Theracurmin inhibits intestinal polyp development in Apc-mutant mice by inhibiting inflammation-related factors.

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).

Stromal iodothyronine deiodinase 2 (DIO2) promotes the growth of intestinal tumors in ApcΔ716 mutant mice.

Iodothyronine deiodinase 2 (DIO2) converts the prohormone thyroxine (T4) to bioactive T3 in peripheral tissues and thereby regulates local thyroid hormone (TH) levels. Although epidemiologic studies suggest the contribution of TH to the progression of colorectal cancer (CRC), the role of DIO2 in CRC remains elusive. Here we show that Dio2 is highly expressed in intestinal polyps of ApcΔ716 mice, a mouse model of familial adenomatous polyposis and early stage sporadic CRC. Laser capture microdissection and in situ hybridization analysis show almost exclusive expression of Dio2 in the stroma of ApcΔ716 polyps in the proximity of the COX-2-positive areas. Treatment with iopanoic acid, a deiodinase inhibitor, or chemical thyroidectomy suppresses tumor formation in ApcΔ716 mice, accompanied by reduced tumor cell proliferation and angiogenesis. Dio2 expression in ApcΔ716 polyps is strongly suppressed by treatment with the COX-2 inhibitor meloxicam. Analysis of The Cancer Genome Atlas data shows upregulation of DIO2 in CRC clinical samples and a close association of its expression pattern with the stromal component, consistently with almost exclusive expression of DIO2 in the stroma of human CRC as revealed by in situ hybridization. These results indicate essential roles of stromal DIO2 and thyroid hormone signaling in promoting the growth of intestinal tumors.

Chemoprevention in Patients with Peutz-Jeghers Syndrome: Lessons Learned.

LESSONS LEARNED: Motivating patients to enroll in chemopreventive studies is challenging.Chemoprevention with toxic drugs is not feasible. BACKGROUND: LKB1 mutations are the underlying genetic abnormality causing Peutz-Jeghers syndrome (PJS) and are a potential target for everolimus. In this phase II study, the efficacy of everolimus on polyp and tumor growth in PJS patients was investigated. METHODS: Adult patients with a proven LKB1 mutation and who were suitable for everolimus treatment were included in two different PJS cohorts: (a) patients with unresectable malignancies and (b) patients with high-risk polyps. Treatment in both groups was oral everolimus, 10 mg daily. Response rates were primary endpoints for both cohorts. RESULTS: Between October 2011 and April 2016, only two patients were enrolled, one in each cohort. A 49-year-old patient with advanced pancreatic cancer in cohort 1 was progressive after 2 months. A 52-year-old male patient in cohort 2 experienced severe toxicity and refused treatment after 4 months, even though endoscopy suggested stabilization of polyps. Adverse events included dental inflammations, mucositis, and rash. In 2016, the trial was aborted for lack of accrual, despite extensive accrual efforts in an area where PJS is highly prevalent and care is highly centralized. CONCLUSION: Due to accrual problems, no conclusions can be drawn about the value of everolimus in PJS treatment, questioning the feasibility of this agent for chemoprevention.

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.

Chemoprevention of colorectal cancer by targeting APC-deficient cells for apoptosis.

Cancer chemoprevention uses natural, synthetic, or biological substances to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. It holds promise for overcoming problems associated with the treatment of late-stage cancers. However, the broad application of chemoprevention is compromised at present by limited effectiveness and potential toxicity. To overcome these challenges, here we developed a new chemoprevention approach that specifically targets premalignant tumour cells for apoptosis. We show that a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of beta-catenin lead to the repression of cellular caspase-8 inhibitor c-FLIP (also known as CFLAR) expression through activation of c-Myc, and that all-trans-retinyl acetate (RAc) independently upregulates tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors and suppresses decoy receptors. Thus, the combination of TRAIL and RAc induces apoptosis in APC-deficient premalignant cells without affecting normal cells in vitro. In addition, we show that short-term and non-continuous TRAIL and RAc treatment induce apoptosis specifically in intestinal polyps, strongly inhibit tumour growth, and prolong survival in multiple intestinal neoplasms C57BL/6J-Apc(Min)/J (Apc(Min)) mice. With our approach, we further demonstrate that TRAIL and RAc induce significant cell death in human colon polyps, providing a potentially selective approach for colorectal cancer chemoprevention by targeting APC-deficient cells for apoptosis.

Antitumor activity of the MEK inhibitor trametinib on intestinal polyp formation in Apc(Δ716) mice involves stromal COX-2.

Extracellular signal-regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using Apc(Δ716) mice, a mouse model of familial adenomatous polyposis and early-stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight-week treatment of Apc(Δ716) mice with trametinib, a small-molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX-2 level in Apc(Δ716) tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX-2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in Apc(Δ716) tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in Apc(Δ716) mice, at least in part, through COX-2 induction in tumor stromal cells.

