The prognostic impact of consensus molecular subtypes (CMS) and its predictive effects for bevacizumab benefit in metastatic colorectal cancer: molecular analysis of the AGITG MAX clinical trial.

Background: The consensus molecular subtypes (CMS) is a transcriptome-based classification of colorectal cancer (CRC) initially described in early-stage cohorts, but the associations of CMS with treatment outcomes in the metastatic setting are yet to be established. This study aimed to evaluate the prognostic impact of CMS classification and its predictive effects for bevacizumab benefit in metastatic CRC by correlative analysis of the AGITG MAX trial. Patients and methods: The MAX trial previously reported improved progression-free survival (PFS) for the addition of bevacizumab (B) to chemotherapy [capecitabine (C)±mitomycin (M)]. Archival primary tumours from 237 patients (50% of trial population) underwent gene expression profiling and classification into CMS groups. CMS groups were correlated to PFS and overall survival (OS). The interaction of CMS with treatment was assessed by proportional hazards model. Results: The distribution of CMS in MAX were CMS1 18%, CMS2 47%, CMS3 12%, CMS4 23%. CMS1 was the predominant subtype in right-sided primary tumours, while CMS2 was the predominant subtype in left-sided. CMS was prognostic of OS (P = 0.008), with CMS2 associated with the best outcome and CMS1 the worst. CMS remained an independent prognostic factor in a multivariate analysis. There was a significant interaction between CMS and treatment (P-interaction = 0.03), for PFS, with hazard ratios (95% CI) for CB+CBM versus C arms in CMS1, 2, 3 and 4: 0.83 (0.43-1.62), 0.50 (0.33-0.76), 0.31 (0.13-0.75) and 1.24 (0.68-2.25), respectively. Conclusions: This exploratory study found that CMS stratified OS outcomes in metastatic CRC regardless of first-line treatment, with prognostic effects of CMS groups distinct from those previously reported in early-stage cohorts. In CMS associations with treatment, CMS2 and possibly CMS3 tumours may preferentially benefit from the addition of bevacizumab to first-line capecitabine-based chemotherapy, compared with other CMS groups. Validation of these findings in additional cohorts is warranted. Clinical trial number: This is a molecular sub-study of MAX clinical trial (NCT00294359).

Vascular endothelial growth factor D expression is a potential biomarker of bevacizumab benefit in colorectal cancer.

BACKGROUND: Bevacizumab prolongs progression-free survival (PFS) in patients with metastatic colorectal cancer. We analysed the protein expression levels of vascular endothelial growth factor (VEGF) ligands and receptors to determine their prognostic and predictive effects. METHODS: We graded expression of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-R1, and VEGF-R2 to assess whether overexpression predicted bevacizumab resistance in samples from 268 of 471 patients randomised to capecitabine (C), capecitabine and bevacizumab (CB), or CB and mitomycin (CBM) in the MAX trial and extended the analysis to the CAIRO-2 population. RESULTS: Patients with low expression of VEGF-D (0, 1þ) benefited from bevacizumab treatment (PFS hazard ratio (HR) (C vs CBþCBM), 0.21; 95% CI, 0.08­0.55; overall survival (OS) HR, 0.35; 95% CI, 0.13­0.90). Patients with higher VEGF-D expression received less benefit (VEGF-D 2þ PFS HR, 0.67; 95% CI, 0.45­1.00; OS HR, 0.82; 95% CI, 0.52­1.30; VEGF-D 3þ PFS HR, 0.77; 95% CI, 0.50­1.17; OS HR, 1.28; 95% CI, 0.79­2.09) (P interaction o0.05). In CAIRO-2, there was no difference in PFS or OS according to VEGF-D expression. CONCLUSIONS: The predictive value of VEGF-D expression for bevacizumab may depend on the chemotherapy backbone used. Further evaluation is required before clinical utilisation.

Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer.

