Estrogen receptor-ß protects against colitis-associated neoplasia in mice.

Estrogen receptor-beta (ERß) has been suggested to exert anti-inflammatory and anti-tumorigenic effects in the colon, providing a translational potential to prevent and/or treat inflammatory bowel disease (IBD) and its progression to colitis-associated colorectal cancer (CAC). However, the specific direct role of ERß in CAC has not yet been tested. We assessed the effects of ERß deficiency in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model using ERß knockout (ßERKO) mice and wild-type (WT) littermates. These mice were injected with AOM followed by 1 week of DSS treatment, and sacrificed on weeks 9 or 16. ßERKO mice developed more severe clinical colitis compared to WT mice, as evidenced by significantly higher disease activity index after DSS treatment, weight to length ratio of the colons, inflammation score and grade of dysplasia. ERß-deficient colons presented greater number and size of polyps at weeks 9 and 16, respectively, and were characterized by a significant increase in interleukin (IL)-6, IL-17, tumor necrosis factor alpha and interferon-gamma mRNA levels. Furthermore, higher protein expression levels of nuclear factor-kappa B, inducible nitric oxide synthase, ß-catenin, proliferating cell nuclear antigen, mucin-1 and significantly lower caveolin-1 and mucin-2 protein levels were shown in ßERKO mice compared to WT mice. These data suggest a possible anti-inflammatory and anti-neoplastic mechanism of action of ERß in CAC. These results demonstrate for the first time that ERß provides protection in the AOM/DSS-induced CAC model in mice, suggesting a preventive and/or therapeutic potential for the use of ERß-selective agonists in IBD.

Gastrin-releasing peptide signaling alters colon cancer invasiveness via heterochromatin protein 1Hsß.

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in colon cancer where they are associated with improved patient survival rates. However, the mechanism of action whereby these proteins mediate their beneficial effects is not known. Heterochromatin protein 1 is an epigenetic modifier of gene transcription for which three different isoforms exist in humans: HP1(Hsα), HP1(Hsß), and HP1(Hsγ). In breast cancer and melanoma, respectively, HP1(Hsα) and HP1(Hsß) have been shown to modulate the aggressiveness of tumor cells in vivo. In contrast, the role of HP1 in colon cancer has not been elucidated, and a mechanism of regulating the expression of any HP1 isoform in any context has not yet been identified. In this article we demonstrate that abrogating GRP/GRPR signaling specifically down-regulates HP1(Hsß) expression and that inhibiting GRPR signaling, or ablating HP1(Hsß) expression, increases colon cancer cell invasiveness in vitro. These findings identify for the first time a signaling pathway regulating heterochromatin protein expression and suggest a mechanism whereby aberrantly expressed GRPR might alter the outcome of patients with colorectal cancer.

Rotavirus infection of murine small intestine causes colonic secretion via age restricted galanin-1 receptor expression.

BACKGROUND & AIMS: Mechanisms for age restriction of rotavirus diarrhea are unclear. Because rotavirus primarily infects small intestine, colonic contribution has not been widely studied. Recent data suggest that colonic secretion postbacterial infection is mediated by galanin-1 receptors (Gal1-R). We evaluated age-dependent expression of Gal1-R in Rhesus rotavirus (RRV)-infected mice and its contribution to fluid secretion. METHODS: Twenty-four hours after infection of C57BL/6J mice (wild type or Gal1-R knockout) with RRV or vehicle, closed small intestinal and colon loops were constructed. Net fluid content of the loops was calculated (milligrams/centimeters) at 2 hours post-treatment with galanin, galanin antibody, or lidocaine. Gal1-R expression was quantified by automated chromogen analysis. RESULTS: Viral antigen was detected in small intestinal epithelial cells but not in colon. Developmental Gal1-R was widely expressed in the small intestine but minimally in the colon. Postinfection, markedly increased Gal1-R was seen in the colon but not after day 25. Galanin caused a significantly higher increase in the net fluid content of infected colon than small intestine. Treatment with lidocaine reduced net fluid secretion in the small intestine and the colon. Mean diarrheal scores were significantly reduced in Gal1-R knockout mice compared with wild type (1.19 +/- 0.31, n = 22 vs 3.36 +/- 0.50, n = 35, P = .0001). CONCLUSIONS: These data show that RRV infection of the small intestine increases colonic secretion through Gal1-R and provide a promising start toward understanding the age restriction of rotavirus diarrhea.

