Different responses of Fe transporters in Caco-2/HT29-MTX cocultures than in independent Caco-2 cell cultures

The human intestinal epithelium is composed of several cell types, mainly enterocytes and goblet (mucin-secreting) cells. This study compares the cellular response of Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX co-culture models for Fe bioavailability. Caco-2 cells in vitro differentiate into enterocyte-like cells and HT29-MTX cell lineage into a mucin-secreting cellular population. Cell cultures were exposed to digests of Fe⁺³, Fe⁺³/ascorbic acid, cooked fish (high-available Fe) or white beans (low-available Fe). Cell responses as shown by mRNA expression of the main Fe transporters, DMT1 and DcytB, and cell ferritin formation were monitored. In Caco-2/HT29-MTX co-cultures, the mucin layer lowered the pool of free Fe to diffuse towards the cell brush border membrane of enterocytes, which was accompanied of an upregulation of DMT1 mRNA expression. In contrast, cultures exposed to digests of fish or white beans showed no significant differences in the regulation of Fe transporters.     

Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion

Human intestinal cell models are widely used to study host-enteric pathogen interactions, with different cell lines exhibiting specific characteristics and functions in the gut epithelium. In particular, the presence of mucus may play an important role in adhesion and invasion of pathogens. The aim of this study was to evaluate the suitability of the mucus-secreting HT29-MTX intestinal epithelial cell model to test adhesion and invasion of Salmonella strains and compare with data obtained with the more commonly used Caco-2 and HT-29 models. Adhesion of Salmonella to HT29-MTX cell model was significantly higher, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface, compared to the non- and low-mucus producing Caco-2 and HT-29 cell models, respectively. In addition, invasion percentages of some clinical Salmonella strains to HT29-MTX cultures were remarkably higher than to Caco-2 and HT-29 cells suggesting that these Salmonellae have subverted the mucus to enhance pathogenicity. The transepithelial electrical resistances of the infected HT29-MTX cell model decreased broadly and were highly correlated with invasion ability of the strain. Staining of S. Typhimurium-infected cell epithelium confirmed the higher invasion by Salmonella and subsequent disruption of tight junctions of HT29-MTX cell model compared with the Caco-2 and HT-29 cell models. Data from this study suggest that the HT29-MTX cell model, with more physiologically relevant characteristics with the mucus layer formation, could be better suited for studying cells–pathogens interactions.     

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

Cross-Talk between Ciliary Epithelium and Trabecular Meshwork Cells In-Vitro: A New Insight into Glaucoma

Purpose

It is assumed that the non-pigmented ciliary epithelium plays a role in regulating intraocular pressure via its neuroendocrine activities. To test this hypothesis, we investigated the effect on a human trabecular meshwork (TM) cell line (NTM) of co-culture with a human non-pigmented ciliary epithelium cell line (ODM-2).

Methods

The cellular cross-talk between ODM-2 and NTM cells was studied in a co-culture system in which the two cell types were co-cultured for 5 to 60min or 2, 4 and 8h and then removed from the co-culture and analyzed. Analyses of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and of the activity of TM phosphatases and matrix metalloproteins (MMPs) were performed. Acid and alkaline phosphatase activity was determined by the DiFMUP (6, 8-difluoro-4-methylumbelliferyl phosphate) assay. MMP levels were determined by gelatin zymography.

Results

Exposure of NTM cells to ODM-2 cells led to the activation of the MAPK signal transduction pathways in NTM cells within 5min of co-culture. Phosphorylation of ERK1/ERK2 and p38 peaked at 10 and 15min and then decreased over time. Interaction between ODM-2 and NTM cells promoted the expression of MMP-9 in the NTM cells after 4h of co-culture.

Conclusions

Our findings provide support for the hypothesis that crosstalk does indeed take place between ODM-2 and NTM cells. Future studies should be designed to determine the relationship between the MMP system, MAPK kinases and phosphatases. Manipulation of these signaling molecules and the related NTM signal transduction pathways may provide targets for developing improved treatments for glaucoma.     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.     

Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer

To compare the differences in MUC2 and MUC5AC mRNA among four colon cancer cell lines and to identify the best in vitro models for studying mucin expression, quantitative real-time polymerase chain reaction was used to measure the expression of MUC2 and MUC5AC mRNA in Caco-2, HT29, LoVo, and LS174T cell lines. The levels of MUC2 mRNA expression in the four colon cancer cell lines ranked in order of mRNA abundance were: LS174T > LoVo > HT-29 > Caco-2. In contrast to MUC2, the abundances of MUC5AC mRNA were in the order: Caco-2 > HT-29 > LS174T > LoVo. Caco-2 (highest level of MUC5AC mRNA) and LS174T (highest level of MUC2 mRNA) were used to investigate the phenotypes. Morphologically, Caco-2 cells were larger with low electron density mucus-storing vacuoles, many cell surface microvilli, and no obvious intercellular spaces between cells, compared to LS174T cells. The proliferative and invasive capacities of LS174T cells were significantly higher than those of Caco-2 cells. Caco-2 and LS174T cells provide excellent in vitro models for studying mucin expression in colon cancer.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.     

Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8–125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62–1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells

Background  

Although probiotic bacteria and their metabolites alter enterocyte gene expression, rapid, non-genomic responses have not been examined. The present study measured accumulation of tracer (2 μM) glucose by Caco-2 cells after exposure for 10 min or less to a chemically defined medium (CDM) with different monosaccharides before and after anaerobic culture of probiotic Lactobacilli.     

Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells

The role of Cox-2 in NSAID-induced apoptosis is debated. We studied the role of Cox-2 inhibition in apoptosis induced by a selective Cox-2 inhibitor, SC236 (a structural analogue of celecoxib) in two colon cancer cell lines, HT29 (expressing Cox-2 protein) and HCT116 (not expressing Cox-2 protein). Apoptosis was quantified by flow cytometry. SC236 0–75 M decreased cell numbers and induced apoptosis to identical levels in HT29 and HCT116 cells. However, SC236, concentrations >75 M reduced Cox-2 protein expression in HT29 cells and induced greater levels of apoptosis in HT29 than in HCT116 cells. In contrast, sulindac sulfide (SSD) (which inhibits Cox-1 and Cox-2) 0–200 M or sulindac sulfone (SSN) 0–500 M (without significant activity against Cox-1 or Cox-2) caused identical decreases in cell number and increases in apoptosis in HT29 and HCT116 cells. Neither SSD nor SSN altered the expression of Cox-2 in HT29 cells. To determine that the higher levels of apoptosis in HT29 cells with SC236 >75 M were related to decreased Cox-2 protein levels, we decreased Cox-2 protein expression in HT29 cells with curcumin (diferuloylmethane) and studied its effect on SC236-induced apoptosis. Curcumin augmented apoptosis induced by SC236 in HT29 cells but not in Cox-2 lacking HCT116 cells. In conclusion, selective Cox-2 inhibitors can induce apoptosis independent of Cox-2 expression. However they may selectively target cells that express Cox-2 by decreasing their Cox-2 protein expression.     

Cellular Asymmetric Catalysis by UDP-glucuronosyltransferase 1A8 Shows Functional Localization to the Basolateral Plasma Membrane

UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney, and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (−)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pretreatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells, and (−)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application, and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and Western blotting showed the presence of UGT1A in the basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that some of the UGT1A8 enzyme is not residing in the endoplasmic reticulum but spans the plasma membrane, resulting in increased accessibility to compounds outside the cell. This facilitates more efficient conjugation of substrate and is additionally coupled with rapid efflux by functionally associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell.     

Validation of NCM460 cell model as control in antitumor strategies targeting colon adenocarcinoma metabolic reprogramming: Trichostatin A as a case study

Background

Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected.

Methods

Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-¹³C₂]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism.

Results

We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells.

Conclusions

We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming.

General significance

Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.     

Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium

Aims:  To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved.
Methods and results:  The adhesion of probiotic ( Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp . lactis Bb12), commensal ( B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria ( E. coli and L. monocytogenes ) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2·6–27·3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower ( P  <   0·05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0·5–2·3%) cultures and Caco-2/HT29-MTX (0·6–3·2%) cocultures, while these values were increased in Caco-2 cultures plus mucin.
Conclusions:  The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2.
Significance and impact of the study:  The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.     

UDP-Glucuronosyltransferase 1A Compromises Intracellular Accumulation and Anti-Cancer Effect of Tanshinone IIA in Human Colon Cancer Cells

Background and Purpose

NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA.

Experimental Approach

We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity.

Key Results

Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect.

Conclusions and Implications

UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs’ substrates.     

Polyamine depletion partially reduces the radiation-induced cell death via cell cycle delay mediated by thioredoxin

In previous studies, polyamine depletion by DFMO (α-difluoromethylornithine)-treatment reduced H₂O₂-induced apoptotic cell death by reduction of ferric ion uptake. In the present study, we analyzed the reduction of radiation-induced cell death by polyamine depletion. Exposure of HT29 cells to radiation induced severe cell death, but when cells were pretreated with DFMO, a specific inhibitor of polyamine biosynthesis, radiation-induced cell death was reduced to 50–60% of control. Cell cycle analysis showed that, in these cells, the time to reach the G₂/M phase arrest was delayed for 20–24 h compared to the control cells, at which stage the fate of cells exposed to ionizing radiation is determined. DFMO-treated cells also showed a low level of thioredoxin, which is a high-level determinant of the cellular fate. To investigate the relationship between the G₂/M phase arrest and the reduction of thioredoxin caused by polyamine depletion, we also analyzed thioredoxin-antisensed (asTRX) HT29 cells as for DFMO-treated cells. In asTRX-transfected cells, the γ-irradiation-induced G₂/M phase arrest was also significantly delayed and radiation-induced cell death was profoundly reduced, as in the DFMO-treated cells. Both sets of cells showed a decrease of cyclin D1 and an increment of HSP25, which are involved in radiation-induced cell cycle progress. Overall, these results suggest that polyamines are essential for normal cell death of HT29 cells triggered by γ-radiation and that this is partially mediated by the regulation of thioredoxin expression.