Apoptosis in colorectal tumour cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate

The short chain fatty acids acetate, propionate and butyrate are produced when dietary fibre is fermented by the colonic bacteria. We have previously shown that sodium butyrate induces apoptosis in 3 colorectal tumour cell lines. We have extended our study to 3 adenoma and 4 carcinoma cell lines and investigated whether propionate and acetate also induce apoptosis. All 3 short chain fatty acids induced apoptosis at physiological concentrations, but of the 3, butyrate was the most effective. Since these fatty acids are produced as a result of bacterial fermentation of dietary fibre, this may in part explain the correlation between a high-fibre diet and low colorectal cancer incidence. Sodium butyrate induced apoptosis in all 7 of the cell lines studied; however, 2 of the 4 carcinoma cell lines (PC/JW/FI and S/KS/FI) were more resistant to butyrate-induced apoptosis than the 3 adenoma cell lines, suggesting that at least some carcinomas may evolve mechanisms to protect the cells from the induction of apoptosis. The bile acid deoxycholic acid has previously been reported as a possible tumour promoter in the large intestine and its levels are reduced by dietary fibre. Concentrations of between 10 nM and 0.1 mM had no effect on either the proliferation or apoptosis of colonic tumour cells in vitro. However, a significant induction of apoptosis was obtained at a concentration of 0.5 mM. These results may have significance for the aetiology of colorectal cancer.     

Stable reintroduction of wild-type P53 (MTmp53ts) causes the induction of apoptosis and neuroendocrine-like differentiation in human ductal pancreatic carcinoma cells

Pancreatic ductal adenocarcinoma is one of the major causes of cancer mortality in the industrialized world, having among the poorest prognosis of any malignancy. Mutations or alterations in the p53 tumor suppressor gene/protein are observed in 50-70% of these cancers, yet little information is available regarding the phenotypic effects of restoration of wild-type (wt) p53 function in pancreatic ductal carcinoma cells. The consequences of stable reintroduction of wt p53 on apoptosis and differentiation was examined in a poorly differentiated pancreatic carcinoma cell line (Panc-1), possessing only mutant (mt) p53 (codon 273 mutation). Cells were transfected with a temperature-sensitive mouse p53val135 (tsp53) vector under additional control of a genetically-modified metallothionein promoter. This tsp53 has a 'mt' phenotype at 37.5 degrees C, and a 'wt' phenotype at 32.5 degrees C and the presence of 100 microM ZnCl2. Stable expression of wt p53 caused upregulation of the p21/WAF1 gene, and G1 growth arrest as shown by flow cytometry and BrdU labeling. Additionally, apoptosis was induced 8-12 post-induction in the majority of the cells (60-70%), as demonstrated by morphological changes, in situ TdT labeling and internucleosomal laddering. However, a subpopulation (30%) of the transfectants survived this apoptotic fate. Unlike the epithelial parental Panc-1 cells, these cells exhibited the appearance of a neuroendocrine-like phenotype with extensive branch-like processes, and marked cytoplasmic and cytoskeletal immunostaining for tau-2, synaptophysin, and chromogranin A. These studies suggest that stable and regulated expression of wt p53 can have multiple phenotypic consequences (apoptosis and altered differentiation to a neuroendocrine-like phenotype) in poorly-differentiated pancreatic carcinoma cells.     

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Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.     

bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis

Butyrate, a short chain fatty acid produced in the colon as a result of fermentation of dietary fibre by symbiotic bacteria, induces apoptosis in colonic tumour cell lines. Three human colonic adenoma cell lines (AA/C1, RG/C2 and BH/C1) and one carcinoma cell line (S/KS/FI) were used to determine the effects of butyrate on the expression of bcl-2, bax and bak to examine the possible role of these proteins in the induction of apoptosis. RG/C2 and BH/C1 cells express p-26-bcl-2 and butyrate treatment decreased p26-bcl-2 levels in association with apoptosis, whereas bax and bak levels remained constant. AA/C1 and S/KS/FI cells have no detectable p26-bcl-2. In S/KS/FI cells, bax or bak levels did not change in response to butyrate. However, in AA/C1 cells, butyrate-induced apoptosis was associated with increased bak levels. Therefore, in AA/C1 cells butyrate-induced apoptosis appears to be mediated through bak. Furthermore, butyrate also induced apoptosis and increased bak levels in AA/C1 cells transfected with a bcl-2 expression vector which expressed high levels of p26-bcl-2. For S/KS/FI cells, two bcl-2 transfectants gave different results. bcl-2 protected against apoptosis in one transfectant in which bak levels were not elevated in response to butyrate, whereas it did not protect in the other transfectant in which bak levels were increased after butyrate treatment. The results suggest that expression of constitutively high levels of p26-bcl-2 only conferred protection against apoptosis when bak levels were not elevated in response to butyrate and that expression of constitutively high levels of p26-bcl-2 does not counter the effects of bak. Different mechanisms appear to be involved in cell death signalling in different tumours since butyrate may induce apoptosis via elevated levels of bak or reduced levels of p26-bcl-2.     