Lubiprostone activates Cl- secretion via cAMP signaling and increases membrane CFTR in the human colon carcinoma cell line, T84.

BACKGROUND: Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. AIM: To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. METHODS: Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. RESULTS: Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. CONCLUSIONS: Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

Aberrant crypt focus size predicts distal polyp histopathology.

Aberrant crypt foci (ACF) are the earliest histopathologic lesion associated with colorectal cancer. ACFs are commonly used as a surrogate marker for colorectal cancer chemoprevention studies in rodents and, more recently, in humans. However, ACF prevalence in unselected populations is not known, nor which ACF features are important for predicting polyp histopathology. To address these questions, we did magnification chromo-colonoscopy on all patients undergoing routine colorectal cancer screening over a 31-month period. ACFs were classified by location, size (small, <20 crypts/ACF; medium, 20-100 crypts/ACF; large, >100 crypts/ACF), and whether they were elevated above the tissue plane. Overall, 802 magnification chromo-colonoscopies with ACF enumeration were done. Mean patient age was 58.6 +/- 8.5 years, of whom 56% were female, 58% were African American, 21% were Caucasian, and 16% were Latino. Total ACF number, along with increasing ACF size and elevation, correlated with the presence of distal hyperplastic polyps and were higher in African Americans. In contrast, ever-smaller ACFs correlated with the presence of distal adenomas and were independent of age and race. The odds ratio for patients with >or=6 small ACFs and adenomas was 6.02 (95% confidence interval, 2.64-13.70) compared with patients with or=1 large ACF and hyperplastic polyps was 5.88 (95% confidence interval, 3.00-11.67) compared with patients with none. Small flat ACFs correlate with the presence of distal adenomas, whereas large raised ACFs correlate with the presence of hyperplastic polyps.

GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

CYP27A1 and CYP24 expression as a function of malignant transformation in the colon.

Vitamin D deficiency is strongly associated with the risk of developing colorectal cancer (CRC). Because of the propensity of bioactive 1,25-dihydroxyvitamin D3 to cause toxic hypercalcemia, considerable effort has been directed to identifying safer drugs while retaining the efficacy of the parent compound. However, vitamin D precursors do not present toxicity concerns and may be sufficient for CRC chemoprevention or chemotherapy, providing the appropriate enzymes are present in colonic epithelia. We previously showed that CYP27B1 is present at equally high levels in the colon and CRC irrespective of differentiation but was not present in metastases. In this study we used quantitative immunohistochemistry to show that CYP27A1, converting D3 to 25-hydroxycholecalciferol, is present in increasing concentrations in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. Whereas total cellular CYP27A1 remains high in CRC and lymph node metastases, the amount of enzyme present in the nuclei decreases with tumor cell dedifferentiation while rising in the cytoplasm. Similarly, increasing amounts of the deactivating enzyme CYP24 are present in the nuclei of normal colonic epithelia, ACFs, and adenomatous polyps. Although the amount of total CYP24 decreases slightly in CRC as a function of tumor cell dedifferentiation and metastasis, location of this enzyme shifts almost entirely from the nuclear compartment to the cytoplasmic compartment. These data indicate that non-toxic vitamin D precursors should be sufficient for CRC chemoprevention, but that neither vitamin D nor its precursors may be sufficient for CRC chemotherapy.

Chemopreventive efficacy of 25-hydroxyvitamin D3 in colon cancer.

Recently, it has been reported that 25-hydroxyvitamin D3-1alpha-hydroxylase [1alpha(OH)ase, CYP27B1], required to convert non-toxic 25-hyxdroxyvitamin D3 [25(OH)D(3)] to its active metabolite [1alpha,25(OH)(2)D(3)], is present in the epithelial cells of the human colon. In the present study, the potential chemoprotective role of 25(OH)D(3) was evaluated for colon cancer using the HT-29, human colon cancer cell line. Colon cancer cells were treated with 25(OH)D(3) (500nM or 1muM), 1alpha,25(OH)(2)D(3) (500nM), cholecalciferol (D3, 1muM) or vehicle and cell number determined at days 2 and 5 post-treatment. Results showed that both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) induced dose- and time-dependent anti-proliferative effects on the HT-29 cells, with maximum inhibition noted at day 5. Western blot analyses revealed an up-regulation of VDR and 1alpha(OH)ase expression following 24h of treatment with 25(OH)D(3), and 1alpha,25(OH)(2)D(3). These results are consistent with the expression of VDR and 1alpha(OH)ase in samples of normal colonic tissue, aberrant crypt foci (ACFs) and colon adenocarcinomas. The VDR expression was sequentially increased from normal to pre-cancerous lesions to well-differentiated tumors and then decreased in poorly differentiated tumors. Expression of 1alpha(OH)ase was equally expressed in normal, pre-cancerous lesions and malignant human colon tissues. The increased expression of 1alpha(OH)ase in colon cancer cells treated with the pro-hormone and its anti-proliferative effects, suggest that 25(OH)D(3) may offer possible therapeutic and chemopreventive option in colon cancer.

