Human Colonic Cell Line HT-29 Research Articles

Artemisinin analog SM934 alleviates epithelial barrier dysfunction via inhibiting apoptosis and caspase-1-mediated pyroptosis in experimental colitis

Intestinal barrier disruption due to the intestinal epithelial cells’ (IECs) death is one of the critical pathological features of inflammatory bowel diseases (IBDs). SM934, an artemisinin analog, has previously been proven to ameliorate colitis induced by dextran sulfate sodium (DSS) in mice by suppressing inflammation response. In this study, we investigated the protective effects of SM934 on the epithelial barrier and the underlying mechanism in trinitrobenzene sulfonic acid (TNBS)-induced colitis mice. We demonstrated that SM934 restored the body weight and colon length, and improved the intestine pathology. Furthermore, SM934 treatment preserved the intestinal barrier function via decreasing the intestinal permeability, maintaining epithelial tight junction (TJ) protein expressions, and preventing apoptosis of epithelial cells, which were observed both in the colon tissue and the tumor necrosis factor-α (TNF-α)-induced human colonic epithelial cell line HT-29. Specifically, SM934 reduced the pyroptosis of IECs exposed to pathogenic signaling and inhibited pyroptosis-related factors such as NOD-like receptor family pyrin domain containing 3 (NLRP3), adapter apoptosis-associated speck-like protein (ASC), cysteine protease-1 (caspase-1), gasdermin (GSDMD), interleukin-18 (IL-18), and high-mobility group box 1 (HMGB1) both in colon tissue and lipopolysaccharide (LPS) and adenosine triphosphate (ATP) co-stimulated HT-29 cells in vitro. Moreover, SM934 interdicted pyroptosis via blocking the transduction of mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways. In conclusion, SM934 protected TNBS-induced colitis against intestinal barrier disruption by inhibiting the apoptosis and pyroptosis of epithelial cells via the NLRP3/NF-κB/MAPK signal axis, and intestinal barrier protection in company with an anti-inflammatory strategy might yield greater benefits in IBD treatment.

Evaluation of Radiosensitive Effect of Tolmetin in Radiotherapy on Human Colonic Carcinoma Cell Line HT-29

Background: Radiotherapy is one of the cancer treatment methods. Although radioresistance and normal tissue toxicity limit the radiation therapy in certain anatomical locations, using some substances can be useful to increase radiosensitivity on cancer cells without cytotoxicity effect on normal cells. Objective: This study aimed to evaluate the radiosensitizing effect of tolmetin in radiotherapy treatment on human colon cancer cell line HT-29. Methods In this study, human colon cancer cells HT-29 in different groups were irradiated with 4 Gy x-ray, and tolmetin was administered in different concentrations (75, 100, and 150 μM). Then, the groups were compared with each other and with the control group by Micronucleus Assay (MN) and Nuclear Division Index (NDI). NDI investigated the cytotoxicity, and MN indicated the genotoxicity. Results: In the group receiving radiation, micronuclei increased significantly compared to the control group. In the group receiving tolmetin with a concentration of 75 and 100 μM, the number of micronuclei also increased compared to the control group. In all groups treated with tolmetin that also received radiation, a significant increase in micronuclei was observed, which was more noticeable at concentrations of 100 and 150 μM. At the same time, tolmetin at the studied concentrations did not change the NDI index. Conclusion: The present study demonstrates that tolmetin has a radiosensitizing effect on HT-29 colon cancer cells, which depends on the tolmetin concentration. In addition, tolmetin has no cytotoxic effect on this cell line.

Strategy for the vibrational circular dichroism study of a glycosylflavonoid evaluated as its peracetate. The case of bioactive 7-O-β-D-glucopyranosylchrysin

As it is difficult to study polyhydroxylated natural products by vibrational circular dichroism (VCD) due to their very limited solubility in infrared transparent solvents, 7-O-β-D-glucopyranosylchrysin (1), isolated from Mimosa rosei, was acetylated to provide 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-glucopyranosyl)-5-acetylchrysin (2). Density functional theory (DFT) VCD spectra of 2 were calculated using the B3LYP, B3PW91, and PBEPBE functionals and the DGDZVP basis set to ascertain a good level of theory for calculating this kind of compounds. Statistical comparison of the experimental and calculated VCD spectra revealed that the frequently employed B3LYP and B3PW91 functionals provided good confidence levels with similar statistical results, allowing band assignments for the pyranoside portion of 2. This is the first report on a satisfactory statistical validation of a VCD measurement for such a glycopyranoside. In addition, in vitro cytotoxicity studies of 1 and 2 against the human colon cell line HT-29 and the human melanoma cell line UACC-62 by the MTT method were assayed. Moreover, the immunomodulatory response of 1 and 2 was evaluated by measuring TNF-α, IL-1β, and IL-10 levels by the ELISA technique. The results revealed low cytotoxicity of both compounds against cancer cells. However, flavonoid 1 upregulated the IL-1β production in lipopolysaccharide (LPS)-induced THP-1 macrophage, thus suggesting the potential of this molecule as an immunostimulant for cancer treatment.