Mitochondrial membrane potential (delta psi(mt)) in the coordination of p53-independent proliferation and apoptosis pathways in human colonic carcinoma cells

We have previously defined depressed mitochondrial function as a determinant in colon cancer risk and progression and established that metabolism of butyrate, a short-chain fatty acid generated during the fermentation of fiber by endogenous intestinal bacteria, induces mitochondrial function-dependent growth arrest and apoptosis of colonic carcinoma cells in vitro. Here, we dissect the relationships among mitochondrial function, growth arrest, and apoptosis, reporting that initiation and maintenance of butyrate-mediated p53-independent p21WAF1/Cip1 induction and subsequent G0/G1 arrest require an intact mitochondrial membrane potential (delta psi(mt)) and that the process of dissipation of the delta psi(mt) is then essential for initiation of a butyrate-induced apoptotic cascade. Thus, we hypothesize that mitochondria play a pivotal role in coordinating proliferation and apoptosis pathways, a coordination that must be tightly regulated in rapidly renewing tissues, such as the colonic mucosa.     

Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments.     

Application of alginate gel as a vehicle for liposomes. II. Erosion of alginate gel beads and the release of loaded liposomes

Short chain fatty acids (SCFAs) stimulate electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes. It is often assumed that activation of Na/H exchange is via an intracellular acidification caused by SCFA uptake. These lecture notes review shortcomings in this model of SCFA-stimulated sodium absorption, revealed by recent reports in the literature. This is supplemented by information generated in our laboratory using both a tissue culture model of colonocytes (HT29-C1 cells) and a native tissue preparation (mouse distal colonic mucosa). In both preparations, evidence suggests that physiologic SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane of colonocytes. Finally, direct observation of such extracellular microdomains with confocal microscopy is used to support a new model, in which pH microdomains play an important role in regulating both SCFA fluxes and sodium absorption.     

Transepithelial SCFA fluxes link intracellular and extracellular pH regulation of mouse colonocytes

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.     

Bcl-2 deregulation leads to inhibition of sodium butyrate-induced apoptosis in human colorectal carcinoma cells

Epidemiological studies have linked dietary fiber to the prevention of human colorectal cancer and suggest that short chain fatty acids such as butyric acid, which is produced by fermentation of dietary fiber in the large intestine, may be an important mediator of the protective effects of fiber. We investigated the role of Bcl-2 deregulation on the sensitivity of colorectal carcinoma cells to undergo butyrate-induced apoptosis. Here we report an inverse relationship between the levels of Bcl-2 and the sensitivity of colorectal carcinoma cell lines to undergo apoptosis in response to butyrate. Overexpression of Bcl-2 in colorectal carcinoma DiFi cells resulted in suppression of butyrate-induced apoptosis and enhanced cell survival in response to butyrate. Butyrate-induced apoptosis was accompanied by inhibition of expression of a 30 kDa protein (p30, immunorecognized by anti-Bcl-2 mAb) and this cellular effect of butyrate was inhibited by Bcl-2 overexpression. These findings suggest that deregulation of Bcl-2 in human colorectal carcinoma cells confers resistance to induction of apoptosis by butyrate, a dietary micronutrient.     

Soluble tumor necrosis factor (sTNF) receptors: a possible prognostic marker for bone marrow transplantation-related complications

BACKGROUND: Studies on colon carcinogenesis suggest that the short-chain fatty acid butyrate may be protective, whereas the secondary bile acid deoxycholate may promote tumor development. Crypt surface hyperproliferation is regarded as a biomarker of colon cancer risk and can be modulated in vitro by the differentiation inducer butyrate and the tumor promoter deoxycholate. We hypothesized that butyrate decreases and deoxycholate increases crypt surface proliferation in vivo and that these effects are mediated by changes in the expression of the protooncogenes c-Fos and c-Jun, which are known to regulate proliferation and differentiation. METHODS: Twenty-five adult Sprague-Dawley rats underwent colonic isolation and 24-hour intraluminal instillation of 10 mmol/L sodium chloride, 10 mmol/ L sodium butyrate, or 10 mmol/L sodium deoxycholate. Proliferation of the whole crypt and five crypt compartments from base to surface was assessed by proliferating cell nuclear antigen immunohistochemistry. The ?h value, an index of "premalignant" hyperproliferation, was calculated as the ratio of labeled cells in the two surface compartments divided by the labeled cells in the entire crypt. Expression of c-Fos and c-Jun was evaluated by Western blot. RESULTS: Crypt surface proliferation and the ?h value were significantly decreased by butyrate and increased by deoxycholate. Butyrate increased colonic expression of c-Jun, whereas deoxycholate significantly induced c-Fos. CONCLUSIONS: The in vivo effects on surface proliferation are consistent with a potential protective [corrected] role for butyrate and a promotive role for deoxycholate in colon carcinogenesis. The concurrently observed effects on colonic c-Jun and c-Fos expression represent a novel finding and suggest that direct or indirect modulation of protooncogene expression may be the mechanism by which these dietary byproducts regulate proliferation in vivo.     

Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis

The administration of sodium butyrate at 0.75 mM induced the functional differentiation of U-937 human promonocytic leukemia cells with negligible cell mortality. However, the drug rapidly caused cell death with characteristics of apoptosis when used at concentrations of 5 mM and above. In addition, butyrate stimulated the expression of the stress-responsive heat-shock protein 70 (HSP70) gene when applied at both differentiation-inducing and apoptosis-inducing concentrations. The induction of HSP70 by butyrate was inhibited by the simultaneous addition of cAMP-increasing agents (dibutyryl cAMP or the combination of forskolin plus theophylline). However, these agents did not prevent differentiation and only partially reduced apoptosis. Moreover, the DNA topoisomerase II inhibitor etoposide, which provoked U-937 cell differentiation and apoptosis with the same or greater efficiency than butyrate, failed to stimulate HSP70 expression. Finally, it was observed that cAMP-increasing agents also abrogated the induction of HSP70 and reduced the apoptosis caused by cadmium chloride, a typical inducer of the stress response. Taken together, these results indicate that HSP70 expression is not required for differentiation of promonocytic cells, as earlier proposed, and that butyrate probably triggers the stress response in these cells.     

Regulation and function of Bcl-2 during differentiation-induced cell death in HL-60 promyelocytic cells

Polymorphonuclear leukocytes are generated by differentiation of early myeloid precursors. Once fully differentiated, blood neutrophils are programmed to die rapidly and are removed by tissue macrophages. In normal myeloid cells, the death mechanism seems to be coupled to the differentiation pathway and is accomplished by a process termed apoptosis. In the present study, we have examined the role of Bcl-2 in the differentiation pathways of the promyelocytic cell line HL-60. Treatment of HL-60 with retinoic acid or phorbol ester, which induced neutrophil or macrophage-like cell differentiation, respectively, resulted in progressive loss of cellular viability and internucleosomal DNA degradation. In HL-60, differentiation and apoptosis were coupled to down-regulation of the Bcl-2 protein. Overexpression of Bcl-2 by gene transfer inhibited apoptosis triggered by terminal differentiation of HL-60. Yet, Bcl-2 did not alter the expression of surface markers or other phenotypic changes that are induced upon myeloid differentiation. In contrast to HL-60, another immature myeloid cell line, K562, did not produce Bcl-2 but expressed a related protein, Bcl-xL, that functions as a repressor of apoptotic cell death. K562 has been shown to be relatively resistant to a variety of apoptotic stimuli. Incubation of HL-60 and K562 with inhibitors of macromolecular synthesis induced apoptosis, which appeared earlier in HL-60 than in K562. Interestingly, Bcl-2 overexpression protected K562 cells from apoptosis induced by inhibitor of macromolecular synthesis but it had little or no effect on HL-60 cells. We conclude that although differentiation and apoptosis proceed simultaneously, they can be uncoupled by expression of Bcl-2. Down-regulation of Bcl-2 appears to be part of the differentiation pathway and may serve to facilitate the apoptotic response.     

Gonadotropin-I and -II subunit gene expression of male striped bass (Morone saxatilis) after gonadotropin-releasing hormone analogue injection: quantitation using an optimized ribonuclease protection assay

We have demonstrated that sodium butyrate induces differentiation in human hepatoma cells; however, recent studies have shown that this agent causes apoptosis in some types of cancer cells. In this study, we examined whether sodium butyrate causes apoptosis in the human hepatoma cell lines, HCC-M and HCC-T. The growth of human hepatoma cells was dose-dependently reduced by sodium butyrate. Flow cytometric analysis showed cell-cycle arrest at the G1 phase in the sodium butyrate-treated cells. Apoptotic change was never found in treated cells at concentration levels of less than 5 mmol/L. Sodium butyrate decreased p53 expression and increased p21WAF-1 expression in HCC-T and HCC-M cells having the wild-type p53 gene. Western blot analysis showed that Bcl-2 was expressed in the HCC-T and HCC-M cells, and its expression was increased after exposure to sodium butyrate. Antisense oligodeoxynucleotide against bcl-2 easily caused apoptosis. These results indicate that sodium butyrate hardly induces apoptotic change in the human hepatoma cell lines, HCC-T and HCC-M, with the increase of Bcl-2 expression. Cell-cycle arrest in the G1 phase caused by sodium butyrate was suggested to be induced by the increase in p21WAF-1 expression, but this change did not link with the p53 increase.     