BACKGROUND: Telomeres are TTAGGG tandem repeats capping chromosomal ends and partially controlled by the telomerase enzyme. The EGFR pathway putatively regulates telomerase function, prompting an investigation of telomere length (TL) and its association with anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). METHODS: Colorectal cancer cell lines were treated with multiple drugs and sensitivity determined. Clinical information was gathered from 75 patients who had received anti-EGFR drugs. Telomere length was measured using a validated qRT-PCR technique. RESULTS: In CRC cell lines, TL independently predicted cetuximab sensitivity. Cells with shorter TL had growth inhibition of 18.6±3.41% as compared with 41.39±8.58% in longer TL (P=0.02). These in vitro findings were validated clinically, in a robust multivariate model. Among patients with KRas WT tumours, those with longer TL had a superior median progression-free survival (PFS) of 24.9 weeks than those with shorter TL; median 11.1 weeks, HR 0.31; P=0.048. CONCLUSION: Telomere length could be a potential unique biomarker predictive of clinical benefit (PFS) of mCRC patients treated with anti-EGFR therapy. This is the novel demonstration of a complex hitherto undescribed interaction, placing anti-EGFR therapy, EGFR pathway, and the telomerase complex within a clinical context.

PR55α-containing protein phosphatase 2A complexes promote cancer cell migration and invasion through regulation of AP-1 transcriptional activity.

The proto-oncogene c-Jun is a component of activator protein-1 (AP-1) transcription factor complexes that regulates processes essential for embryonic development, tissue homeostasis and malignant transformation. Induction of gene expression by c-Jun involves stimulation of its transactivation ability and upregulation of DNA binding capacity. While it is well established that the former requires JNK-mediated phosphorylation of S63/S73, the mechanism(s) through which binding of c-Jun to its endogenous target genes is regulated remains poorly characterized. Here we show that interaction of c-Jun with chromatin is positively regulated by protein phosphatase 2A (PP2A) complexes targeted to c-Jun by the PR55α regulatory subunit. PR55α-PP2A specifically dephosphorylates T239 of c-Jun, promoting its binding to genes regulating tumour cell migration and invasion. PR55α-PP2A also enhanced transcription of these genes, without affecting phosphorylation of c-Jun on S63. These findings suggest a critical role for interplay between JNK and PP2A pathways determining the functional activity of c-Jun/AP-1 in tumour cells.

ß-catenin negatively regulates expression of the prostaglandin transporter PGT in the normal intestinal epithelium and colorectal tumour cells: a role in the chemopreventive efficacy of aspirin?

BACKGROUND: Levels of the pro-tumorigenic prostaglandin PGE(2) are increased in colorectal cancer, previously attributed to increased synthesis through COX-2 upregulation and, more recently, to decreased catabolism. The functionally linked genes 15-prostaglandin dehydrogenase (15-PGDH) and the prostaglandin transporter PGT co-operate in prostaglandin degradation and are downregulated in colorectal cancer. We previously reported repression of 15-PGDH expression by the Wnt/ß-catenin pathway, commonly deregulated during early colorectal neoplasia. Here we asked whether ß-catenin also regulates PGT expression. METHODS: The effect of ß-catenin deletion in vivo was addressed by PGT immunostaining of ß-catenin(-/lox)-villin-cre-ERT2 mouse tissue. The effect of siRNA-mediated ß-catenin knockdown and dnTCF4 induction in vitro was addressed by semi-quantitative and quantitative real-time RT-PCR and immunoblotting. RESULTS: This study shows for the first time that deletion of ß-catenin in murine intestinal epithelium in vivo upregulates PGT protein, especially in the crypt epithelium. Furthermore, ß-catenin knockdown in vitro increases PGT expression in both colorectal adenoma- and carcinoma-derived cell lines, as does dnTCF4 induction in LS174T cells. CONCLUSIONS: These data suggest that ß-catenin employs a two-pronged approach to inhibiting prostaglandin turnover during colorectal neoplasia by repressing PGT expression in addition to 15-PGDH. Furthermore, our data highlight a potential mechanism that may contribute to the non-selective NSAID aspirin's chemopreventive efficacy.

A 19S proteasomal subunit cooperates with an ERK MAPK-regulated degron to regulate accumulation of Fra-1 in tumour cells.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms-association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS-ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS-ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing.

Heterogeneity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling.