Treatment with anti-interleukin-6 receptor antibody ameliorates intestinal polyposis in Apc(Min/+) mice under high-fat diet conditions.

The prevalence of colorectal malignancies is increasing in the world. The parallel increase of metabolic syndrome gives a speculation between these two conditions, although the precise mechanism is still unclear. Interleukin-6 (IL-6) is a cytokine known to correlate with obesity and serve as a proinflammatory adipokine. In the present study, we investigated the effect of IL-6 signaling blockade on intestinal polyp formation in obesity using a mouse model of adenomatous polyposis coli (Apc). Male C57BL/6J-Apc(Min/+) mice were fed a high-fat diet from 5 weeks of age, and the overweight mice thus obtained were given a weekly intraperitoneal injection of anti-mouse IL-6 receptor antibody (MR16-1) from 6 to 15 weeks of age, while control mice received IgG or phosphate-buffered saline (PBS). The total number of intestinal polyps was significantly decreased in the MR16-1-injected group (53.1 ± 6.8) relative to the control groups (PBS-injected, 81.3 ± 6.1; rat IgG-injected, 74.7 ± 4.8, p = 0.01), and in particular the number of polyps larger than 2 mm in diameter was markedly decreased. In addition, the mean diameter of polyps in the MR16-1-injected group was significantly smaller than that in the control groups. On the other hand, no significant differences in body weight, epididymal fat pad mass, or the plasma levels of glucose, insulin and triglyceride were observed among the three groups. Thus, treatment with anti-IL-6 receptor antibody suppressed polyp growth in obese Apc(Min/+) mice fed the high-fat diet. We suggest that IL-6 signaling may be responsible for the obesity-associated colorectal tumorigenesis.

Role of microRNAs in resveratrol-mediated mitigation of colitis-associated tumorigenesis in Apc(Min/+) mice.

The pleiotropic effects of resveratrol include anti-inflammatory, antioxidant, and anticancer activities, and thus unique possibilities exist to explore mechanistic pathways of chemoprevention. The aim of this study was to investigate the role of microRNA (miRNA) alterations induced by resveratrol in the context of chemopreventive mechanisms against dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis in the Apc(Min/+) mouse. To that end, Apc(Min/+) mice were exposed to 2% DSS to enhance intestinal inflammation and polyp development. Concurrently, mice received either vehicle or resveratrol treatment via oral gavage for 5 weeks. Interestingly, treatment of DSS-exposed mice with resveratrol resulted in decreased number and size of polyps, fewer histologic signs of cell damage, and decreased proliferating epithelial cells in intestinal mucosa compared with vehicle. Resveratrol treatment dramatically reversed the effects of DSS on the numbers of specific inflammatory CD4(+) T cells, CD8(+) T cells, B cells, natural killer T cells, and myeloid-derived suppressor cells in mesenteric lymph nodes. Resveratrol treatment also decreased interleukin-6 (IL-6) and tumor necrosis factor-α protein levels and reduced IL-6 and cyclooxygenase-2 mRNA expression. Microarray analysis revealed 104 miRNAs exhibiting >1.5-fold differences in expression in the intestinal tissue of resveratrol-treated mice. Among them, two miRNAs with anti-inflammatory properties, miRNA-101b and miRNA-455, were validated to be upregulated with resveratrol treatment by reverse-transcription polymerase chain reaction. Pathway analysis revealed that numerous differentially regulated miRNAs targeted mRNAs associated with inflammatory processes with known roles in intestinal tumorigenesis. These results suggest that resveratrol mediates anti-inflammatory properties and suppresses intestinal tumorigenesis through miRNA modulation.

Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine.

Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.

Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

A pilot, randomized, placebo-controlled, double-blind phase 0/biomarker study on effect of artepillin C-rich extract of Brazilian propolis in frequent colorectal adenoma polyp patients.

