Arctigenin Inhibits Etoposide Resistance in HT-29 Colon Cancer Cells during Microenvironmental Stress.

Microenvironmental stress, which is naturally observed in solid tumors, has been implicated in anticancer drug resistance. This tumor-specific stress causes the degradation of topoisomerase IIα, rendering cells resistant to topoisomerase IIα-targeted anticancer agents. In addition, microenvironmental stress can induce the overexpression of 78kDa glucose regulated protein (GRP78), which can subsequently block the activation of apoptosis induced by treatment with anticancer agents. Therefore, inhibition of topoisomerase IIα degradation and reduction in GRP78 expression may be effective strategies for inhibiting anticancer drug resistance. In this study, we investigated the active compound arctigenin, which inhibited microenvironmental stress-induced etoposide resistance in HT-29 cells. Arctigenin was also highly toxic to etoposide-resistant HT-29 cells, with an IC50 value of 10 µM for colony formation. We further showed that arctigenin inhibited the degradation of topoisomerase IIα and reduced the expression of GRP78. Thus, these results suggest that arctigenin is a novel therapeutic agent that inhibits resistance to etoposide associated with microenvironmental stress conditions.

Induced cortical tension restores functional junctions in adhesion-defective carcinoma cells.

Normal epithelial cells are stably connected to each other via the apical junctional complex (AJC). AJCs, however, tend to be disrupted during tumor progression, and this process is implicated in cancer dissemination. Here, using colon carcinoma cells that fail to form AJCs, we investigated molecular defects behind this failure through a search for chemical compounds that could restore AJCs, and found that microtubule-polymerization inhibitors (MTIs) were effective. MTIs activated GEF-H1/RhoA signaling, causing actomyosin contraction at the apical cortex. This contraction transmitted force to the cadherin-catenin complex, resulting in a mechanosensitive recruitment of vinculin to cell junctions. This process, in turn, recruited PDZ-RhoGEF to the junctions, leading to the RhoA/ROCK/LIM kinase/cofilin-dependent stabilization of the junctions. RhoGAP depletion mimicked these MTI-mediated processes. Cells that normally organize AJCs did not show such MTI/RhoA sensitivity. Thus, advanced carcinoma cells require elevated RhoA activity for establishing robust junctions, which triggers tension-sensitive reorganization of actin/adhesion regulators.

[Establishment and biological characterization of 5-fluorouracil-resistant human colon cancer HT-29/5-FU cell line].

OBJECTIVE: To establish 5-fluorouracil (5-FU)-resistant human colon cancer HT-29 cell line (HT-29/5-FU) in vitro and observe its biological properties. METHODS: The HT-29/5-FU cell line was established by continuously exposing the HT-29 cells to ascending doses of 5-FU. The morphology and colony formation rate were detected. The growth curve and the chemosensitivity of HT-29/5-FU cell line were determined by MTT assay. Western blotting was used to analyze the expressions of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). Cell cycle and apoptosis were measured by flow cytometry. RESULTS: The resistance of HT-29/5-FU cells to 5-FU was 3.59-fold greater than that of HT-29 cells. The morphology of HT-29/5-FU cells differed from that of HT-29 cells. Compared with HT-29 cells, HT-29/5-FU cells showed remarkable reduction of cell proliferation and colony formation, higher expressions of TS and DPD, higher percentage of cells in the S phase, and stronger ability of resistance to apoptosis induced by 5-FU. CONCLUSION: The biological characters of HT-29/5-FU cell line may play an important role in 5-FU-resistant mechanisms, which may also contribute to elucidate the potential mechanisms of tumor drug resistance as well as resistance reversal.

The Australian fruit Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) inhibits telomerase, increases histone deacetylase activity and decreases proliferation of colon cancer cells.

Fruit antioxidants have many health benefits including prevention of cancer development. The native Australian bush fruit Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) has a high content of anthocyanin-rich phenolics, with an antioxidant capacity at levels higher than most fruits. In the present study the molecular mechanisms of the anti-proliferative activity of Illawarra plum on colorectal cancer cells were investigated. Non-tumorigenic young adult mouse colonic (YAMC) cells and tumorigenic human colonic (HT-29) cells were treated with a polyphenolic-rich Illawarra plum extract (0-1000 microg/ml). Illawarra plum had anti-proliferative properties in only the cancer cells, with growth suppressed in a dose- and time-dependent manner. Treatment of HT-29 cells with Illawarra plum extract (500 mg/ml; 24 h) was also associated with a 2-fold increase in apoptosis, and a cell cycle delay in the S phase (P < 0.01). Assessment of biomarkers for DNA damage revealed that plum treatment caused a 93% down-regulation of telomerase activity (P < 0.001) and a decrease in telomere length (up to 75%; P < 0.01). Treatment with Illawarra plum extract also induced morphological alterations to HT-29 cells that were suggestive of induction of autophagy, as the formation of cytoplasmic vacuoles was observed in many cells. This could be induced by the increased (6-fold) histone deacetylase (HDAC) activity (P < 0.001) and the trend for increased expression of the class III HDAC sirtuin 1. The present study has shown that Illawarra plum extract is able to reduce the proliferation of colon cancer cells by altering the cell cycle, increasing apoptosis and possibly inducing autophagy. The active ingredients in Illawarra plum may provide an alternative chemoprevention strategy to conventional chemotherapy.