Inhibition of Notch signaling is effective in inhibiting colon tumorigenesis, but targeting specific components of the pathway may provide more effective strategies. Here we show that the expression of Jagged1, a ligand for canonical Notch signaling, was restricted to enteroendocrine cells or undetectable in the mucosa of the human small and large intestine, respectively. In contrast, increased expression characterized half of human colon tumors, although not all tumors with elevated Wnt signaling displayed elevated Jagged1. Increased Jagged1 was also present in intestinal tumors of Apc(1638N/+) and Apc(Min/+) mice, but to a higher level and more frequently in the former, and in 90% of mouse tumors Notch signaling was elevated when Jagged1 was elevated. In the human HT29Cl16E colonic carcinoma cell line, induction of goblet cell differentiation by contact inhibition of growth depended on the loss of Jagged1-mediated Notch activation, with signaling through Notch1 and Notch2 acting redundantly. Therefore, targeting of Jagged1 could be effective in downregulating Notch signaling in a subset of tumors, but may avoid the limiting gastrointestinal toxicity caused by pharmacological inhibition of Notch signaling.

Molecular mechanisms of action and prediction of response to oxaliplatin in colorectal cancer cells.

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin.

c-Myc overexpression sensitises colon cancer cells to camptothecin-induced apoptosis.

The proto-oncogene c-Myc is overexpressed in 70% of colorectal tumours and can modulate proliferation and apoptosis after cytotoxic insult. Using an isogenic cell system, we demonstrate that c-Myc overexpression in colon carcinoma LoVo cells resulted in sensitisation to camptothecin-induced apoptosis, thus identifying c-Myc as a potential marker predicting response of colorectal tumour cells to camptothecin. Both camptothecin exposure and c-Myc overexpression in LoVo cells resulted in elevation of p53 protein levels, suggesting a role of p53 in the c-Myc-imposed sensitisation to the apoptotic effects of camptothecin. This was confirmed by the ability of PFT-alpha, a specific inhibitor of p53, to attenuate camptothecin-induced apoptosis. p53 can induce the expression of p21(Waf1/Cip1), an antiproliferative protein that can facilitate DNA repair and drug resistance. Importantly, although camptothecin treatment markedly increased p21(Waf1/Cip1) levels in parental LoVo cells, this effect was abrogated in c-Myc-overexpressing derivatives. Targeted inactivation of p21(Waf1/Cip1) in HCT116 colon cancer cells resulted in significantly increased levels of apoptosis following treatment with camptothecin, demonstrating the importance of p21(Waf1/Cip1) in the response to this agent. Finally, cDNA microarray analysis was used to identify genes that are modulated in expression by c-Myc upregulation that could serve as additional markers predicting response to camptothecin. Thirty-four sequences were altered in expression over four-fold in two isogenic c-Myc-overexpressing clones compared to parental LoVo cells. Moreover, the expression of 10 of these genes was confirmed to be significantly correlated with response to camptothecin in a panel of 30 colorectal cancer cell lines.

Down-regulation of beta-catenin TCF signaling is linked to colonic epithelial cell differentiation.

The beta-catenin TCF pathway is implicated in the regulation of colonic epithelial cell proliferation, but its role in the regulation of cell differentiation is unknown. The colon carcinoma cell line, Caco-2, spontaneously undergoes G(0)/G(1) cell cycle arrest and differentiates along the absorptive cell lineage over 21 days in culture. In parallel, we show that beta-catenin-TCF activity and complex formation are significantly down-regulated. The down-regulation of beta-catenin-TCF signaling was independent of APC, which we characterized as having a nonsense mutation in codon 1367 in Caco-2 cells, but was associated with a decrease in TCF-4 protein levels. Total beta-catenin levels increased during Caco-2 cell differentiation, although this was attributable to an increase in the membrane, E-cadherin-associated, fraction of beta-catenin. Importantly, down-regulation of beta-catenin-TCF signaling in undifferentiated Caco-2 cells by three different mechanisms, ectopic expression of E-cadherin, wild-type APC, or dominant negative TCF-4, resulted in an increase in the promoter activities of two genes that are well-established markers of cell differentiation, alkaline phosphatase and intestinal fatty acid binding protein. These studies demonstrate, therefore, that in addition to its established role in the regulation of cell proliferation, down-regulation of the beta-catenin-TCF pathway is associated with the promotion of a more-differentiated phenotype in colonic epithelial cells.

