Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.     

Redistribution of cyclic GMP in response to sodium butyrate in colon cells

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.     

Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5

Butyrate induces differentiation and alters cell proliferation in intestinal-epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium-dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose-free inosine-containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase-IV (DPP-IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS-TC2, BCS-TC2.2, HT-29, and Caco-2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco-2 cells. Inosine-differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco-2 cells. AnxA2 down-regulation is not due to proteasome activation and seems to be related to the butyrate-induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth-state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell-to-cell contacts. Butyrate induces changes in subcellular localization towards a vesicle-associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up-regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up-regulation of AnxA1 could contribute to the reported anti-inflammatory effects of butyrate in colon inflammatory diseases.     

Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP).

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 - 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.     

Effect of butyrate on the expression of the human preprourokinase gene introduced into Chinese hamster ovary cells

A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K1 cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase production. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the other hand, was inhibited by butyrate concentrations greater than 2.5 mM. The response of cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the intracellular level of urokinase.     

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

短链脂肪酸丁酸抑制动脉粥样硬化形成及其分子机制

本研究通过观察丁酸对动脉粥样硬化斑块形成以及肠道组织结构和功能的影响,探讨丁酸防治动脉粥样硬化的效应及可能机制.选取8周龄雄性载脂蛋白E基因敲除(apolipoprotein E-knockout,ApoE-/-)小鼠,随机分成对照组(高脂高胆固醇饲料+饮水中给予200 mmol/L氯化钠,n = 10)和丁酸组(高脂...     

Butyrate- but not TGFbeta1-induced apoptosis of colorectal adenoma cells is associated with increased expression of the differentiation markers E-cadherin and alkaline phosphatase

Sodium butyrate and transforming growth factor beta (TGFbeta1) are growth inhibitory to colonic adenoma cell lines. Butyrate induces apoptosis, whereas in some adenoma cell lines, TGFbeta1 can be growth inhibitory without apoptosis. In this report, we show that the adenoma cell line PC/BH/C1 undergoes apoptosis in response to TGFbeta1. Butyrate induced cell death is preceded by the induction of two markers of colonic differentiation--alkaline phosphatase (ALP) activity and E-cadherin protein expression. However, TGFbeta1-induced apoptosis was not accompanied by induction of these differentiation markers. It is possible that the apoptosis induced by TGFbeta1 in the adenoma cell line PC/BH/C1 is due to conflicting signals, as downregulation of c-myc protein in response to TGFbeta1 occurs only slowly in this cell line. Development of resistance to TGFbeta1 in colonic tumours may involve two separate stages--resistance to growth inhibition and resistance to TGFbeta1-induced apoptosis. Our results indicate that sodium butyrate induces apoptosis via differentiation, but TGFbeta1 induces apoptosis by a differentiation-independent mechanism. As for butyrate, the induction of E-cadherin expression is a potentially important chemopreventative action, since increased E-cadherin expression has been correlated with decreased metastatic potential. This is the first report of induction of E-cadherin by a naturally occurring factor in the diet. Butyrate may reduce tumour growth and invasion, not only as a result of the induction of apoptosis, but also through increased expression of E-cadherin.     

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.     

Butyrate upregulates stromelysin-1 production by intestinal mesenchymal cells

Nutritional factors and resident bacteria participate in the pathogenesis of intestinal inflammation. However, the ways in which bacteria and complex diets might modulate matrix metalloproteinase (MMP) production are unknown. We hypothesized that butyrate might enhance production of MMPs, thus amplifying their response to signals in inflammatory conditions. Human mesenchymal cells were incubated with butyrate and then stimulated with cytokines. MMPs and inhibitors were studied by Western blotting and quantitative RT-PCR. Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE. We showed that butyrate selectively enhanced the protein production and mRNA expression of stromelysin-1 in tumor necrosis factor-alpha- or interleukin-1beta-stimulated mesenchymal cells. Butyrate alone did not induce any change in MMP production or mRNA expression. It increased the acetylation of histones in mesenchymal cells. Furthermore, acetylation of histones (induced by trichostatin A) reproduced the effects of butyrate. Although butyrate is a major source of nutrient for the colonic epithelial cells, it modulates intestinal inflammation through the secretion of stromelysin-1 in stimulated stromal cells via the inhibition of histone deacetylase.     

