The Schlafen family: complex roles in different cell types and virus replication.

The Schlafen (slfn) gene family members express broadly, but the research has mainly focused on human slfn (h-slfn) and mouse slfn (m-slfn). The slfn members can be divided into three groups, and each group has its own characteristics and functions. Although the effects of slfns are still poorly understood, it has been confirmed that slfns are involved in the defense of immune system and regulate immune cells' proliferation and differentiation. In some malignant tumors, the slfn proteins can inhibit the growth and invasion of cancer cells, promote cancer cells sensibility to chemotherapeutics, and can be a promising new therapeutic target. In addition, the slfn proteins also disturb replication and virulence of viruses. In this review, we summarize the characteristics of the Schlafen family's structures and functions with the aim to achieve a more comprehensive understanding of slfns.

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.

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -ß, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3Cpro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.

Surface glycosaminoglycans protect eukaryotic cells against membrane-driven peptide bacteriocins.

Enzymatic elimination of surface glycosaminoglycans or inhibition of their sulfation provokes sensitizing of HT-29 and HeLa cells toward the peptide bacteriocins nisin A, plantaricin C, and pediocin PA-1/AcH. The effect can be partially reversed by heparin, which also lowers the susceptibility of Lactococcus lactis to nisin A. These data indicate that the negative charge of the glycosaminoglycan sulfate residues binds the positively charged bacteriocins, thus protecting eukaryotic cells from plasma membrane damage.

PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

Nuclear factor-κB (NFκB) and peroxisome proliferator activated receptor-γ (PPARγ) are both transcription factors that perform distinct but overlapping roles in cellular regulation. Here we report that PPARγ acts as an E3 ubiquitin ligase, physically interacting with p65 to induce its ubiquitination and degradation. The ligand-binding domain of PPARγ interacts with the Rel Homology Domain region of NFκB/p65 to undergo robust ubiquitination and degradation that was independent of PPARγ transcriptional activity. Moreover, the ligand-binding domain of PPARγ delivered Lys48-linked polyubiquitin, resulting in the ubiquitination and degradation of p65. Lys28 was found to be critically important for PPARγ-mediated ubiquitination and degradation of p65, as it terminated both NFκB/p65-mediated pro-inflammatory responses and xenograft tumours. These findings demonstrate that PPARγ E3 ubiquitin ligase activity induces Lys48-linked ubiquitination and degradation of p65, and that this function is critical to terminate NFκB signalling pathway-elicited inflammation and cancer.

Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells.

BACKGROUND: Epidemiological and experimental evidence that support the correlation between Type 2 diabetes mellitus (T2D) and increased risks of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and canonical Wnt signaling pathways. Insulin was shown to stimulate Wnt target gene expression, utilizing the effector of the Wnt signaling pathway. Whether insulin affects expression of components of Wnt pathway has not been extensively examined. METHODS: cDNA microarray was utilized to assess the effect of insulin on gene expression profile in the rat intestinal non-cancer IEC-6 cell line, followed by real-time RT-PCR, Western blotting and reporter gene analyses in intestinal cancer and non-cancer cells. RESULTS: Insulin was shown to alter the expression of a dozen of Wnt pathway related genes including TCF-4 (=TCF7L2) and frizzled- (Fzd-4). The stimulatory effect of insulin on TCF-4 expression was then confirmed by real-time RT-PCR, Western blotting and luciferase reporter analyses, while the activation on Fzd-4 was confirmed by real-time PCR. GENERAL SIGNIFICANCE: Our observations suggest that insulin may crosstalk with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.

7-ketocholesterol induces endoplasmic reticulum stress in HT-29 cells.

7-Ketocholesterol (7-Kchol, oxidized cholesterol) is an important mediator of cell death in atherosclerosis mediated by up-regulated Nox 4 gene expression. In the current study using the human colon cancer HT-29 cell line, we have demonstrated that 7-Kchol promotes endoplasmic reticulum (ER) stress via gene up-regulation of ER chaperone and membrane kinases.

Anti-inflammatory activity of probiotic Bifidobacterium: enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells.

AIM: To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms. METHODS: Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted. RESULTS: Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-alpha, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IkappaB-zeta mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM. CONCLUSION: Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.

