.

It is important to establish effective methods for preventing colorectal cancer because the number of colorectal cancer deaths is increasing. Erythromycin one of the macrolide antibiotics, has been shown to exert pleiotropic effects, such as anti-inflammatory and anti-oxidative effects, on mammalian cells. In the present study, we aimed to evaluate the preventive effects of erythromycin on intestinal carcinogenesis. We first confirmed that erythromycin suppresses the transcriptional activity of nuclear factor-κB and activator protein-1 and the expression of its downstream targets, interleukin-6 and cyclooxygenase-2 in human colon cancer cells. Next, we fed 5-week-old male Apc mutant Min mice with diets containing 500 ppm erythromycin for 15 weeks. Erythromycin treatment significantly reduced the number of proximal intestinal polyps to 70.9% of the untreated control value. Moreover, erythromycin reduced the levels of interleukin-6 and cyclooxygenase-2 mRNA expression in intestinal polyps. Although the levels of hepatic NADPH oxidase mRNA were decreased, erythromycin treatment did not affect the levels of oxidative stress markers, reactive carbonyl species, in the liver of Min mice. Our results suggest that erythromycin suppresses intestinal polyp development in Min mice, in part by attenuating local inflammation, and indicate that erythromycin is useful as a chemopreventive agent.

This study aimed to identify gastric mucosal protectants that suppress intestinal tumorigenesis in a mouse model. We chose six gastric mucosal protectants (ecabet sodium hydrate, irsogladine maleate, rebamipide, sofalcone, teprenone and troxipide) and examined their effects on the activity of oxidative stress-related transcriptional factors, including AP-1, NF-jB, NRF2, p53 and STAT3, in Caco-2 cells using a luciferase reporter gene assay. Among the six protectants, irsogladine maleate clearly inhibited NF-jB and AP-1 transcriptional activity. Furthermore, the chemopreventive property of irsogladine maleate was examined in a Min mouse model of familial adenomatous polyposis. Treatment with irsogladine maleate at doses of 5 and 50 ppm significantly reduced the number of intestinal polyps to 69% and 66% of the untreated control value, respectively. In these polyps, mRNA levels of the downstream targets of NF-jB, such as IL-1β and IL-6, were decreased by irsogladine maleate treatment. Moreover, the levels of oxidative stress-related markers, reactive carbonyl species, in the livers of Min mice were clearly decreased following the administration of irsogladine maleate. This study demonstrated that irsogladine maleate suppresses intestinal polyp formation in Min mice partly through the NF-jB signaling pathway, thus reducing oxidative stress.

Establishing effective methods for preventing colorectal cancer by so-called "functional foods" is important because the global burden of colorectal cancer is increasing. Enterococcus faecalis strain EC-12 (EC-12), which belongs to the family of lactic acid bacteria, has been shown to exert pleiotropic effects, such as anti-allergy and anti-infectious effects, on mammalian cells. In the present study, we aimed to evaluate the preventive effects of heat-killed EC-12 on intestinal carcinogenesis. We fed 5-week-old male and female Apc mutant Min mice diets containing 50 or 100 ppm heat-killed EC-12 for 8 weeks. In the 50 ppm treated group, there was 4.3% decrease in the number of polyps in males vs. 30.9% in females, and significant reduction was only achieved in the proximal small intestine of female mice. A similar reduction was observed in the 100 ppm treated group. Moreover, heat-killed EC-12 tended to reduce the levels of c-Myc and cyclin D1 mRNA expression in intestinal polyps. Next, we confirmed that heat-killed EC-12 suppressed the transcriptional activity of the T-cell factor/lymphoid enhancer factor, a transcriptional factor involved in cyclin D1 mRNA expression in intestinal polyps. Our results suggest that heat-killed EC-12 very weakly suppresses intestinal polyp development in Min mice, in part by attenuating β-catenin signaling, and this implies that heat-killed EC-12 could be used as a "functional food".

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Therefore, it is important to establish useful methods for preventing CRC. One prevention strategy involves the use of cancer chemopreventive agents, including functional foods. We focused on the well-known cancer chemopreventive agent curcumin, which is derived from turmeric. However, curcumin has the disadvantage of being poorly soluble in water due to its high hydrophobicity. To overcome this problem, the formation of submicron particles with surface controlled technology has been applied to curcumin to give it remarkably improved water solubility, and this derived compound is named Theracurmin. To date, the preventive effects of Theracurmin on hereditary intestinal carcinogenesis have not been elucidated. Thus, we used Apc-mutant mice, a model of familial adenomatous polyposis, to evaluate the effects of Theracurmin. First, we showed that treatment with 10-20 µM Theracurmin for 24 hours reduced nuclear factor-κB (NF-κB) transcriptional activity in human colon cancer DLD-1 and HCT116 cells. However, treatment with curcumin mixed in water did not change the NF-κB promoter transcriptional activity. As NF-κB is a regulator of inflammation-related factors, we next investigated the downstream targets of NF-κB: monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6. We found that treatment with 500 ppm Theracurmin for 8 weeks inhibited intestinal polyp development and suppressed MCP-1 and IL-6 mRNA expression levels in the parts of the intestine with polyps. This report provides a proof of concept for the ongoing Theracurmin human trial (J-CAP-C study).