Expression of vitamin D receptor and 25-hydroxyvitamin D3-1{alpha}-hydroxylase in normal and malignant human colon.

Considerable evidence exists to support the use of vitamin D to prevent and/or treat colorectal cancer. However, the routine use of bioactive vitamin D, 1,25-dihydroxyvitamin D3, is limited by the side effect of toxic hypercalcemia. Recent studies, however, suggest that colonic epithelial cells express 25-hydroxyvitamin D3-1alpha-hydroxylase, an enzyme that converts nontoxic pro-vitamin D, 25-hydroxycholecalciferol [25(OH)D3], to its bioactive form. Yet, nothing is known as to the cellular expression of 1alpha-hydroxylase and the vitamin D receptor (VDR) in the earliest histopathologic structures associated with malignant transformation such as aberrant crypt foci (ACF) and polyps [addressing the possibility of using nontoxic 25(OH)D3 for chemoprevention], nor is anything known as to the expression of these proteins in colorectal cancer as a function of tumor cell differentiation or metastasis [relevant to using 25(OH)D3 for chemotherapy]. In this study, we show that 1alpha-hydroxylase is present at equal high levels in normal colonic epithelium as in ACFs, polyps, and colorectal cancer irrespective of tumor cell differentiation. In contrast, VDR levels were low in normal colonic epithelial cells; were increased in ACFs, polyps, and well-differentiated tumor cells; and then declined as a function of tumor cell de-differentiation. Both 1alpha-hydroxylase and VDR levels were negligible in tumor cells metastasizing to regional lymph nodes. Overall, these data support using 25(OH)D3 for colorectal cancer chemoprevention but suggest that pro-vitamin D is less likely to be useful for colorectal cancer chemotherapy.

Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract.

Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.

A phase IIa randomized, double-blind trial of erlotinib in inhibiting epidermal growth factor receptor signaling in aberrant crypt foci of the colorectum.

Colorectal cancer progresses through multiple distinct stages that are potentially amenable to chemopreventative intervention. Epidermal growth factor receptor (EGFR) inhibitors are efficacious in advanced tumors including colorectal cancer. There is significant evidence that EGFR also plays important roles in colorectal cancer initiation, and that EGFR inhibitors block tumorigenesis. We performed a double-blind randomized clinical trial to test whether the EGFR inhibitor erlotinib given for up to 30 days had an acceptable safety and efficacy profile to reduce EGFR signaling biomarkers in colorectal aberrant crypt foci (ACF), a subset of which progress to colorectal cancer, and normal rectal tissue. A total of 45 patients were randomized to one of three erlotinib doses (25, 50, and 100 mg) with randomization stratified by nonsteroidal anti-inflammatory drug (NSAID) use. There were no unanticipated adverse events with erlotinib therapy. Erlotinib was detected in both normal rectal mucosa and ACFs. Colorectal ACF phosphorylated ERK (pERK), phosphorylated EGFR (pEGFR), and total EGFR signaling changes from baseline were modest and there was no dose response. Overall, this trial did not meet is primary efficacy endpoint. Colorectal EGFR signaling inhibition by erlotinib is therefore likely insufficient to merit further studies without additional prescreening stratification or potentially longer duration of use.

Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures receptor number.

