Silencing of stat4 gene inhibits cell proliferation and invasion of colorectal cancer cells.

Signal transducers and activators of transcription (STAT) play critical roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases, including colorectal cancer. In the present study, we aimed to evaluate the function of STAT4 in human colorectal cancer (CRC). The expression of STAT4 was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was carried out to investigate the effects of lentivirus-mediated STAT4 shRNA (Lv-shSTAT4) on cell proliferation and invasive potential indicated by MTT and Transwell assays in CRC cell lines (SW480 and Caco2). As a consequence, it was found that the expression of STAT4 protein was significantly increased in CRC tissues compared with that in adjacent non-cancerous tissues (ANCT) (71.1% vs 44.4%, P=0.015), and was related with the Duke’s staging and depth of invasion in CRC patients (P=0.022; P=0.001). Silencing of STAT4 gene suppressed cell proliferation and invasion of CRC cells. Taken together, these findings demonstrate that increased expression of STAT4 is positively correlated with the depth of invasion in CRC patients, and inhibition of STAT4 expression represses the growth and invasion of CRC cells, suggesting that STAT4 may be a promising therapeutic target for the treatment of CRC.

Crosstalk between possible cytostatic and antiinflammatory potential of ketoprofen in the treatment of culture of colon and cervix cancer cell lines.

OBJECTIVES: The aim of this study was to test the cytostatic potential of ketoprofen in the in vitro treatment of cells derived from colon and cervix cancer. BACKGROUND: NF-κB and cyclooxygenase can have a role in different stages of the development and progression of cancer. In recent years, special attention has been paid to the possible cytostatic potential of nonsteroidal anti-inflammatory drugs. There are no published data on the use of ketoprofen in pharmacotherapy of the colon and cervical carcinoma. METHODS: We examined the effect of ketoprofen alone or in combination with cisplatin and 5-fluorouracil on proliferation of the two cell lines, HeLa (human cervical carcinoma cells) and Caco-2 (human colon cancer cells) by MTT test. Measurement of the level of NF-κB was also performed in the cells of both cell lines. RESULTS: The results of present study have shown that at least one of the mechanisms of antiproliferating and/or cytostatic effects of different concentrations of ketoprofen on Caco-2 and HeLa cells could include the transcription factor NF-κB. CONCLUSIONS: Since this transcription factor is controlled by the altered expression of COX-2, the inhibition of this enzyme by ketoprofen may represent a significant step in synergistic cascade of the therapy and prevention of colon and cervical cancer (Tab. 4, Ref. 31).

Collagen XVII expression correlates with the invasion and metastasis of colorectal cancer.

Collagen XVII has a well-established role as an adhesion molecule and a cell surface receptor located in the type I hemidesmosome of stratified epithelia. Its ectodomain is constitutively shed from the cell surface and suggested to regulate the adhesion, migration, and signaling of cutaneous epithelial cells. Collagen XVII was not previously thought to be expressed by colon epithelial cells. Immunohistochemical analysis of tissue microarray samples of 141 cases of colorectal carcinoma showed that collagen XVII is expressed in normal human colonic mucosa and colorectal carcinoma. In colorectal carcinoma, increased collagen XVII expression was significantly associated with higher TNM stage. It also correlated with infiltrative growth pattern and tumor budding as well as lymph node and distant metastasis. Increased collagen XVII expression was associated with decreased disease-free and cancer-specific survival. Immunofluorescence staining of collagen XVII and its well-known binding partner laminin γ2 chain demonstrated a partial colocalization in normal and tumor tissue. In vitro, the overexpression of murine collagen XVII promoted the invasion of CaCo-2 colon carcinoma cells through Matrigel (BD Biosciences; Bedford, MA). To conclude, this study reports for the first time the expression of collagen XVII in colon epithelium and the association of increased collagen XVII immunoexpression with poor outcome in colorectal carcinoma.

EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3ß1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2.

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3ß1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3ß1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

Biochemical and pathological toxic effects induced by the cyanotoxin Cylindrospermopsin on the human cell line Caco-2.