Diosgenin, a steroid saponin of Trigonella foenum graecum (Fenugreek), inhibits azoxymethane-induced aberrant crypt foci formation in F344 rats and induces apoptosis in HT-29 human colon cancer cells.

Trigonella foenum graecum (fenugreek) is traditionally used to treat disorders such as diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Recent studies suggest that fenugreek and its active constituents may possess anticarcinogenic potential. We evaluated the preventive efficacy of dietary fenugreek seed and its major steroidal saponin constituent, diosgenin, on azoxymethane-induced rat colon carcinogenesis during initiation and promotion stages. Preneoplastic colonic lesions or aberrant crypt foci (ACF) were chosen as end points. In addition, we assessed the mechanism of tumor growth inhibition of diosgenin in HT-29 human colon cancer cells. To evaluate the effect of the test agent during the initiation and postinitiation stages, 7-week-old male F344 rats were fed experimental diets containing 0% or 1% fenugreek seed powder (FSP) or 0.05% or 0.1% diosgenin for 1 week and were injected with azoxymethane (15 mg/kg body weight). Effects during the promotional stage were studied by feeding 1% FSP or 0.1% diosgenin 4 weeks after the azoxymethane injections. Rats were sacrificed 8 weeks after azoxymethane injection, and their colons were evaluated for ACF. We found that, by comparison with control, continuous feeding of 1% FSP and 0.05% and 0.1% diosgenin suppressed total colonic ACF up to 32%, 24%, and 42%, respectively (P < or = 0.001 to 0.0001). Dietary FSP at 1% and diosgenin at 0.1% fed only during the promotional stage also inhibited total ACF up to 33% (P < or = 0.001) and 39% (P < or = 0.0001), respectively. Importantly, continuous feeding of 1% FSP or 0.05% or 0.1% diosgenin reduced the number of multicrypt foci by 38%, 20%, and 36% by comparison with the control assay (P < or = 0.001). In addition, 1% FSP or 0.1% diosgenin fed during the promotional stage caused a significant reduction (P < or = 0.001) of multicrypt foci compared with control. Dietary diosgenin at 0.1% and 0.05% inhibited total colonic ACF and multicrypt foci formation in a dose-dependent manner. Results from the in vitro experiments indicated that diosgenin inhibits cell growth and induces apoptosis in the HT-29 human colon cancer cell line in a dose-dependent manner. Furthermore, diosgenin induced apoptosis in HT-29 cells at least in part by inhibition of bcl-2 and by induction of caspase-3 protein expression. On the basis of these findings, the fenugreek constituent diosgenin seems to have potential as a novel colon cancer preventive agent.

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.

Nuclear localization of proteasomes participates in stress-inducible resistance of solid tumor cells to topoisomerase II-directed drugs.

Physiological cell conditions of solid tumors, such as glucose starvation and hypoxia,induce cellular resistance to topoisomerase II-directed drugs. Here, we show that the induction of drug resistance is mediated by nuclear accumulation of proteasomes, large multicatalytic protease complexes. We found that the nuclear proteasome accumulation during glucose starvation was attenuated by stable expression of a mutant type of proteasome subunit, XAPC7, that lacked the nuclear localization signal (NLS). It is important that the expression of NLS-defective XAPC7 also diminished the induction of resistance to etoposide and doxorubicin, typical topoisomerase II-directed drugs. Under normal conditions, however, the NLS-defective XAPC7 had little effect on either nuclear proteasome distribution or etoposide sensitivity. Our findings demonstrate that stress-induced nuclear proteasome accumulation occurs through up-regulation of the NLS-dependent transport. Inhibition of the nuclear proteasome accumulation can be a novel approach to circumventing resistance to topoisomerase II-directed drugs.

Altered signaling of TNFalpha-TNFR1 and SODD/BAG4 is responsible for radioresistance in human HT-R15 cells.