Fermented wheat aleurone inhibits growth and induces apoptosis in human HT29 colon adenocarcinoma cells.

Fermentation of dietary fibre by the gut microflora may enhance levels of SCFA, which are potentially chemoprotective against colon cancer. Functional food containing wheat aleurone may prevent cancer by influencing cell cycle and cell death. We investigated effects of fermented wheat aleurone on growth and apoptosis of HT29 cells. Wheat aleurone, flour and bran were digested and fermented in vitro. The resulting fermentation supernatants (fs) were analysed for their major metabolites (SCFA, bile acids and ammonia). HT29 cells were treated for 24-72 h with the fs or synthetic mixtures mimicking the fs in SCFA, butyrate or deoxycholic acid (DCA) contents, and the influence on cell growth was determined. Fs aleurone was used to investigate the modulation of apoptosis and cell cycle. The fermented wheat samples contained two- to threefold higher amounts of SCFA than the faeces control (blank), but reduced levels of bile acids and increased concentrations of ammonia. Fs aleurone and flour equally reduced cell growth of HT29 more effectively than the corresponding blank and the SCFA mixtures. The EC(50) (48 h) ranged from 10 % (flour) to 19 % (blank). Markedly after 48 h, fs aleurone (10 %) significantly induced apoptosis and inhibited cell proliferation by arresting the cell cycle in the G0/G1 phase. In conclusion, fermentation of wheat aleurone results in a reduced level of tumour-promoting DCA, but higher levels of potentially chemopreventive SCFA. Fermented wheat aleurone is able to induce apoptosis and to block cell cycle - two essential markers of secondary chemoprevention.

Attenuation of colonic inflammation by PPARgamma in intestinal epithelial cells: effect on Toll-like receptor pathway.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor highly expressed in the colon and playing an anti-inflammatory role through inhibition of the NF-kappaB pathway. Toll-like receptor 4 (TLR4) has been known to mediate LPS-induced cellular signaling through activation of NF-kappaB pathway in intestinal epithelial cells. The aims of this study were to evaluate attenuation of inflammation by PPARgamma in intestinal epithelial cells and to study the possible relation between PPARgamma and TLR4. HT-29 human epithelial cells were stimulated with LPS (20 microg/ml) and PPARgamma ligand, 15d-PGJ2 (10 microM), or with LPS (20 microg/ml) alone for 24 hr. COX-2, IL-8, TLR4, and PPARgamma mRNA expression was assessed by RT-PCR. IL-8 protein levels and TLR4 protein expression were analyzed by ELISA and Western blot, respectively. To evaluate the action mechanisms of PPARgamma ligand, Western blot analysis for IkappaBalpha degradation was performed. Costimulation with LPS and PPARgamma ligand in comparison to LPS stimulation alone (1) decreased COX-2, IL-8 mRNA expression and IL-8 protein secretion, (2) decreased TLR4 mRNA and protein expression, and (3) decreased PPARgamma mRNA expression. PPARgamma ligand delayed LPS-induced IkappaBalpha degradation. These findings suggest that PPAR-gamma ligands suppress inflammation in intestinal epithelial cells. PPARgamma and TLR, these two antagonistic signaling pathways in intestinal epithelial cells may be partially cross-linked.

Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes.

Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.

Proteomic analysis of colonic myofibroblasts and effect on colon cancer cell proliferation.

BACKGROUND: The stromal microenvironment influences many steps of tumor progression through the elaboration of signals from myofibroblasts. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in colonic epithelial proliferation. The purpose of this study was to determine (1) the influence of myofibroblasts on colon cancer cell proliferation and PI3K activity, and (2) the protein alterations associated with myofibroblasts derived from polyp versus normal margins. METHODS: Myofibroblasts were derived from polyps and corresponding normal mucosa. Myofibroblasts were cocultured with colon cancer cells HT29 stably transfected with green fluorescent protein and KM20 cells. Proliferation was quantitated by green fluorescent protein count and cytokeratin enzyme-linked immunosorbent assay. HT29 cells were incubated with conditioned medium from myofibroblasts, and the effect on proliferation and PI3K activity was determined by 5-bromo 2-deoxyuridine incorporation and Akt kinase assay, respectively. Protein profiles were obtained by SELDI-TOF MS analysis. RESULTS: In coculture experiments, all myofibroblasts significantly enhanced HT29 and KM20 cell proliferation. However, polyp myofibroblasts enhanced proliferation of the cancer cells to a greater extent than normal myofibroblasts. Conditioned medium from all myofibroblasts stimulated Akt kinase activity. SELDI-TOF MS profiles showed more than 40 protein peaks for each isolate. One protein was differentially expressed in polyps versus normal cells. CONCLUSIONS: Utilizing a novel proteomic approach, we identify distinct protein profiles in myofibroblasts of polyps compared with stromal cells of normal mucosa. Moreover, myofibroblasts can stimulate indirectly PI3K activity and enhance colon cancer cell proliferation. These findings suggest that targeted therapy to signaling pathways in myofibroblasts may be useful in colorectal cancer chemoprevention and possible treatment.

Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.

Characterization of a spontaneously polarizing HT-29 cell line, HT-29/cl.f8.

A cloned cell line that spontaneously polarizes in standard glucose-containing media was derived from a single cell of the adenocarcinoma cell line HT-29. The cloned line, designated HT-29/cl.f8, has remained stable over 2 yr in culture, maintained high transepithelial resistance (300 ohm cm(2) or higher), and correctly sorted influenza virus and vesicular stomatitis virus to apical or basolateral domains, respectively. The newly cloned cells also displayed apical microvilli, tight junctions, and desmosomes, the morphological characteristics of mature epithelia. The cloned HT-29/cl.f8 cells function as epithelial enterocytes as shown by the apical expression of intestinal alkaline phosphatase, the expression of vimentin and cytokeratin, and lack of expression of mucin. We propose that the newly cloned HT-29/cl.f8 cells offer a viable alternative for studies of enterocyte function that will readily yield interpretable data not complicated by cell alterations due to the presence of drugs or chemicals that induce differentiation.

Early effects of low dose-rate radiation on cultured tumor cells.

Low-dose hyperradiosensitivity (HRS) has been found for several cell types after exposure to low doses, < 0.5 Gy, of high dose-rate (typically 50-150 Gy/h) low-LET radiation. HRS precedes the occurrence of a relative resistance for doses above 0.5-1 Gy. A critical question is whether HRS is of importance in radionuclide therapy where the dose-rate is low but the total dose might be high. An indication that cells exposed to low dose-rate can be kept hyperradiosensitive has recently been published. We have in the present study applied cells without (glioma U373MG) and with (glioma U118MG and colon carcinoma HT29) HRS and studied early effects, up to one week, during low dose-rate (LDR), 0.05-0.09 Gy/hours, exposure (total dose after one week: 11.8 +/- 1.5 Gy). The cells were grown on thin foils above a (32)P source placed in a cell culture chamber. Cell number reductions, cell-cycle disturbances, and changed numbers of apoptotic cells were analyzed after continuous LDR exposures. There seemed to be no relation with HRS when the cell number reduction was considered. The U373MG cells, lacking HRS, had the strongest cell number reduction due to a combination of a G(2) block and increased apoptosis. The U118MG and HT29 cells, both having HRS, had surprisingly low cell number reductions. U118MG had only a G(2) block but no increase in apoptosis. HT29 had both a G(2) block and an increase in apoptosis but the apoptosis change was somewhat smaller than for U373MG. Thus, there seemed to be no obvious relation between HRS and early cellular effects when the cells were analyzed after continuous LDR exposure.

Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology.

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells.

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.

Exposure to a deuterated analogue of phenylbutyrate retards S-phase progression in HT-29 colon cancer cells.

Differentiation agents that induce neoplastic cells to regain a normal phenotype and/or cause growth arrest without significantly affecting normal cells represent an attractive option for cancer treatment. Analogues of short chain fatty acids, such as phenylbutyrate (PB), have been studied as clinically relevant agents. In an attempt to improve its pharmacokinetic profile, structural modifications of PB and other fatty acids have been studied. We hypothesize that strategic isotopic modification of PB would result in a longer half-life and thus translate into a more potent differentiation agent for clinical use. Using a colon cancer model, we demonstrated that 2,2,3,3-tetradeuterated PB (D4PB) significantly increased induction of apoptosis and inhibition of cell proliferation as compared with PB and butyrate. Difference in potency could not be explained by the effect of D4PB on the expression of specific regulatory proteins of the apoptotic cascade or from the inhibitory effect of D4PB on histone deacetylase activity. Interestingly, exposure of HT-29 colon cancer cells to D4PB resulted in a slowing of S transit, in contrast to butyrate and PB, which induced a G2/M cell cycle block. This difference in cell cycle effect may explain the differences seen in the potency of the phenotypic changes seen with treatment with D4PB. Further studies are needed to elucidate the mechanisms underlying effects of D4PB on the cell cycle.