Cylindrospermopsin (CYN), a cyanotoxin produced by several freshwater cyanobacteria, causes human intoxications and animal mortalities. The present study focuses on the cytotoxic effects of CYN on Caco-2 cells at 24 and 48 h. The basal cytotoxicity endpoints studied were total protein content (TP), neutral red uptake (NR) and reduction of the tetrazolium salt (MTS). The effect of non-cytotoxic concentrations of CYN on the generation of intracellular reactive oxygen species (ROS), γ-glutamylcysteine synthetase (GCS) activity and glutathione (GSH) content was also studied and the morphological alterations in the Caco-2 cells subsequent to CYN exposure were recorded. The most sensitive endpoint - the reduction of MTS - showed that the viability of Caco-2 cells after exposure to the highest concentration assayed (40 µg/mL CYN) was reduced by about 90%. Intracellular ROS production increased only when exposed to a concentration of 1.25 µg/mL CYN, while GSH content and GCS activity increased when exposed to 2.5 µg/mL CYN. The main insights provided by the present study are the ultrastructural alterations, which reveal lipid degeneration, mitochondrial damage and nucleolar segregation with altered nuclei. Therefore, it has been demonstrated that CYN can induce toxic effects in Caco-2 cells in a time-concentration dependent manner. Moreover, unlike the cytotoxic and biochemical alterations, which were only evident at higher concentrations, morphological damage at the ultrastructural level was noticeable even at the lowest concentration used.

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells.

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.

Transcriptional profiling of Clostridium difficile and Caco-2 cells during infection.

Clostridium difficile is well recognized as the most common infectious cause of nosocomial diarrhea. The incidence and severity of C. difficile infection (CDI) is increasing worldwide. Here, we evaluated simultaneously the transcriptional changes in the human colorectal epithelial Caco-2 cells and in C. difficile after infection. A total of 271 transcripts in Caco-2 cells and 207 transcripts in C. difficile were significantly differentially expressed at 1 time point during CDI. We used the gene ontology annotations and protein-protein network interactions to underline a framework of target molecules that could potentially play a key role during CDI. These genes included those associated with cellular metabolism, transcription, transport, cell communication, and signal transduction. Our data identified certain key factors that have previously been reported to be involved in CDI, as well as novel determinants that may participate in a complex mechanism underlying the host response to infection, bacterial adaptation, and pathogenesis.

Isoflavones with unusually modified B-rings and their evaluation as antiproliferative agents.

Six novel isoflavone derivatives along with four known isoflavones were isolated from a culture of a highly nickel-resistant strain of Streptomyces mirabilis from a former uranium mining area. The structures of 7-hydroxy-3',5'-dihydroxyisoflavone (5), 5,7-dihydroxy-3',5'-dihydroxyisoflavone (6), 2'-hydroxy-3'-methoxygenistein (7), as well as hydroisoflavones A-C (8-10) were elucidated by MS and NMR analyses. Compounds 8-10 feature yet unprecedented types of non-aromatic, hydroxylated B rings, which result from plant isoflavone biotransformation. All new compounds display weak cytotoxic but potent antiproliferative activities. The anti-oestrogenic properties of 8 against MCF-7 human breast cancer cell line (GI(50): 6 microM) is even higher than the reference compound genistein.

Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A.

Heat-induced cell death appears to be a cell-specific event. Chronic heat stress was lethal to human colon cancer cells (Caco-2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ-T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45-51 degrees C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco-2 cells was not mediated via oxidative stress since Caco-2 cells were much more resistant than HeLa and other cancer cells to H(2)O(2)-induced cell death. Acute heat stress caused a striking loss of eukaryotic initiation factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat-induced loss of eIF5A is likely to be due to changes in the protein stability. The half-life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco-2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco-2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat-induced loss of eIF5A and prevented heat-induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress.

Ganglioside composition of differentiated Caco-2 cells resembles human colostrum and neonatal rat intestine.