Radioresistance in pre-irradiated human HT-R15 cells is associated with changes of the TNFR1-dependent death pathway. HT-R15 cells are characterized by elevated protein levels of TNFR1 and TNF and by increased sensitivity to exogenous TNFalpha compared to their parental HT-29 cells. Alterations are also observed downstream in the signaling cascade, such as the activation of NF-kappaB or the overexpression of the death domain kinase RIP and reduced caspase 8 activity. However, these changes seem to be a consequence of defective upstream TNFalpha signaling rather than the primary cause of cellular resistance in HT-R15 cells. Of major importance for resistance in HT-R15 cells is the silencer of death domain, SODD/BAG4. Following gamma-irradiation, the membrane-associated 49 kDa SODD decreases in the parental but not in the resistant cells, whereas after TNFalpha treatment, SODD expression declines only in the resistant cells. A 42 kDa cytoplasmic SODD protein is detected, which is elevated only in the resistant cells. This SODD protein is not involved in the regulation of cell survival after radiation or TNFalpha treatment but rather in altered TNFR1 shedding. Inhibition of TNFR1 release by the metalloprotease inhibitor BB-2516 results in a significant increase of the 42 kDa SODD protein without affecting cell survival in sensitive or resistant HT cells. Moreover, TNFR1 release into the culture medium is augmented in the resistant cells. These results suggest that defective TNFalpha signaling and/or altered silencing by SODD/BAG4 in HT-R15 cells are involved in the radiation resistance of HT-R15 cells and also affect the paracrine functions of TNFR1. Resistance is circumvented by TNFalpha treatment, independent of cytoplasmic TNFalpha/TNFR1 functions.

Early oncogene mRNA expression in HT-29 cells treated with the endogenous colon mitosis inhibitor pyroglutamyl-histidyl-glycine.

BACKGROUND: The tripeptide pyroGlu-His-GlyOH (pEHG), isolated from intestinal extracts (1), suppresses growth of human colonic epithelial cells and human colorectal adenocarcinoma cells (HT-29) both in vitro and in vivo. The present study represents the first attempt to relate alterations in relevant gene expression to the effect on cell growth. MATERIALS AND METHODS: Northern blot with RNA, extracted from HT-29 cells exposed to pEHG, was hybridised with c-fos and c-myc probes. RESULTS: c-fos gene expression was doubled in HT-29 cells after 20 minutes of incubation with pEHG and decreased to half the initial value after 2 hours. The increase at 20 minutes was concentration-dependent at a peptide concentration range from 10(-6)-10(-12) M. The expression of the oncogene c-myc showed only marginal alternations at the concentrations and times tested. CONCLUSION: The colon mitosis-inhibiting peptide pEHG increases gene expression of c-fos, but not that of c-myc.

Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus.

BACKGROUND: Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines. MATERIALS AND METHODS: We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice. RESULTS: Stable HT-29 clones with > 97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression. CONCLUSION: This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.

Evidence that APC regulates survivin expression: a possible mechanism contributing to the stem cell origin of colon cancer.

Because colorectal cancers (CRCs) frequently display APC mutation, inhibition of apoptosis, and increased expression of the antiapoptotic protein survivin, we hypothesized that APC mutation inhibits apoptosis by allowing constitutive survivin expression. Using HT-29 CRC cell lines having inducible wild-type APC (wt-APC) or transfected dominant-negative TCF-4, we show that wt-APC down-regulates survivin expression via APC/beta-catenin/TCF-4 signaling. Using normal colonic epithelium, we found survivin by immunostaining/reverse transcription-PCR to be preferentially expressed in the lower crypt (which inversely correlates with wt-APC's expression pattern). Thus, wt-APC, by progressively decreasing survivin and increasing apoptosis from crypt bottom to top, may limit the population size of stem cells and other proliferative cells in the lower crypt; mutant APC may allow expansion of these populations, thereby initiating tumorigenesis.

Overexpression of folate binding protein alpha is one of the mechanism explaining the adaptation of HT29 cells to high concentration of methotrexate.

The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate (MTX). Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10(-4) MTX HT29 cells. The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells. In contrast to 10(-7) M MTX cells, growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both DHFR and FBPalpha. Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells.

In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor.