Contribution of Epithelial Apoptosis and Subepithelial Immune Responses in Campylobacter jejuni-Induced Barrier Disruption.

Campylobacter jejuni is a widespread zoonotic pathogen and the leading bacterial cause of foodborne gastroenteritis in humans. Previous infection studies showed disruption of intercellular contacts, induction of epithelial apoptosis, and immune activation, all three contributing to intestinal barrier dysfunction leading to diarrhea. The present study aims to determine the impact of subepithelial immune cells on intestinal barrier dysfunction during Campylobacter jejuni infection and the underlying pathological mechanisms. Infection was performed in a co-culture of confluent monolayers of the human colon cell line HT-29/B6-GR/MR and THP-1 immune cells. Twenty-two hours after infection, transepithelial electrical resistance (TER) was decreased by 58 ± 6% compared to controls. The infection resulted in an increase in permeability for fluorescein (332 Da; 4.5-fold) and for FITC-dextran (4 kDa; 3.5-fold), respectively. In contrast, incubation of the co-culture with the pan-caspase inhibitor Q-VD-OPh during the infection resulted in a complete recovery of the decrease in TER and a normalization of flux values. Fluorescence microscopy showed apoptotic fragmentation in infected cell monolayers resulting in a 5-fold increase of the apoptotic ratio, accompanied by an increased caspase-3 cleavage and caspase-3/7 activity, which both were not present after Q-VD-OPh treatment. Western blot analysis revealed increased claudin-1 and claudin-2 protein expression. Inhibition of apoptosis induction did not normalize these tight junction changes. TNFα concentration was increased during the infection in the co-culture. In conclusion, Campylobacter jejuni infection and the consequent subepithelial immune activation cause intestinal barrier dysfunction mainly through caspase-3-dependent epithelial apoptosis. Concomitant tight junction changes were caspase-independent. Anti-apoptotic and immune-modulatory substances appear to be promising agents for treatment of campylobacteriosis.

WIPF2 promotes Shigella flexneri actin-based motility and cell-to-cell spread.

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin-based motility and formation of membrane protrusions at cell-cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell-to-cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation-promoting factor N-WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N-WASP exists as a complex with WIP. The involvement of WIP in N-WASP-dependent actin-based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT-29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non-motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N-WASP to the bacterial pole, and among N-WASP-positive bacteria, a decrease in actin tail-positive bacteria, suggesting that WIPF2 is required for N-WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

A new host cell internalisation pathway for SadA-expressing staphylococci triggered by excreted neurochemicals.

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

Characterization of the bacteriophages binding to human matrix molecules

Recent literature has suggested a novel symbiotic relationship between bacteriophage and metazoan host that provides antimicrobial defense protecting mucosal surface by binding to host matrix mucin glycoproteins. Here, we isolated and studied different bacteriophages that specifically interact with human extracellular matrix molecules such as fibronectin, gelatin, heparin and demonstrated their potency for protection to host against microbial infections. We showed that subpopulations of bacteriophages that work against clinical isolates of Escherichia coli can bind to pure gelatin, fibronectin and heparin and reduced bacterial load in human colon cell line HT29. The bacteriophages were characterized with respect to their genome sizes, melting curve patterns and host tropism (cross-reactivity with different hosts). Since, the bacteriophages are non-toxic to the host and can effectively reduce bacterial load in HT29 cell line their therapeutic potency against bacterial infection could be explored.