OBJECTIVE: Brazilian propolis, a folk medicine, is used worldwide as an alternative medicine to prevent colon cancer. The objective of the study was to test in a small pilot biomarker study in a high-risk group the safety and efficacy of propolis for colon cancer prevention, which has not been evaluated in humans. METHODS: Subjects with adenoma polyps recently removed from the colon were randomly assigned to a propolis group of 15 and a placebo group of 16. In a double-blind study, the propolis group received capsules containing 165 µmol artepillin C and 150 µmol other polyphenols per day for 3 months. Prior to and at the end of the experiments, their blood was analyzed using biochemical tests, and specimens from the normal-appearing sigmoid colon mucosa were biopsied endoscopically to examine the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and mRNA expressions of proliferating cell nuclear antigen, cyclin D1, and Bax. RESULTS: Propolis extract significantly increased the mRNA level of cyclin D1 in the sigmoid colon mucosa, and the other biomarkers remained unchanged. Blood biochemical tests showed significantly higher activity of creatine phosphokinase (CPK), 143 ± 52 units/ml in the propolis group and 104 ± 38 units/ml in the placebo group (p = 0.026), at the end of the study. The increase in CPK activity in the propolis group was due to the increase of the myocardial band form of CPK. On the other hand, laxative treatment prior to endoscopic biopsy significantly increased 8-OHdG levels. CONCLUSIONS: The results from our pilot study did not provide evidence that Brazilian propolis was effective in preventing changes occurring during early stages of colon cancer. In contrast, propolis may have detrimental side effects on muscle tissue, including myocardial cells.

Quercetin's effects on intestinal polyp multiplicity and macrophage number in the Apc(Min/+) mouse.

Numerous in vitro studies argue for quercetin's chemopreventive potential in colon cancer; however, experimental studies in rodents are limited. Macrophages play a role in tumorigenesis, but the effects of quercetin on macrophage infiltration in colon cancer is unknown. We examined the effects of quercetin on intestinal polyp multiplicity and macrophage number in Apc(Min/+) mice. Apc(Min/+) mice were assigned to placebo or quercetin (n = 8/group) groups. Mice were given a placebo or quercetin (0.02%) diet from 4-20 wk of age, after which intestines were analyzed for polyp number and size in the small intestine (Sections 1-4) and colon (Section 5) and for macrophage number in the small intestine (Sections 1 and 3). Spleen weight was determined as a marker of systemic inflammation. Quercetin decreased total intestinal polyps by 67% (P < 0.05). Specifically, quercetin reduced intestinal polyps in categories >2 mm (69%) and 1-2 mm (79%; P < 0.05), and in Sections 2 (75%), 3 (80%), and 4 (79%; P < 0.05). Quercetin also decreased macrophage number in Sections 1 (57%) and 3 (81%), and spleen weight (P < 0.05). These data suggest that quercetin can reduce polyp number and size distribution in the Apc(Min/+) mouse and that these effects may be related to a reduction in macrophage infiltration.

HASPIN kinase inhibitor CHR-6494 suppresses intestinal polyp development, cachexia, and hypogonadism in Apcmin/+ mice.

HASPIN has been identified as a nuclear Ser/Thr kinase specifically expressed in haploid germ cells. HASPIN kinase inhibitors were recently isolated, and their antitumor activity reported. Colorectal cancer occurs with high incidence worldwide. In this study, we examined whether HASPIN inhibitor CHR-6494 suppresses cancer progression in Apc mice, a familial colon tumor disease model. Mice were treated by intraperitoneal injection of CHR-6494 for 50 days. Following the treatment period, intestinal polyps were counted and testosterone and spermatogenesis levels were observed. Intraperitoneal administration of CHR-6494 significantly inhibited intestinal polyp development and recovered body weight in Apc mice. Although spermatogenesis was inhibited with increasing age in Apc mice, CHR-6494 significantly improved blood testosterone levels and spermatogenesis. Our results suggest that HASPIN inhibitors may be useful as anti-cancer agents and for the treatment of hypogonadism in colorectal cancer patients.

Targeting cyclooxygenase-2 and the epidermal growth factor receptor for the prevention and treatment of intestinal cancer.

Clinical and animal studies indicate a role for cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) in the development and progression of intestinal polyps and cancers. Although this combination of enzyme inhibition has shown synergy in intestinal polyp and tumor models, the exact mechanism for these effects remains undefined. Therefore, we sought to define the molecular mechanisms through which this process occurs. We observed a significant reduction in the number and size of small intestinal polyps in APC(min+/-) mice treated with either celecoxib (a selective COX-2 inhibitor) or erlotinib (Tarceva, an EGFR inhibitor). However, in combination, there was an overall prevention in the formation of polyps by over 96%. Furthermore, we observed a 70% reduction of colorectal xenograft tumors in mice treated with the combination and microarray analysis revealed genes involved in cell cycle progression were negatively regulated. Although we did not observe significant changes in mRNAs of genes with known apoptotic function, there was a significant increase of apoptosis in tumors from animals treated with the combination. The inhibition of EGFR also induced the down-regulation of COX-2 and further inhibited prostaglandin E2 formation. We observed similar effects on the prevention of intestinal adenomas and reduction of xenograft tumor volume when nonselective COX inhibitors were used in combination with erlotinib. Together, these findings suggest that the inhibition of both COX-2 and EGFR may provide a better therapeutic strategy than either single agent through a combination of decreased cellular proliferation and prostaglandin signaling as well as increased apoptosis.