TR6, a member of the tumor necrosis factor (TNF) receptor superfamily, has recently been shown to bind to Fas ligand (FasL) and inhibit FasL-mediated cell killing in vitro. In the current study, we demonstrate that TR6 can block the lethal activity of FasL in multiple in vitro systems, and extend this finding to an in vivo model of hepatitis. The binding of human TR6 to human FasL was verified with BIAcore chip technology. Human primary hepatocytes, HT-29 cells and Jurkat cells were assayed for viability to demonstrate TR6 inhibition of FasL-mediated cytotoxicity in vitro. Human TR6 was also shown to cross-react with membrane-bound mouse FasL, since the in vitro cytotoxic activity of L929 cells transfected with murine FasL was inhibited in the presence of human TR6. In vivo, FasL-induced acute, lethal, fulminant hepatic apoptosis resulting in death within 2 h of intravenous injection into Fas+ mice, but not Fas- MRL/lpr mice. Pretreatment of mice with TR6 blocked FasL-induced mortality, presumably by attenuating FasL-induced hepatic apoptosis. Thus, in both in vitro and in vivo systems, TR6 acts as a functional FasL decoy receptor and may be clinically useful in the treatment of hepatitis and other diseases associated with FasL-mediated tissue injury.

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

Changes in the noninvasive, in vivo electrical impedance of three xenografts during the necrotic cell-response sequence.

PURPOSE: To investigate the noninvasive, in vivo use of electrical impedance spectroscopy (EIS) as a method for observing the real-time, cellular-level responses of a volume of tissue to therapies. Here, we studied the EIS response during the development and progression of hyperthermia-induced coagulative necrosis in three diverse human xenografts. METHODS AND MATERIALS: A necrotic cell response sequence was selectively induced in three types of subcutaneously-grown human tumor xenografts by applying hyperthermia at 44.5 degrees C. The electrical impedance of the tumors was measured from 100 Hz to 10 MHZ, noninvasively, in vivo during the treatments. From the full spectrum EIS, ratios between resistivities at selected frequencies (p-ratios) were used as indicators of the changes in the electrical impedance spectra of each tumor's cell population. RESULTS: The rho-ratios consistently demonstrated characteristic, early, rapid increases which coincided with cell and organelle swelling typical of early necrosis. These increases subsequently slowed, but no decrease began before the end of treatment, unlike previous, similarly treated, thermo-sensitive EMT6 mouse tumors. This was consistent with the xenograft histology, which revealed ubiquitous, early-stage coagulative necrosis, with no gross plasma membrane damage at the end of treatment. The extent of both the necrosis and p-ratio changes were similar to those seen early in the EMT6 tumor treatment. Within several days after treatment, the xenograft volumes regressed nearly completely, suggesting completion of the cell populations' necrotic response (lysing) during this period. Consistent with this, extended EIS measurements over a 24-h posttreatment period allowed tracking of the necrotic response sequence through this lysing phase for one type of xenograft. CONCLUSION: The change in the electrical impedance of a volume of tumor tissue which occurs during and/or after a hyperthermia treatment can be correlated with the extent of necrosis observed histologically in the cell population.

Ropivacaine inhibits serum-induced proliferation of colon adenocarcinoma cells in vitro.

Ropivacaine, a new long-acting local anesthetic, is currently being investigated for the treatment of ulcerative colitis. In view of the increased incidence of dysplasia and neoplasia associated with ulcerative colitis, it is important that the medical treatment of these patients does not stimulate cell proliferation further. This study was performed to evaluate the effect of ropivacaine on the proliferation of human colon adenocarcinoma cells (HT-29 and Caco-2) in vitro. A serum-induced proliferation assay of human colon adenocarcinoma cells was used. Ropivacaine inhibited the growth of HT-29 and Caco-2 cells in a dose-dependent manner. Fifty percent inhibition of growth was found at a ropivacaine concentration of 250 microM when the HT-29 cells were cultured in 1% fetal calf serum and of 550 microM when the HT-29 cells were cultured in 10% serum. The effective concentrations are within the range of the therapeutic concentrations obtained in the colon of patients treated rectally with ropivacaine. Lidocaine, hydrocortisone, and 5-aminosalicylic acid were found to be less potent than ropivacaine in inhibiting proliferation. Ropivacaine caused a dose-dependent membrane depolarization that appeared to correlate with the inhibited cell proliferation, whereas the effect was not related to inhibition of leukotriene B4 or prostaglandin E2. In conclusion, the antiproliferative activity of ropivacaine, combined with previously reported anti-inflammatory activities, makes this drug an interesting new alternative for the local treatment of ulcerative colitis.