We previously demonstrated that quantitative immunohistochemistry (Q-IHC) performed by measuring the cumulative signal strength of the digital file encoding an image can be used to determine the absolute amount of chromogen present per pixel. We now show that Q-IHC so performed can be used to accurately determine the amount of peptide hormone receptor of interest in archived tissues. To do this we transfected Balb 3T3 fibroblasts with the cDNA encoding the human receptor for gastrin-releasing peptide (GRP), and selected six cell lines stably expressing between 10(2) and 10(6) receptors/cell. These cell lines were fixed in formalin, embedded in paraffin, and treated with antipeptide antibodies against the GRP receptor, followed by DAB chromogen to identify bound antibody. Images were acquired using a 4.9 million pixel digital scanning 24-bit RGB camera, saved in TIFF format, and used for subsequent analysis. Q-IHC was performed after digitally dissecting out the relevant portion of the image for analysis, and processing using a program written in C (available at http://www.uic.edu/com/dom/gastro/Freedownloads.html). Under the conditions defined here, chromogen quantity as determined by Q-IHC tightly correlated with GRP receptor number (r(2)=0.867) in these cell lines. Using the conversion factor identified as a result of these studies, we then determined GRP receptor number on eight randomly selected, archived human colon cancers. Overall GRP receptor expression in colon cancer depended on the degree to which cells within any particular tumor were differentiated, with well-differentiated cells expressing the greatest numbers of receptors (approximately 55,000 +/- 10,000 sites/cell). These studies indicate that Q-IHC can be used to determine receptor quantity in archived tissues and other samples of limited quantity.

Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407.

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.

Galanin contributes to the excess colonic fluid secretion observed in dextran sulfate sodium murine colitis.

Galanin is present in enteric nerves lining the gastrointestinal (GI) tract where it is normally involved in regulating intestinal motility by binding to the galanin-1 receptor (Gal1R) subtype expressed by smooth muscle cells. In contrast, although epithelial cells lining the colon do not normally express Gal1R, this protein is up-regulated by the inflammation-associated transcription factor NF-kappaB. We previously showed that the murine colitis induced by dextran sulfate sodium (DSS) was associated with increased Gal1R expression as well as by increased colonic fluid secretion. Although Gal1R up-regulation by colonic epithelial cells results in increased intestinal Cl- secretion, the relative contributions of galanin to this excess colonic fluid secretion could not be determined. We therefore created a mouse genetically incapable of synthesizing Gal1R (GAL1R-/- mice). We herein demonstrate that both wild-type and GAL1R-/- mice developed identical histologic lesions in response to DSS. This was characterized by a marked inflammatory infiltrate, activation of NF-kappaB in both enterocytes and enteric nerves, and a threefold increase in neuronal galanin. Colonic fluid secretion, while increased, was approximately half that in GAL1R-/- mice as compared with their wild-type littermates. Overall, then, these findings strongly suggest that approximately half of the increase in colonic fluid secretion in DSS colitis is due to up-regulation of the Gal1R.

DDB2 suppresses epithelial-to-mesenchymal transition in colon cancer.

Colon cancer is one of the deadliest cancers worldwide because of its metastasis to other essential organs. Metastasis of colon cancer involves a complex set of events, including epithelial-to-mesenchymal transition (EMT) that increases invasiveness of the tumor cells. Here, we show that the xeroderma pigmentosum group E (XPE) gene product, damaged DNA-binding protein (DDB)-2, is downregulated in high-grade colon cancers, and it plays a dominant role in the suppression of EMT of the colon cancer cells. Depletion of DDB2 promotes mesenchymal phenotype, whereas expression of DDB2 promotes epithelial phenotype. DDB2 constitutively represses genes that are the key activators of EMT, indicating that DDB2 is a master regulator of EMT of the colon cancer cells. Moreover, we observed evidence that DDB2 functions as a barrier for EMT induced by hypoxia and TGF-ß. Also, we provide evidence that DDB2 inhibits metastasis of colon cancer. The results presented here identify a transcriptional regulatory pathway of DDB2 that is directly linked to the mechanisms that suppress metastasis of colon cancer.

Regulation of CYP24 splicing by 1,25-dihydroxyvitamin D3 in human colon cancer cells.