Gangliosides are glycosphingolipids found in cell membranes and human milk with important roles in cell proliferation, differentiation, growth, adhesion, migration, signalling and apoptosis. Similar changes in ganglioside composition occur during embryonic development, lactation and cancer cell differentiation. It is not known, however, whether ganglioside compositional changes that occur in differentiating colon cancer cells reflect changes that occur during intestinal development. The Caco-2 cell line is commonly used to study physiological and pathophysiological processes in the small intestine and colon. Therefore, to examine this question, undifferentiated and differentiated Caco-2 cells were grown and total lipid was extracted from cell supernatant fractions using the Folch method. The upper aqueous phase containing gangliosides was collected and purified. Total gangliosides were measured as ganglioside-bound N-acetyl neuraminic acid, while individual ganglioside content was quantified via a colorimetric assay for sialic acid and scanning densitometry. The total ganglioside content of differentiated Caco-2 cells was 2.5 times higher compared with undifferentiated cells. Differentiated Caco-2 cells had significantly more (N-acetylneuraminyl) 2-galactosylglucosyl ceramide (GD3) and polar gangliosides, and a lower N-acetylneuraminylgalactosylglucosylceramide (GM3):GD3 ratio than undifferentiated cells. The present study demonstrates that the total ganglioside content and individual ganglioside composition of differentiated Caco-2 cells are similar to those of human colostrum and neonatal rat intestine. Differentiated Caco-2 cells may therefore be an alternative model for studying physiological and pathological processes in the small intestine and colon, and may help to elucidate possible functions for specific gangliosides in development and differentiation. Further research using more sensitive techniques of ganglioside analysis is needed to confirm these findings.

Antitumoral effect of phenazine N5,N10-dioxide derivatives on Caco-2 cells.

We studied the in vitro antitumoral effect of a series of phenazine di- N-oxide derivatives, named 2-chloroacetylamino-7(8)-nitrophenazine N(5), N(10)-dioxide (1), 2-amino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (2), 2-chloroacetylamino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (3), and 2-amino-7(8)-methoxyphenazine N(5), N(10)-dioxide (4), on Caco-2 cells. These phenazine N(5), N(10)-dioxide derivatives belong to our in-house chemical library. The products were selected according to their stereoelectronic characteristics and taking into account their differential cytotoxicity against V79 cells. Human colorectal adenocarcinoma cell line Caco-2 was used to study the cell growth inhibition capacity of these compounds, their capacity of altering the cell cycle and possible induction of apoptosis, DNA fragmentation, and genotoxic damage. The IC 50 after 24 h of incubation was lower for 1, 2, and 3 (4.8, 46.8, and 8.2 microM, respectively) than for 4 (474.7 microM). Compound 1 induced arrest in the G2/M phase at 24 and 48 h of treatment and apoptosis at the highest doses at 24 h of treatment. These facts were corroborated with caspase 3, caspase 9, and cytochrome c activation and DNA fragmentation at 24 h of treatment. The derivatives studied induced neither significant single strand breaks nor oxidative damage at the different studied times. We concluded that among the series of N(5), N(10)-dioxide phenazine derivatives analyzed, 1, which contains a nitro moiety and a chloroacetamide group, is the most promising as an antitumoral compound.

Proanthocyanidin from grape seeds inactivates the PI3-kinase/PKB pathway and induces apoptosis in a colon cancer cell line.

The aim of this investigation was to evaluate the chemopreventative/antiproliferative potential of a grape seed proanthocyanidin extract (GSPE) against colon cancer cells (CaCo2 cells) and to investigate its mechanism of action. GSPE (10-100 microg/ml) significantly inhibited cell viability and increased apoptosis in CaCo2 cells, but did not alter viability in the normal colon cell line (NCM460). The increased apoptosis observed in GSPE-treated CaCo2 cells correlated with an attenuation of PI3-kinase (p110 and p85 subunits) and decreased PKB Ser(473) phosphorylation. GSPE might thus exert its beneficial effects by means of increased apoptosis and suppression of the important PI3-kinase survival-related pathway.

Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease.