Expression of 5-lipoxygenase by human colorectal carcinoma Caco-2 cells during butyrate-induced cell differentiation

Butyrate, a short-chain fatty acid, modulates proliferation and differentiation of normal and neoplastic colonocytes. We examined the expression of 5-lipoxygenase (5-LO) and its metabolites in human colorectal carcinoma (Caco-2) cells, exposed to differentiation-inducing doses of butyrate. Treatment with butyrate significantly increased 5-lipoxygenase mRNA and protein in comparison to nontreated cells. Cyclooxygenases (COX)-1 and COX-2 mRNA were not significantly influenced by the treatment. However, 5-LO activity, low in nontreated cells, increased only minimally after butyrate, and its metabolic product (5-HETE) was detectable neither in control nor in treated cells. In contrast, 15-HETE (a product of 15-LO, which is also upregulated by butyrate) rose significantly. We conclude that, although being overexpressed by butyrate on mRNA and protein level, 5-LO remains inactive in differentiating Caco-2 cells. This is likely to be due either to some associated actions of butyrate, or to 5-LO-inhibition by 15-HETE, concomitantly induced by butyrate treatment.     

Protein Kinase-zeta inhibits collagen I-dependent and anchorage-independent growth and enhances apoptosis of human Caco-2 cells

Colonic carcinogenesis is accompanied by abnormalities in multiple signal transduction components, including alterations in protein kinase C (PKC). The expression level of PKC-zeta, an atypical PKC isoform, increases from the crypt base to the luminal surface and parallels crypt cell differentiation in normal colon. In prior studies in the azoxymethane model of colon cancer, we showed that PKC-zeta was down-regulated in rat colonic tumors. In this study, we showed that PKC-zeta is expressed predominantly in colonic epithelial and not stromal cells, and loss of PKC-zeta occurs as early as the adenoma stage in human colonic carcinogenesis. To assess the regulation of growth and differentiation by PKC-zeta, we altered this isoform in human Caco-2 colon cancer cells using stable constitutive or inducible expression vectors, specific peptide inhibitors or small interfering RNA. In ecdysone-regulated transfectants grown on collagen I, ponasterone A significantly induced PKC-zeta expression to 135% of empty vector cells, but did not alter nontargeted PKC isoforms. This up-regulation was accompanied by a 2-fold increase in basal and 4-fold increase in insulin-stimulated PKC-zeta biochemical activity. Furthermore, PKC-zeta up-regulation caused >50% inhibition of cell proliferation on collagen I (P < 0.05). Increased PKC-zeta also significantly enhanced Caco-2 cell differentiation, nearly doubling alkaline phosphatase activity, while inducing a 3-fold increase in the rate of apoptosis (P < 0.05). In contrast, knockdown of this isoform by small interfering RNA or kinase inhibition by myristoylated pseudosubstrate significantly and dose-dependently increased Caco-2 cell growth on collagen I. In transformation assays, constitutively up-regulated wild-type PKC-zeta significantly inhibited Caco-2 cell growth in soft agar, whereas a kinase-dead mutant caused a 3-fold increase in soft agar growth (P < 0.05). Taken together, these studies indicate that PKC-zeta inhibits colon cancer cell growth and enhances differentiation and apoptosis, while inhibiting the transformed phenotype of these cells. The observed down-regulation of this growth-suppressing PKC isoform in colonic carcinogenesis would be predicted to contribute to tumorigenesis.     

Butyrate enemas enhance both cholinergic and nitrergic phenotype of myenteric neurons and neuromuscular transmission in newborn rat colon

Postnatal changes in the enteric nervous system (ENS) are involved in the establishment of colonic motility. In adult rats, butyrate induced neuroplastic changes in the ENS, leading to enhanced colonic motility. Whether butyrate can induce similar changes during the postnatal period remains unknown. Enemas (Na-butyrate) were performed daily in rat pups between postnatal day (PND) 7 and PND 17. Effects of butyrate were evaluated on morphological and histological parameters in the distal colon at PND 21. The neurochemical phenotype of colonic submucosal and myenteric neurons was analyzed using antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS). Colonic motility and neuromuscular transmission was assessed in vivo and ex vivo. Butyrate (2.5 mM) enemas had no impact on pup growth and histological parameters compared with control. Butyrate did not modify the number of Hu-immunoreactive (IR) neurons per ganglia. A significant increase in the proportion (per Hu-IR neurons) of nNOS-IR myenteric and submucosal neurons and ChAT-IR myenteric neurons was observed in the distal colon after butyrate enemas compared with control. In addition, butyrate induced a significant increase in both nitrergic and cholinergic components of the neuromuscular transmission compared with control. Finally, butyrate increased distal colonic transit time compared with control. We concluded that butyrate enemas induced neuroplastic changes in myenteric and submucosal neurons, leading to changes in gastrointestinal functions. Our results support exploration of butyrate as potential therapy for motility disorders in preterm infants with delayed maturation of the ENS.