Resistance to butyrate-induced cell differentiation and apoptosis during spontaneous Caco-2 cell differentiation.

BACKGROUND & AIMS: The short-chain fatty acid butyrate induces cell cycle arrest, differentiation, and apoptosis in colon cancer cells, but often induces opposite effects in normal colonic epithelial cells. We determined whether response to butyrate is dependent on the basal differentiation status of colonic epithelial cells. METHODS: Caco-2 cells at progressive stages of differentiation were treated with butyrate, and endpoints were measured. RESULTS: Response of Caco-2 cells to butyrate was dependent on their differentiation status. Butyrate maximally stimulated cell cycle arrest, apoptosis, alkaline phosphatase activity, transepithelial resistance, cell migration, urokinase receptor expression, and interleukin 8 secretion in undifferentiated Caco-2 cells, whereas differentiated Caco-2 cells were essentially resistant to these effects. Consistently, butyrate selectively induced histone hyperacetylation in undifferentiated Caco-2 cells. This resistance was also observed during HT29cl.19A cell differentiation, but not in the nondifferentiating SW620 cell line. Finally, the rate of butyrate use significantly increased as Caco-2 cells underwent spontaneous differentiation. CONCLUSIONS: Colonic epithelial cells become progressively more refractory to the effects of butyrate during absorptive cell differentiation. We postulate that this resistance is caused by the rapid use of butyrate by differentiated Caco-2 cells, which likely results in low intracellular concentrations and subsequently in its inability to inhibit histone deacetylase.

Dissociation of staurosporine-induced apoptosis from G2-M arrest in SW620 human colonic carcinoma cells: initiation of the apoptotic cascade is associated with elevation of the mitochondrial membrane potential (deltapsim).

We have identified an alternative apoptotic cascade induced in SW620 human colonic carcinoma cells by the protein kinase antagonist staurosporine (stsp). Consistent with its effect in other colonic epithelial cells, stsp induced G2-M arrest and apoptosis of SW620 cells. However, despite the paradigm that growth arrest triggers apoptotic cascades, apoptosis was detected before G2-M arrest. Reports have linked dissipation of the mitochondrial membrane potential (deltapsim) to the initiation of apoptosis and have linked elevation of the deltapsim to the escape from apoptosis However, neither apoptosis nor cell cycle arrest were altered by the collapse of the deltapsim, and increased deltapsim enhanced the initiation of apoptosis but blocked G2-M arrest. Although reactive oxygen species (ROS) have been implicated in some colonic epithelial cell and stsp-induced cascades, neither antioxidants nor the inhibition of RNA or protein synthesis altered apoptosis of SW620 cells. Finally, cytosolic cytochrome c has been linked to activation of caspase-3 and dissipation of the deltapsim. However, caspase-3 activation preceded the accumulation of cytochrome c in the cytosol and was accompanied by transient elevations in both the deltapsim and mitochondria-associated cytochrome c. Therefore, we have identified a distinct apoptotic cascade in SW620 cells that was induced independently of growth arrest, dissipation of the deltapsim, ROS production, or synthesis of de novo RNA or protein, and we have linked its efficient initiation to early elevation of the deltapsim.

Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer.

The short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, is a physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063 sequences demonstrated a complex cascade of reprogramming of SW620 colonic epithelial cells upon treatment with butyrate characterized by the progressive recruitment of gene sets as a function of time. Comparison with the effects of trichostatin A, in conjunction with differences in the kinetics of alteration of histone acetylation induced by butyrate and trichostatin A, identified subsets of induced and repressed genes likely coordinately regulated by altered histone acetylation. The butyrate response was also compared in detail with that of sulindac, a nonsteroidal anti-inflammatory drug with significant chemopreventive activity for colon cancer, and curcumin, a component of mustard and curry structurally and functionally related to sulindac that also has chemopreventive activity. Although gene clusters were identified that showed similar responses to butyrate and sulindac, the data were characterized by the extensive differences in the effects of the two agents. This was striking for functional classes of genes involved in signaling pathways and in cell cycle progression, although butyrate and sulindac induce a similar G0-G1 arrest, elevation of beta-catenin-Tcf signaling, and apoptotic cascade. As regards cell cycle arrest, the underlying mechanism in response to butyrate was most similar to that of the Caco-2 cell line that had spontaneously undergone a G0-G1 arrest and least similar to the G2-M arrest stimulated by curcumin. Thus, high-throughput microarray analysis of gene expression profiles can be used to characterize and distinguish the mechanisms of response of colonic epithelial cells to physiological and pharmacological inducers of cell maturation. This has important implications for characterization of chemopreventive agents and recognition of potential toxicity and synergies. The data bases, gene clusters, and analyses are available at http:// sequence.aecom.yu.edu/genome/.