BACKGROUND & AIMS: Tissue transglutaminase (tTG) autoantibodies are markers of celiac disease, and the enzyme is required for several crucial biological processes. The aim of this study was to determine whether these autoantibodies are involved in the pathogenesis of the mucosal lesion typical of celiac disease. METHODS: Using rhodamine-conjugated phalloidin staining, we evaluated whether tTG antibodies, both commercially available and cloned from patients with celiac disease, cause cytoskeletal changes in Caco-2, MCF7, and NIH 3T3 cells. We monitored cell levels of bromodeoxyuridine incorporation to determine whether tTG autoantibodies are able to induce NIH 3T3 fibroblasts and epithelial mucosal cells into S phase. RESULTS: Treatment with tTG antibodies caused a dose-dependent increase of membrane ruffling in Caco-2, MCF7, and NIH 3T3 cells. It also dose-dependently induced G(0)-synchronized NIH 3T3 fibroblasts into S phase but did not affect the rate of apoptosis. Similarly, tTG antibodies induced S-phase entry of epithelial cells in cultured intestinal biopsy specimens from patients with celiac disease. They did not affect biopsy specimens from patients without celiac disease. CONCLUSIONS: Our results suggest that tTG autoantibodies per se, by interacting with the extracellular membrane-bound transglutaminase, may play an important role in epithelial cell proliferation in celiac disease.

Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro.

Colorectal cancer is one of the most common cancers in Western countries. The World Health Organisation identifies diet as a critical risk factor in the development and progression of this disease and the protective role of high levels of fruit and vegetable consumption. Several studies have shown that apples contain several phenolic compounds that are potent anti-oxidants in humans. However, little is known about other beneficial properties of apple phenolics in cancer. We have used the HT29, HT115 and CaCo-2 cell lines as in vitro models to examine the effect of apple phenolics (0.01-0.1% apple extract) on key stages of colorectal carcinogenesis, namely; DNA damage (Comet assay), colonic barrier function (TER assay), cell cycle progression (DNA content assay) and invasion (Matrigel assay). Our results indicate that a crude extract of apple phenolics can protect against DNA damage, improve barrier function and inhibit invasion (p<0.05). The anti-invasive effects of the extract were enhanced with twenty-four hour pretreatment of cells (p<0.05). We have shown that a crude apple extract from waste, rich in phenolic compounds, beneficially influences key stages of carcinogenesis in colon cells in vitro.

Genotoxic potential associated with low levels of the Fusarium mycotoxins nivalenol and fusarenon X in a human intestinal cell line.

This study aims to assess the genotoxic potential of nivalenol (NIV) and fusarenon X (FusX), produced by various Fusarium on cereals. Toxins were applied in time and dose-dependent experiments to the human enterocyte-like Caco-2 cell-line, both in dividing (undifferentiated) and in 10-12 days post-confluent cells (differentiated). Genotoxicity was evaluated through the alkaline Comet assay in a concentration range defined for each toxin as below the cytotoxicity threshold IC(10), determined by the MTS and the neutral red assays, to prevent false positive results because of DNA damage stemming from necrosis. Thus, genotoxicity was explored in the sub-cytotoxic 0-0.5 microM and 0-0.05 microM ranges respectively for NIV and FusX as the latter was found about 10-fold more cytotoxic than NIV. For both toxins, a 3h exposure did not cause any DNA damage, unlike after 24 and 72 h exposure in post confluent Caco-2 cells where DNA damage was significantly observed with a dose-dependent relationship. In dividing cells, only FusX increases DNA strand breaks in the 0.01-0.05 microM range after 72 h. These results demonstrated the existence of a genotoxic potential for NIV and FusX at low exposure levels and could contribute to the risk assessment process of these toxins that are of growing concern.

New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage.

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development.

Isothiocyanate iberin modulates phase II enzymes, posttranslational modification of histones and inhibits growth of Caco-2 cells by inducing apoptosis.