Resveratrol dimer trans-ε-viniferin prevents rotaviral diarrhea in mice by inhibition of the intestinal calcium-activated chloride channel

We previously identified, by a natural-product screen, resveratrol oligomers as inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Here, we report the resveratrol dimer trans-ε-viniferin (TV) and tetramer r-2-viniferin (RV) as inhibitors of the intestinal calcium-activated chloride channel (CaCC) and demonstrate their antisecretory efficacy in a neonatal mouse model of rotaviral diarrhea. Short-circuit measurements show inhibition of CaCC current in the human colonic cell line HT-29 by TV and RV with IC50∼1 and 20μM, respectively. TV primarily inhibited the physiologically relevant, long-term CaCC current following agonist stimulation, without effect on cytoplasmic Ca2+ signaling. TV and RV inhibited short-circuit current in mouse colon as well. In a neonatal mouse model of rotaviral secretory diarrhea produced by oral inoculation with rotavirus, 2μg TV or 11μg RV inhibited secretory diarrhea by >50%, without effect on the rotaviral infection. Our results support the antisecretory efficacy of non-toxic, natural-product resveratrol oligomers for diarrheas produced by CaCC activation. Because these compounds also inhibit the CFTR chloride channel, they may be useful for antisecretory therapy of a wide range of diarrheas.

Inhibitory Effects of Probiotic Lactobacillus on the Growth of Human Colonic Carcinoma Cell Line HT-29.

This study was conducted to investigate the inhibitory effect of Lactobacillus cells and supernatants on the growth of the human colon cancer cell line HT-29. Our study results indicated that the PM153 strain exhibits the best adhesion ability and the highest survival in the gastrointestinal tract simulation experiment. Furthermore, after an 8-h co-culture of PM153 and HT-29 cells, the PM153 strain can induce the secretion of nitric oxide from the HT-29 cells. In addition, after the co-culture of the BCRC17010 strain (109 cfu/mL) and HT-29 cells, the Bax/Bcl-2 ratio in the HT-29 cells was 1.19, which showed a significant difference from the other control and LAB groups (p < 0.05), which therefore led to the inference that the BCRC17010 strain exerts a pro-apoptotic effect on the HT-29 cells. Upon co-culture with HT-29 cells for 4, 8 and 12 h, the BCRC14625 strain (109 cfu/mL) demonstrated a significant increase in lactate dehydrogenase (LDH) activity (p < 0.05), causing harm to the HT-29 cell membrane; further, after an 8-h co-culture with the HT-29 cells, it induced the secretion of nitric oxide (NO) from the HT-29 cells. Some lactic acid bacteria (LAB) strains have ability to inhibit the growth of the colorectal cancer cell line HT-29 Bax/Bcl-2 pathway or NO production. In summary, we demonstrated that the BCRC17010 strain, good abilities of adhesion and increased LDH release, was the best probiotic potential for inhibition of HT-29 growth amongst the seven LAB strains tested in vitro.

1,2-Benzisothiazole Derivatives Bearing 4-, 5-, or 6-Alkyl/arylcarboxamide Moieties Inhibit Carbonic Anhydrase Isoform IX (CAIX) and Cell Proliferation under Hypoxic Conditions.

Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated.

Arcobacter butzleri isolates exhibit pathogenic potential in intestinal epithelial cell models.

The pathogenic potential of Arcobacter butzleri isolates on human (HT-29/B6) and porcine epithelial (IPEC-J2) cells was investigated by invitro assays. Five of six A.butzleri isolates were able to adhere and invade HT-29/B6 cells while only four isolates adhered and two invaded IPEC-J2 cells. Two non- or poorly invasive A.butzleri isolates were highly cytotoxic to differentiated HT-29/B6 cells but none to IPEC-J2 cells as determined by WST-assays. Epithelial integrity of cell monolayers, monitored by measurement of the transepithelial electrical resistance (TER), was decreased by all A.butzleri isolates in HT-29/B6 and IPEC-J2 cells to 30-15% and 90-50% respectively. The A.butzleri strain-specific pathomechanisms observed with the human colon cell line HT-29/B6, like adhesion, invasion and cytotoxicity might all contribute to epithelial barrier dysfunction, which could explain a leak-flux type of diarrhoea in humans. In contrast, porcine cells seem to be less susceptible to A.butzleri. Arcobacter butzleri has enteric pathogenic potential, characterized by defined interactions with human epithelial cells and strain-specific pathomechanisms.

A novel inflammatory regulator TIPE2 inhibits TLR4-mediated development of colon cancer via caspase-8.

