Novel small molecule inhibiting CDCP1-PKCδ pathway reduces tumor metastasis and proliferation.

CUB domain-containing protein-1 (CDCP1) is a trans-membrane protein predominantly expressed in various cancer cells and involved in tumor progression. CDCP1 is phosphorylated at tyrosine residues in the intracellular domain by Src family kinases and recruits PKCδ to the plasma membrane through tyrosine phosphorylation-dependent association with the C2 domain of PKCδ, which in turn induces a survival signal in an anchorage-independent condition. In this study, we used our cell-free screening system to identify a small compound, glycoconjugated palladium complex (Pd-Oqn), which significantly inhibited the interaction between the C2 domain of PKCδ and phosphorylated CDCP1. Immunoprecipitation assays demonstrated that Pd-Oqn hindered the intercellular interaction of phosphorylated CDCP1 with PKCδ and also suppressed the phosphorylation of PKCδ but not that of ERK or AKT. In addition, Pd-Oqn inhibited the colony formation of gastric adenocarcinoma 44As3 cells in soft agar as well as their invasion. In mouse models, Pd-Oqn markedly reduced the peritoneal dissemination of gastric adenocarcinoma cells and the tumor growth of pancreatic cancer orthotopic xenografts. These results suggest that the novel compound Pd-Oqn reduces tumor metastasis and growth by inhibiting the association between CDCP1 and PKCδ, thus potentially representing a promising candidate among therapeutic reagents targeting protein-protein interaction.

Impact of Acetazolamide, a Carbonic Anhydrase Inhibitor, on the Development of Intestinal Polyps in Min Mice.

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.

Preventive Effects of Heat-Killed Enterococcus faecalis Strain EC-12 on Mouse Intestinal Tumor Development.

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 ß-catenin signaling, and this implies that heat-killed EC-12 could be used as a "functional food".

Chemopreventive effects of a low-side-effect antibiotic drug, erythromycin, on mouse intestinal tumors.

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.

Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin.

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 ß-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.

Sesamol suppresses cyclooxygenase-2 transcriptional activity in colon cancer cells and modifies intestinal polyp development in Apc (Min/+) mice.

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 ß-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.

Novel screening system revealed that intracellular cholesterol trafficking can be a good target for colon cancer prevention.

In conventional research methods for cancer prevention, cell proliferation and apoptosis have been intensively targeted rather than the protection of normal or benign tumor cells from malignant transformation. In this study, we aimed to identify candidate colon cancer chemopreventive drugs based on the transcriptional activities of TCF/LEF, NF-κB and NRF2, that play important roles in the process of malignant transformation. We screened a "validated library" consisting of 1280 approved drugs to identify hit compounds that decreased TCF/LEF and NF-κB transcriptional activity and increased NRF2 transcriptional activity. Based on the evaluation of these 3 transcriptional activities, 8 compounds were identified as candidate chemopreventive drugs for colorectal cancer. One of those, itraconazole, is a clinically used anti-fungal drug and was examined in the Min mouse model of familial adenomatous polyposis. Treatment with itraconazole significantly suppressed intestinal polyp formation and the effects of itraconazole on transcriptional activities may be exerted partly through inhibition of intracellular cholesterol trafficking. This screen represents one of the first attempts to identify chemopreventive agents using integrated criteria consisting of the inhibition of TCF/LEF, NF-κB and induction of NRF2 transcriptional activity.

Mieap suppresses murine intestinal tumor via its mitochondrial quality control.

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.