Excessive prostaglandin production by cyclooxygenase-2 in stromal and epithelial cells is a causative factor of colorectal carcinogenesis. Thus, compounds which inhibit cyclooxygenase-2 transcriptional activity in colon epithelial cells could be candidates for anti-carcinogenic agents. A cyclooxygenase-2 transcriptional activity in the human colon cancer cell line DLD-1 has been measured using a β-galactosidase reporter gene system. Using this system, we demonstrated that the decrease in basal cyclooxygenase-2 transcriptional activities at 100 µM sesamol, one of the lignans in sesame seeds, was 50%. Other compounds in sesame seeds such as sesamin, sesamolin, ferulic acid, and syringic acid did not exhibit significant suppression of cyclooxygenase-2 transcriptional activity at up to 100 µM. In a following experiment, 6-week-old male Min mice, Apc-deficient mice, were divided into a non-treated and 500 ppm sesamol groups. At the age of 15 weeks, it was found that treatment with sesamol decreased the number of polyps in the middle part of small intestine to 66.1% of the untreated value. Moreover, sesamol suppressed cyclooxygenase-2 and cytosolic prostaglandin E2 synthase mRNA in the polyp parts. The present findings may demonstrate the novel anti-carcinogenetic property of sesamol, and imply that agents that can suppress cyclooxygenase-2 expression may be useful cancer chemopreventive agents.

It has been reported that activation of NF-E2 p45-related factor-2 (NRF2), a transcription factor, induces a variety of antioxidant enzymes, and plays an important role in preventing carcinogenesis. AHCC is a standardized extract of cultured Lentinula edodes mycelia and it has been demonstrated to improve cancer. However, the effects of AHCC on NRF2 have not been examined, and the effects on intestinal adenoma development are not yet fully understood. We first investigated the effects of AHCC (1-5 mg/ml) on NRF2 activity in human colon cancer cell lines by a luciferase reporter gene assay, and found NRF2 transcriptional activities were increased ~12.6-fold. In addition, AHCC dose-dependently increased HO-1 and NQO-1 mRNA levels, and decreased interleukine-6 mRNA levels. Next, we administered 1,000 ppm AHCC for 8 weeks in the diet of Apc mutant Min mice, and found that AHCC significantly reduced the total number of intestinal polyps to 57.7% and to 67.6% of the control value in male and female Min mice, respectively, with suppression of interleukine-6 in the polyp part. These data suggest that AHCC possesses an ability to suppress cellular oxidative stress through activation of NRF2, thereby lowering intestinal polyp development in Min mice.

Intestinal bacteria play an important role in human health. This prospective cohort study aimed to investigate the relationship between the abundance of different intestinal bacteria and the risk of developing colorectal cancer (CRC). Fecal samples from CRC patients (n = 157) were collected at the start of the study wherein patients subsequently underwent endoscopy to remove polyps. Gut bacteria were isolated by using specific culture methods and the fecal counts of various bacteria were quantified by reverse-transcription-quantitative-PCR (RT-qPCR) assays. The obtained data were subjected to cohort analysis in relation to the incidence of colorectal adenomas after 4 years of intervention. No relationship was detected between the counts of major intestinal bacteria and the incidence of colorectal adenomas. However, interestingly, a significant negative correlation was noted between colorectal adenoma incidence and the counts of bacteria grown on Columbia blood agar base (COBA) (P = 0.007). The risk ratio of colorectal adenomas was 0.58 (95% CI: 0.35-0.96) in the group with the highest bacterial count compared to the lowest. Bacteria grown on COBA were more abundant in older patients, non-smoking patients, and patients with a lower body mass index. The RT-qPCR results revealed a significantly lower colorectal adenoma incidence in subjects with higher enterococcal count as compared to subjects with a lower count, with a risk ratio of 0.47 (95% CI: 0.30-0.76). Correlation of a higher enterococci count with a lower risk of CRC development suggests that certain Enterococcus strains may have adenoma suppressive effects.