Divergent phenotypic patterns and commitment to apoptosis of Caco-2 cells during spontaneous and butyrate-induced differentiation.

Caco-2 cells differentiate spontaneously when cultured in confluence and on exposure to the physiologically relevant short-chain fatty acid, butyrate. This study aimed to compare the phenotype induced by these pathways and their relations to cell turnover. Caco-2 cells were treated with butyrate at a nontoxic concentration of 2 mM for 3 days, or allowed to spontaneously differentiate for 0-21 days. Brush border hydrolase activities and carcinoembryonic antigen (CEA) expression, transepithelial resistance and dome formation, expression of components of the urokinase system, and cell turnover by flow cytometry, and the degree of DNA fragmentation were quantified. Butyrate induced increases in alkaline phosphatase activity and CEA expression but not the activities of other hydrolases, while culture alone induced progressive increases in the activities/expression of all markers. Butyrate induced a significantly greater increase in transepithelial resistance (TER) than occurred during culture alone but the densities of domes were similar. Butyrate induced a ninefold increase in urokinase receptor expression and twofold increase in urokinase activity, while culture alone induced a significantly smaller increase in receptor expression, an increase in plasminogen activator inhibitor-1 but no change in activity. While both stimuli induced cell cycle arrest, only butyrate increased the proportion of cells undergoing apoptosis. In conclusion, differentiation of Caco-2 cells can proceed along multiple pathways but does not necessarily lead to apoptosis. The phenotypic changes during spontaneous differentiation mimic those that occur in normal colonic epithelial cells in vivo during their migration from the crypt base to neck, while butyrate-induced effects more closely follow those occurring when normal colonic epithelial cells migrate from crypt neck to the surface compartment.

Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells.

LIM1215 colon cancer cells were used as a model of human colonic epithelium to examine the effects of butyrate on protein kinase C (PKC) activity and isoform expression. On Western blot analysis, LIM1215 cells express the PKC isoforms alpha, beta, epsilon, zeta, and lambda, but not gamma, straight theta, or micro. Treatment with 2 mM butyrate for 48 h reduced cellular PKC activity up to 50% and specifically reduced the expression of PKC alpha and PKC epsilon. Similar results were obtained using Caco-2 colon cancer cells. These effects were neither a consequence of the induction of differentiation itself nor the result of direct or indirect activation of PKC. Although dependent on gene transcription and protein synthesis, the effect was not due to a reduction in the synthesis of PKC protein. Butyrate's effect was independent of its beta-oxidation but was mimicked, at least in part, by trichostatin A, an inhibitor of histone deacetylase.

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Relationship of hydrolase activities to epithelial cell turnover in distal colonic mucosa of normal rats.

BACKGROUND: The relationships between changes induced by diet in colonic epithelial kinetics and in the activities of brush border hydrolases are poorly defined. The aims of this study are to define these relationships, as changes in kinetics would be expected to influence differentiation, and to determine whether the type of ingested dietary indigestible carbohydrates influences hydrolase activities. METHODS: Groups of eight rats were fed a low fibre diet +/- supplements of different types of indigestible carbohydrates for 4 weeks. Alkaline phosphatase (ALP) and dipeptidyl peptidase IV (DPPIV) activities and epithelial kinetics were measured in distal colonic mucosa. RESULTS: Median ALP activities correlated positively and DPPIV activity negatively with the median proportion of cells entering metaphase (r = 0.58 and -0.58, respectively; P < 0.05) and number of metaphase arrests per crypt column across the diets (r = 0.59 and 0.58, respectively; P < 0.05). Stepwise regression analysis showed that both hydrolases independently predicted these kinetic indices (R2 > 63% for each). Mucosal ALP activities were markedly elevated during consumption of raw potato starch, guar gum and methylcellulose, while only potato starch caused a significant elevation of DPPIV activities. CONCLUSIONS: The type of indigestible carbohydrate in the diet influences colonic mucosal hydrolase activities. The opposite relationship between kinetics and each of the two hydrolases indicates that these hydrolases do not reflect the same event; dipeptidyl peptidase IV might relate to differentiation status while ALP could also be influenced by epithelial irritation due to changes in luminal conditions.