In vivo fluorescence diagnostics and photodynamic therapy of gastrointestinal superficial polyps with aminolevulinic acid. A clinical and spectroscopic study.

In the present study initial results of clinical study related to the treatment of patients having different types of precancerous lesions in the area of esophagus, stomach and intestine by photodynamic therapy (PDT) based on aminolevulinic acid (ALA-PDT) are reported. The procedure was performed by laser fibre system with the light guides introduced through biopsy channel of an endoscope. In addition, in vivo fluorescent diagnostics and spectral analyses of biopsies were performed. Each patient had a positive response to therapy. In two cases there was a total response and in other five cases more than sixty percent of suspected area was removed. Additionally, sigilocellular carcinoma of stomach was revealed in one case. It appears from the results of this study, that the treatment of precancerous lesions with ALA-PDT could be successful treatment modality.

Chemopreventive effects of dietary folate on intestinal polyps in Apc+/-Msh2-/- mice.

Epidemiological and animal studies (reviewed in Y. I. Kim, J. Nutr. Biochemistry, 10: 66-88, 1999; J. B. Mason and T. Levesque, Oncology, 10: 1727-1743, 1996) suggest that dietary folate intake is inversely related to the risk of colorectal cancer. However, the optimal timing of folate intervention and mechanisms by which folate modulates colorectal carcinogenesis have not been clearly established. A recently developed murine model of intestinal tumorigenesis, which carries a heterozygous mutation in the Apc gene and a null mutation in the Msh2 gene (Apc+/-Msh2-/-), was used to determine the effect of dietary folate on intestinal tumorigenesis. Apc+/- Msh2-/- mice were randomized to receive either 0 or 8 mg of folate/kg diet starting at either 3 or 6 weeks of age. The 3- and 6-week diet starts represent intervention before and after the establishment of neoplastic foci, respectively. At 11 weeks of age, mice were killed, and the small intestines and colons were analyzed for adenomas and aberrant crypt foci (ACF). Serum folate concentrations were determined by a standard microbiological assay. Genomic DNA methylation was assessed by in vitro [3H]methyl incorporation into hepatic DNA and by a methyl-sensitive restriction digestion method. Microsatellite instability was determined in matched normal and polyp DNA from the small intestine and colon at 5 loci. Serum folate concentrations accurately reflected dietary folate levels (P < 0.005). Folate supplementation, started before the establishment of neoplastic foci, significantly decreased the number of small intestinal adenomas (by 2.7-fold; P = 0.004) and colonic ACF (by 2.8-fold; P = 0.028) and colonic adenomas (by 2.8-fold; P = 0.1) compared with a moderate degree of folate deficiency. In contrast, a moderately folate-deficient diet, started after the establishment of neoplastic foci, significantly reduced the number of small intestinal adenomas (by 4.2-fold; P = 0.001) but had no effect on colonic ACF and adenomas compared with folate supplementation. Genomic DNA methylation and microsatellite instability do not seem to play a major role in folate-modulated intestinal and colonic tumorigenesis in this model. In conclusion, in this murine model, dietary folate supplementation significantly protects against small intestinal and colorectal tumorigenesis if it is provided before the establishment of neoplastic foci However, if it is provided after the establishment of neoplastic foci, dietary folate seems to have an opposite effect. These data suggest that the timing of folate intervention is critical in providing an effective and safe chemopreventive effect on intestinal tumorigenesis. Notwithstanding the limitations associated with this model, our data suggest that the optimal timing of folate intervention must be established before folate supplementation can be used as a safe chemopreventive agent against colorectal cancer.

Suppression of intestinal polyposis in Mdr1-deficient ApcMin/+ mice.

Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor complex has been considered crucial for the initiation of intestinal tumorigenesis. The human multidrug resistance (MDR)1 (ABCB1) gene contains multiple beta-catenin-T-cell factor4-binding elements in its promoter and is one of the immediate targets of the complex. In the current study, we have further substantiated the biological involvement of MDR1 in intestinal tumorigenesis based on the following evidence: (a) aberrant induction of the Mdr1a (Abcb1a) gene product, P-glycoprotein, associated with nuclear accumulation of the beta-catenin protein, was observed even in nascent microscopic adenomas of Min mice; (b) Mdr1-deficient Min (Apc(Min/+)Mdr1a/b(-/-)) mice developed significantly fewer intestinal polyps than did Apc(Min/+)Mdr1a/b(+/+) mice; and (c) Inhibitors of P-glycoprotein, verapamil, and cyclosporin A had a suppressive effect on the in vitro polypoid growth of IEC6 expressing stabilized (DeltaN89) beta-catenin protein. Inhibitors of P-glycoprotein may be included in a novel class of chemopreventive agents against colorectal carcinogenesis.