TIPE2 is a novel inflammation regulator, and the role of TIPE2 in colitis-induced colon cancer is not clear. The aim of this study was to test whether TIPE2 inhibits TLR4 pathway in colon cancer patients and to explore potential mechanism of TIPE2 in colon cancer by caspase-8. Expression of TIPE2 and TLR4 in human colon cancer tissues and cell line HT-29 was detected by immunohistochemistry or real-time PCR. TIPE2 mRNA was suppressed by siRNA transfection and the transfection efficiency was proved by fluorescence microscopy and real-time PCR. TLR4 pathway was activated by treating the cells with 1 μg/ml LPS for 4 h. Caspase-8 activities were tested by colorimetric assay in four HT-29 cell groups. TIPE2 was expressed in the cytoplasm of colon cancer tissues and HT-29 cells. TIPE2 expression was more pronounced in colon cancer tissues compared to normal controls and it was related with lymph node metastasis and Dukes stage of colon cancer. TIPE2 expression was positively correlated with that of TLR4 in colon cancer (r=0.7354). TIPE2 expression was knocked down successfully by siRNA transfection. Caspase-8 activity was elevated both in TIPE2 knockdown cells and in TLR4 activated cells compared to wild-type cells (P< 0.05). And the caspase-8 activity was further increased in TIPE2 knockdown cells after TLR4 was activated (P < 0.05). TIPE2 can inhibit caspase-8 activity in colon cancer cells. TIPE2 can regulate TLR4 inflammatory effect and inhibit further amplification of cascade reaction via caspase-8, which provides one new therapeutic target for clinical treatment schedule.

Determination of Cell Death Induced by Lovastatin on Human Colon Cell Line HT29 Using the Comet Assay

Apoptosis or programmed cell death is an essential process for elimination of damaged cells. Also, induction of apoptosis is fundamental for treating cancer. Screening for agents that induce apoptosis in tumor cells help in the development of novel agents for cancer treatment. Numerous studies suggest that the exposure of tumor cells to statins can lead to cell death via two separate processes: apoptosis or necrosis. Severe fragmentation of DNA during apoptosis can be readily measured by the neutral comet assay. Migration of DNA fragments of apoptotic cells by the electrical field can produce comet-like images. The aim of this study was to determine the type of cell death induced by lovastatin on human colon tumor cells by using the neutral comet assay and to evaluate the utility of this method for detection of apoptosis. HT29 cells were grown in DMEM medium then exposed to different concentrations of lovastatin, and DNA fragmentation associated with apoptosis was detected by the neutral comet assay method. Lovastatin induced an apoptotic response in the HT29 cell line and produced a comet pattern similar to the positive control. This study showed that lovastatin can induce apoptosis in the HT29 cell line and confirmed the utility of comet assay for detection of apoptosis.

PTH-008 Photodynamic Antimicrobial Chemotherapy (PACT) Selectively Kills Clostridium Difficile over Colon Cells and is Effective Against 5 Hypervirulent Strains of the Pathogen

IntroductionClostridium difficile (CD) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea in the developed world. Since 2003, a new lineage of strains with more severe virulence has emerged,...

996 Photodynamic Antimicrobial Chemotherapy (PACT) Selectively Kills Clostridium difficile Over Colon Cells and Is Effective Against 5 Hypervirulent Strains of the Pathogen

Introduction Clostridium difficile (CD) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea in the developed world. Since 2003, a new lineage of strains with more severe virulence has emerged, leading to an increased number of outbreaks of disease in North America and Europe and raising the impellent need for an effective therapy. Photodynamic Antimicrobial Chemotherapy (PACT) utilises the ability of light-activated photosensitisers (PS) to produce free radical species lethal to the target pathogens. To date, no pathogens have developed resistance to PACT. This study aimed to develop and evaluate PACT for the treatment of CD . Methods High throughput screening of 15 photosensitiser (PS) drugs were performed in aerobic conditions against the hypervirulent R20291 strain of CD . These included both clinically approved PS drugs and experimental PS’s engineered for CD . Lead candidate agents were then tested against C. difficile strain R20291 in microaerophilic and anaerobic conditions, against 4 of the other most clinically significant hypervirulent CD strains, each belonging to a different ribotype, and against the human colonic cell line HT-29 at effective antimicrobial doses to exclude background colonic cytotoxicity. Results Nine PS were successful in killing 99.99% of R20291 at a concentration of 10 μM after exposure to laser light at 665 nm at an intensity of 24 mJ/cm2. Remarkably, three of them (S4, CE6 and PS4) also reduced bacterial growth by 99.9% in absence of oxygen at the concentration of 50 μM and no PS-associated toxicity was observed in the absence of light. PACT was found to be similarly effective against all 5 hypervirulent CD strains. Three PS were not toxic to HT-29 cells at effective antimicrobial concentrations. Conclusion We have found PACT effectively kills the 5 most clinically relevant hypervirulent CD strains. PACT efficacy traditionally is thought to require oxygen to generate reactive oxygen species. We have shown PACT to be effective in anaerobic conditions mimicking the colonic microenvironment in which CD reside. As PACT was not toxic to human HT-29 cells at effective antimicrobial doses, this would permit selective targeting of the pathogen in the site of infection. It is believed the research being undertaken could be an important step towards the eradication of C. difficile colitis. Disclosure of Interest None Declared.