Ceramide involvement in homocamptothecin- and camptothecin-induced cytotoxicity and apoptosis in colon HT29 cells.

Topoisomerase I inhibitors of the camptothecin (CPT) family have emerged as potent clinical chemotherapeutic agents in first-line treatment of solid colorectal cancer and in second-line for 5-fluorouracil resistant patients. CPT and homocamptothecin (hCPT), derivative with enhanced lactone stability, induced growth inhibition in HT29 cells via p53-independent apoptosis. hCPT- and CPT-induced apoptosis was dependent on caspase-3 but not caspase-1. We report here substantial evidence that ceramide, resulting from de novo pathway or catabolism modulation, acted as a second messenger of these antitumor drugs in HT29 cells and leads to the activation of caspase-3. In addition, hCPT and CPT may favor ceramide signaling by disturbing sites of synthesis (Golgi) and trafficking of glucosylceramide from Golgi to lipid droplets. This work contributes to the understanding of the mechanism of action of CPTs, and suggests that inhibitors of glycosylation or activators of de novo metabolism could be of clinical interest in enhancing the effects of CPTs.

Atypical protein kinase C zeta as a target for chemosensitization of tumor cells.

Exposure of tumor cells to cytotoxic agents simultaneously activates a variety of intracellular signaling pathways. Some of these pathways involve enzymes from the protein kinase C (PKC) family of serine/threonine kinases. This family includes isoenzymes that negatively influence cell death, whereas other demonstrate an opposite effect. The present study analyzes the role of the zeta atypical PKC isoform in tumor cell response to cytotoxic agents. Using a histone H1 phosphorylation assay, we showed that both tumor necrosis factor alpha and etoposide activate PKCzeta in U937 human leukemic cells. Stable transfection of a kinase-dead, dominant-negative PKCzeta mutant in U937 cells decreases Bcl-2 expression while increasing the expression of Bax and several procaspases. This transfection also prevents etoposide-induced nuclear factor-kappaB nuclear translocation and accumulation of X-linked inhibitor of apoptosis protein. PKCzeta inhibition accelerates the occurrence of apoptosis in leukemic cells exposed to etoposide and tumor necrosis factor alpha. This sensitization was confirmed in vitro by use of a clonogenic assay. In addition, PKCzeta inhibition sensitized tumor cells grown in nude mice to etoposide. These results indicate that PKCzeta isoform is a protective signals that is activated in tumor cells exposed to a cytotoxic agent. This inducible resistance factor thus appears an attractive target for chemosensitization of tumor cells.

Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell line.

The tumor cell line HT-29 was derived from a primary adenocarcinoma of the rectosigmoid colon. HT-29 is hypertriploid (3n+) and has accumulated numerous chromosomal structural aberrations. To identify material involved in chromosome rearrangements, we performed a comprehensive cytogenetic analysis using G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). The combination of molecular cytogenetic techniques enabled us to define the first comprehensive karyotype for HT-29. Seventeen marker chromosomes were found in 75-100% of metaphase cells, generally in a single copy per cell. We confirmed the composition of eight previously described markers, refined the classification of seven others, and identified two novel marker chromosomes. Notable aberrations included a reciprocal translocation between chromosomes 6 and 14 and an unusual, large derivative chromosome 8 composed entirely of 8q material. The telomere status, evaluated by FISH, revealed telomeric signals at the termini of all chromosomes. No interstitial telomeric sequences were observed in any cell. Although numerous chromosomal aberrations are present in HT-29, the cell line appears to have retained a high level of genomic stability during passage in culture since undergoing transformation. The excellent resolving power of SKY, coupled with additional information obtained from molecular cytogenetic analyses, will improve our ability to identify genetic lesions characteristic of cancer.

Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation.

BACKGROUND: Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth-related actions in a phorbol ester-like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations. METHODS: Human colon cancer cells (Caco-2 and HT-29) were treated with deoxycholate or its two structural isomers, 3-beta-12-alpha-dihydroxy-5-beta-cholan-24-oic acid and 3-alpha-12-beta-dihydroxy-5-beta-cholan-24-oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology. RESULTS: Within the concentration range of up to 20 microM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth-promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 microM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth. CONCLUSION: Low-dose deoxycholate stimulates colon cancer cell proliferation while > 100 micromol L(-1) of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.

Modulation of HMG-N2 binding to chromatin by butyrate-induced acetylation in human colon adenocarcinoma cells.

Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG-N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG-N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG-N2 mRNA and of total or acetylated HMG-N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG-N2-EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG-N2. Levels of HMG-N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG-N2 mRNA levels after treatment with butyrate. Analysis of HMG-N2-EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG-N2-EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG-N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG-N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG-N2 proteins, may be partly responsible for the wide range of butyrate-associated effects.

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by alpha2beta1 integrin low clustering through focal adhesion kinase.

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.