Effect of butyrate on paracellular permeability in rat distal colonic mucosa ex vivo.

BACKGROUND AND AIMS: The effects of butyrate on colonic epithelial barrier function are poorly understood. The aim of this study was to examine the short-term effects of butyrate on paracellular permeability of rat distal colonic epithelium. METHODS: Mucosa mounted in Ussing chambers was treated with butyrate (1-10 mmol/L) for 4 h. Transepithelial conductance, [51Cr]-EDTA flux, mucosal brush border hydrolase activity and epithelial kinetics, using proliferating cell nuclear antigen (PCNA) staining, were measured. RESULTS: On exposure to butyrate (10 mmol/L, but not 1 or 5 mmol/L), transepithelial conductance was 65 +/- 2% higher (mean +/- SEM; n = 8, P < 0.05, paired t-test) and the rate coefficient for [51Cr]-EDTA flux was 65 +/- 25% higher (P = 0.03) than those of control tissue. Histologically, the epithelium exhibited no signs of injury, but butyrate-treated tissue exhibited interstitial oedema consistent with water uptake in association with butyrate absorption. Butyrate caused a reduction in crypt column height to 30.6 +/- 1.6 cells from 33.4 +/- 1.8 cells in controls (n = 10, P < 0.03), but the number of cells per crypt column staining with PCNA was unchanged. Butyrate significantly reduced the mucosal activities of alkaline phosphatase by 40 +/- 16%, maltase by 54 +/- 12% and dipeptidyl peptidase IV by 41 +/- 14%. CONCLUSIONS: Acute exposure to butyrate increased paracellular permeability in rat distal colon. The mechanism involved may relate to the loss of differentiated surface epithelial cells, or as a physiological response to Na+-coupled butyrate uptake.

Colonic epithelial cell activation and the paradoxical effects of butyrate.

Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism that dictates a cell's response to butyrate is its state of activation. First, the responses to 24 h exposure to butyrate (1-2 mM) of normal and neoplastic human colonic epithelial cells activated by their isolation and primary culture, and of colon cancer cell lines, LIM1215 and Caco-2, were examined. In primary cultures of normal and cancer cells, butyrate had no effect on alkaline phosphatase activities but significantly suppressed urokinase receptor expression by a mean +/- SEM of 30 +/- 12% and 36 +/- 9%, respectively. Interleukin-8 secretion was suppressed by 44 +/- 7% in normal cells (P < 0.05) but was unchanged in cancer cells. In contrast, the cell lines significantly increased alkaline phosphatase activities by >50%, urokinase receptor expression >2-fold and interleukin-8 secretion >3-fold in response to butyrate. Secondly, the effect of butyrate on Caco-2 cells was examined with or without prior exposure to a specific activating stimulus [tumour necrosis factor alpha (TNF alpha)]. Interleukin-8 secretion increased by 145 +/- 23% and 132 +/- 17% on 24 h exposure to 2 mM butyrate or 0.1 microM TNF alpha alone, respectively. However, in cells pre-treated with TNF alpha, butyrate significantly inhibited secretion by 34 +/- 7% below unstimulated levels. The response to butyrate of urokinase receptor, whose expression was not stimulated by TNF alpha, was unchanged. These effects were mimicked by trichostatin A, an inhibitor of histone deacetylase, suggesting that butyrate's paradoxical effects may have been operating by the same mechanism. In conclusion, some of the paradoxical effects of butyrate do not appear to represent inherent differences between normal and transformed cells. Rather, the response may be determined by the state of activation of the cells.