Morphological and cytochemical findings in 150 cases of T-lineage acute lymphoblastic leukaemia in adults. German Multicentre ALL Study Group (GMALL)

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic death. We have developed a serum-free suspension culture that allows terminal differentiation and facilitates the investigation of factors affecting chondrocyte apoptosis. We have found that chondrocytes not committed to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some months after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture density and sensitivity to induction of apoptosis with the protein kinase inhibitor, staurosporine, was consistent with the proposal that these chondrocytes, like nearly all cells, require intercellular communication for survival. Chondrocytes that were committed to terminal differentiation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed the hypertrophic phenotype but maintained their viability in culture for only approximately 6 days. Subsequent cell death was very consistent between cultures and shown to occur by an apoptotic process by analysis of DNA fragmentation and cell morphology. Short-term viability of hypertrophic chondrocytes was independent of culture density and relatively resistant to treatment with staurosporine. Induction of the hypertrophic phenotype in immature chondrocytes committed them to cell death and prevention of expression of the hypertrophic phenotype prevented cell death. We conclude that commitment of chondrocytes to terminal differentiation is associated with a commitment to apoptosis and apoptosis of hypertrophic chondrocytes in growth cartilage does not require initiation by external signals.     

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BACKGROUND: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation. METHODS: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin. RESULTS: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F-actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth. CONCLUSION: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype.     

Increased growth of Bifidobacterium and Eubacterium by germinated barley foodstuff, accompanied by enhanced butyrate production in healthy volunteers

Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley mainly consists of low-lignified hemicellulose and glutamine-rich protein. GBF improves the proliferation of intestinal epithelial cells and defecation, through the bacterial production of short chain fatty acids (SCFA), especially butyrate. In this study we investigated the mechanism of production of butyrate by microflora in humans and in vitro. Daily administration of 9 g GBF for 14 successive days significantly increased fecal butyrate content. Fecal Bifidobacterium and Eubacterium were also significantly increased by GBF administration in healthy volunteers. Ten anaerobic micro-organisms selected from intestinal microflora were cultured in vitro in the medium containing GBF as a sole carbon source (GBF medium). After a 3-day incubation, 7 strains (Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Bacteroides ovatus, Clostridium butyricum, and Eubacterium limosum) lowered the medium pH producing SCFA. Eubacterium grown together with Bifidobacterium in GBF medium efficiently produced butyrate. On the other hand, GBF changed the intestinal microflora and increased probiotics such as Bifidobacterium in the intestinal tract. As a result, butyrate was produced by the mutual action of Eubacterium and Bifidobacterium. This butyrate is considered to enhance the proliferation of colonic epithelial cells.     

Induction of antigen presentation by macrophages after phagocytosis of tumour apoptotic cells

Due to their resistance to classical chemotherapies, most human colorectal cancers have a high incidence and a poor prognosis. Immunotherapy using interleukin 2 (IL2) has provided disappointing results in the treatment of these cancers. Recently, however, we have demonstrated that a treatment combining a cell-differentiating agent, sodium butyrate (NaBut) with IL2 resulted in a remission of established peritoneal colorectal carcinomatosis in rats. Separately, neither NaBut nor IL2 treatment cured these tumour-bearing rats. NaBut is known to induce cell differentiation and subsequent apoptosis in epithelial cells, while IL2 stimulates the immune cells capable of participating in tumour rejection. We postulated that the significant therapeutic effect of NaBut/IL2 treatment could be attributed to a NaBut-induced increase in the immunogenicity of the cancer cells. We report here that NaBut induced an apoptotic process in rat colon tumour cells in vivo and in vitro. We observed, in an efficient cure, colocalization of apoptotic bodies and monocytes/macrophages at the periphery of the tumour. We propose that these apoptotic bodies are phagocytosed in vivo by the macrophages. We also showed in vitro that a subpopulation of macrophages involved in the phagocytic clearance of apoptotic cells expresses cell surface molecules associated with antigen presentation and stimulates the proliferation of naive splenocytes. Our data suggest that therapies that recruit massive induction of the apoptotic process in tumour cells could favour tumour antigen presentation via their specific phagocytosis by antigen-presenting cells (APCs). We propose that the development of specific therapies that stimulate both tumour cell apoptosis and the immune system could offer new opportunities in anti-cancer treatments of poorly immunogenic cancer cells.