CYP24 is a well-established vitamin D receptor (VDR) target gene. The active VDR ligand 1,25(OH)2D3 regulates its own catabolism by increasing CYP24 expression. It is well known that in the presence of 1,25(OH)2D3, VDR binds to VDREs in the promoter region of CYP24 and initiates CYP24 transcription. However, little is known about the role of 1,25(OH)2D3 in the posttranscriptional modulation of CYP24. In this study, we investigated the functional significance of 1,25(OH)2D3 in CYP24 RNA splicing in colon cancer cells. Using RT-PCR, we found that 1,25(OH)2D3 actively induces CYP24 splicing in a time-dependent manner and CYP24 splicing pattern could be cell type or tissue specific. The induction of RNA splicing by 1,25(OH)2D3 was mainly CYP24 selective. Treatment of cells with parathyroid hormone inhibited basal CYP24 splicing, but failed to inhibit 1,25(OH)2D3-induced CYP24 splicing. Further experiments demonstrated that new RNA synthesis was required for the induction of CYP24 splicing by vitamin D. In addition, alteration of multiple signaling pathways also affected CYP24 splicing and cellular sensitivity in response to vitamin D appeared to correlate with the induction of CYP24 splicing. These results suggest that 1,25(OH)2D3 not only regulates CYP24 transcription, but also plays an important role in posttranscriptional modulation of CYP24 by inducing its splicing. Our findings reveal an additional regulatory step that makes the vitamin D mediated action more prompt and efficient.

Identification of ChIP-seq mapped targets of HP1ß due to bombesin/GRP receptor activation.

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in human colorectal cancer (CRC) including Caco-2 cells. We have previously shown that GRPR activation results in the up-regulation of HP1ß, an epigenetic modifier of gene transcription. The aim of this study was to identify the genes whose expression is altered by HP1ß subsequent to GRPR activation. We determined HP1ß binding positions throughout the genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After exposure to GRP, we identified 9,625 genomic positions occupied by HP1ß. We performed gene microarray analysis on Caco-2 cells in the absence and presence of a GRPR specific antagonist as well as siRNA to HP1ß. The expression of 97 genes was altered subsequent to GRPR antagonism, while the expression of 473 genes was altered by HP1ß siRNA exposure. When these data were evaluated in concert with our ChIP-seq findings, 9 genes showed evidence of possible altered expression as a function of GRPR signaling via HP1ß. Of these, genomic PCR of immunoprecipitated chromatin demonstrated that GRPR signaling affected the expression of IL1RAPL2, FAM13A, GBE1, PLK3, and SLCO1B3. These findings provide the first evidence by which GRPR aberrantly expressed in CRC might affect tumor progression.

Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia.

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically.

Randomized phase II trial of sulindac, atorvastatin, and prebiotic dietary fiber for colorectal cancer chemoprevention.

Sulindac, atorvastatin, or prebiotic dietary fiber may reduce colorectal cancer (CRC) risk. However, clinical trial data are currently limited. We conducted a randomized, phase II chemoprevention trial involving subjects 40 years or older, with previously resected colon cancer or multiple/advanced colorectal adenomas. Magnification chromoendoscopy (MCE) was performed to identify and characterize rectal aberrant crypt foci (ACF); eligibility criteria required five or more rectal ACFs at baseline. Intervention assignments were as follows: (a) atorvastatin 20 mg qd; (b) sulindac 150 mg bid; (c) oligofructose-enriched inulin (as ORAFTI®Synergy1) 6 gm bid; or (d) control (maltodextrin) 6 gm bid, for 6 months. Percent change in rectal ACF number (%ΔACF) within arm was the primary endpoint. Secondary endpoints included changes in proliferation (Ki67) and apoptosis (caspase-3), as measured from normal mucosa biopsy samples. Among 85 eligible randomized subjects, 76 (86%) completed the trial per protocol. The median (range) of rectal ACF was 9 (5-34) and 8 (0-37) at baseline and postintervention, respectively. The median (SD) for %ΔACF was 5.6 (-69% to 143%), -18.6 (-83% to 160%), -3.6 (-88% to 83%), and -10.0 (-100% to 117%) in the atorvastatin, sulindac, ORAFTI®Synergy1 and control arms, respectively. Neither within-arm (P = 0.12-0.59) nor between-arm (P = 0.30-0.92) comparisons of %ΔACF were statistically significant. The active and control interventions also seemed to have similar effects on mucosal proliferation and apoptosis (P > 0.05 for each comparison). Data from this multicenter, phase II trial do not provide convincing evidence of CRC risk reduction from 6-month interventions with atorvastatin, sulindac, or ORAFTI®Synergy1, although statistical power was limited by the relatively small sample size.