PPARγ as a sensor of lipase activity and a target for the lipase inhibitor orlistat

A PPARγ fluorescence polarization (FP) assay was used to measure the release of fatty acid products from triglyceride emulsions during digestion with pancreatic and yeast lipases in a real-time, homogenous assay. Using the same FP assay we show the anti-obesity drug Orlistat is a PPARγ ligand with an IC50 of 2.84 ± 0.16 μM. Analytical Mass Spectrometry confirms that Orlistat does not bind covalently to PPARγ. The PPARγ FP assay is shown to be a simple method for measuring real-time lipase activity using a number of triglyceride substrates including olive oil and grape seed oil emulsions. Incubation of Orlistat with the human intestinal epithelial cell line Caco-2, at concentrations of 1 - 100 μM, leads to induction of genes regulated by PPARγ. At 100 μM Orlistat, transcription of β-defensin 1 (hDB1) & Adipose Differentiation Related Protein (ADRP) increase by up to 2.6 fold and 6.8 fold, respectively. Although at 1 μM and 100 μM Orlistat did not significantly increase defensin protein synthesis, at 10 μM Orlistat induced a 1.5 fold increase in hDB1 protein secretion in the human colonic adenocarcinoma cell line HT-29. Thus Orlistat is similar to the anti-diabetic drug Rosiglitazone in its ability to induce defensin gene expression. The antimicrobial peptide β-defensin 1 protects against pathogenic micro-organisms in the gut and PPARγ suppresses inflammatory gene expression. These may be beneficial side effects of Orlistat consumption on gut epithelial cells.

Clostridium difficile Toxin A Inhibits Erythropoietin Receptor-Mediated Colonocyte Focal Adhesion Through Inactivation of Janus Kinase-2

Previously, we demonstrated that the erythropoietin receptor (EpoR) is present on fibroblasts, where it regulates focal contact. Here, we assessed whether this action of EpoR is involved in the reduced cell adhesion observed in colonocytes exposed to Clostridium difficile toxin A. EpoR was present and functionally active in cells of the human colonic epithelial cell line HT29 and epithelial cells of human colon tissues. Toxin A significantly decreased activating phosphorylations of EpoR and its downstream signaling molecules JAK-2 (Janus kinase 2) and STAT5 (signal transducer and activator of transcription 5). In vitro kinase assays confirmed that toxin A inhibited JAK 2 kinase activity. Pharmacological inhibition of JAK2 (with AG490) abrogated activating phosphorylations of EpoR and also decreased focal contacts in association with inactivation of paxillin, an essential focal adhesion molecule. In addition, AG490 treatment significantly decreased expression of occludin (a tight junction molecule) and tight junction levels. Taken together, these data suggest that inhibition of JAK2 by toxin A in colonocytes causes inactivation of EpoR, thereby enhancing the inhibition of focal contact formation and loss of tight junctions known to be associated with the enzymatic activity of toxin A.

Effects of quercetin studied in colonic HT‐29/B6 cells and rat intestine in vitro

The aim of this study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line HT-29/B6 and rat small and large intestine in vitro. Rat native ileum and late distal colon were incubated in Ussing chambers, and the total resistance (R(T) ) was measured, and expression of tight junction proteins was characterized in immunoblots. By simulating inflammatory conditions with TNF-α, we examined the barrier-preventive effects of quercetin. Incubation with TNF-α led to a decrease of R(T) in HT-29/B6 cell monolayers, which could be partially inhibited by quercetin. In accordance with cell culture experiments, quercetin increased mucosal resistance of rat ileum and late distal colon. Thus, barrier disturbance in late distal colon specimens induced by TNF-α and IFN-γ could be partially prevented by coincubation with quercetin. These findings demonstrate that quercetin enhances barrier function in rat small and large intestine and possesses protective effects on cytokine-induced barrier damage.

MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice

The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.

Determination of cell death induced by lovastatin on human colon cell line HT29 using comet assay

Determination of cell death induced by lovastatin on human colon cell line HT29 using comet assay