The aim of presented study was to further investigate the concentration-dependent changes induced by isothiocyanate iberin (IBN) in human colon carcinoma Caco-2 cells. The concentrations of IBN below IC(50) value (18 microM, 72 h) triggered the augmentation of mRNA levels for phase II detoxification GSTA1 and UGT1A1 enzymes and antioxidant thioredoxin reductase 1 gene in cells treated for 24 h. In addition a significant increase of acetylated H4 histone was detected. The mRNA induction peaked at IC(50) value and returned to level of control cells at 40 microM concentration of IBN. The cell cycle changes, gamma-H2AX stainability and the increase of phospho-H3 mitotic marker were induced at concentrations above IC(50) value. Appearance of Annexin V positive apoptotic cells and sub-G1 fragmented DNA as well as decrease of mitochondrial transmembrane potential confirmed cytotoxic effect of IBN observed in MTT assay. The predominance of necrotic cells and profound positivity of gamma-H2AX took place at the highest concentration of IBN. Thus, IBN represents the effective member of natural chemopreventive isothiocyanate family with which apoptotic potential can by employed to eliminate tumor cells.

c-Fos is a critical mediator of inflammatory-mediated repression of the apical sodium-dependent bile acid transporter.

BACKGROUND & AIMS: Ileal bile acid malabsorption is present in Crohn's ileitis. The molecular mechanisms of regulation of the apical sodium-dependent bile acid transporter (ASBT) by inflammatory cytokines in vitro and in vivo are investigated. METHODS: Transient transfection studies of the human, mouse, and rat ASBT promoters and Northern analyses were performed in cells treated with the inflammatory cytokines and/or various activator protein-1 constructs. Rat ASBT promoter transgenic, wild-type, and c-fos-null mice were treated with indomethacin to assess the response to acute inflammation of the ileal mucosa. RESULTS: In Caco-2 cells, ASBT messenger RNA expression was reduced 65% after interleukin-1beta treatment, while c-fos and c-jun were up-regulated 2-fold. Human ASBT promoter activity was enhanced by c-jun and repressed by a dominant negative c-jun, c-fos, or a dominant negative c-fos. Meanwhile, c-fos antisense treatment activated the human ASBT promoter 5-fold and not only abrogated interleukin-1beta-mediated repression but led to a paradoxical increase in ASBT promoter activity. Indomethacin-induced acute ileitis led to repression of ASBT in wild-type mice and in the transgenic rat ASBT promoter reporter, while paradoxical activation of ASBT was seen in c-fos-null mice. Indomethacin-induced ileal injury was greater in the c-fos-null mice compared with the wild-type littermates. CONCLUSIONS: Human, rat, and mouse ASBT is inhibited by inflammatory cytokines via direct interactions of c-fos with the ASBT promoter.

The critical role of 15-lipoxygenase-1 in colorectal epithelial cell terminal differentiation and tumorigenesis.

Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis.

Modular effects of estradiol on ethanol-induced apoptosis in human intestinal epithelial cells.

OBJECTIVE: Epidemiological data indicate that females develop alcohol-induced liver disease (ALD) more rapidly and more severely than males. Though the contribution of gut-derived endotoxin to the onset and development of ALD suggests the loss of epithelial cell viability that results in impaired intestinal function due to alcohol exposure, the additional effects of female sex hormones on intestinal cell viability is not known. The aim of this study was to examine the influence of estradiol on the intestinal cell death induced by acute and low concentrations of ethanol in an in vitro system. MATERIAL AND METHODS: Human intestinal epithelial Caco-2 cells were incubated with 0, 5, and 10% ethanol for 3 h. Estradiol stimulation, concentration of 3, 30, and 300 pg/ml occurred in the presence or absence of 10% ethanol for 2 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were quantified using flow cytometry. RESULTS: Treatment with 10% ethanol markedly induced PS externalization, caspase activation, and DNA fragmentation after 2 h incubation. Whereas estradiol itself did not affect cell viability, physiological concentrations of estradiol enhanced PS externalization and DNA fragmentation induced by 10% ethanol, and these were remarkable at 300 pg/ml estradiol. CONCLUSIONS: Ethanol-induced apoptosis was potentiated by physiological concentrations of estradiol, especially at the higher level which is found only in females. Our data suggest that enhanced ethanol-induced intestinal epithelial cell apoptosis in the presence of estradiol could cause greater intestinal permeability, which allows endotoxin to enter the circulation and eventually results in more severe ALD in females.