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PubMed1998

Acylation of naturally occurring and synthetic 1-deoxysphinganines by ceramide synthase. Formation of N-palmitoyl-aminopentol produces a toxic metabolite of hydrolyzed fumonisin, AP1, and a new category of ceramide synthase inhibitor.

Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro- and threo-2-amino, 3-hydroxy- (and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with Vmax/Km of 40-125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4-6 for the threo-isomers. Ceramide synthase also acylates AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a Vmax/Km of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro- and threo-isomers are acylated with the highest apparent Vmax/Km for the erythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins.

Acylation↗
PubMed2004

Activator protein 2alpha associates with adenomatous polyposis coli/beta-catenin and Inhibits beta-catenin/T-cell factor transcriptional activity in colorectal cancer cells.

In most human colorectal cancers, mutations in the adenomatous polyposis coli gene (APC) or CTNNB1 constitutively activate the beta-catenin/T-cell factor (TCF)/lymphoid enhancer factor (LEF) signaling pathway. Here, we show that the transcription factor activator protein (AP)-2alpha inhibited a beta-catenin/TCF-responsive reporter in human embryonic kidney 293 cells and in two human colorectal cancer lines, despite the fact that beta-catenin and TCF-4 protein levels were unchanged in the nucleus. Co-immunoprecipitation studies revealed that AP-2alpha formed a complex with APC and beta-catenin and that AP-2alpha disrupted beta-catenin/TCF-4 interactions in the nucleus. Thus, AP-2alpha.APC.beta-catenin complex formation appears to suppress beta-catenin transactivation by shifting the pool of nuclear beta-catenin toward an inactive form, having reduced binding to TCF/LEF transcription factors. Glutathione S-transferase pull-down assays showed that AP-2alpha physically associated with APC rather than with beta-catenin, and the AP-2alpha binding site was identified in the N terminus of APC, involving both the heptad and armadillo repeat domains, whereas the APC binding site in AP-2alpha was in the basic region of the C-terminal DNA binding domain. These findings provide the first evidence for a specific interaction between the tumor suppressor protein APC and the transcription factor AP-2alpha, and they suggest a link between the Wnt signaling pathway and various other pathways of development and differentiation associated with AP-2alpha.

Cytoskeletal Proteins↗
PubMed2006

A growth-suppressive function for the c-fes protein-tyrosine kinase in colorectal cancer.

The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.

Cell Line↗
PubMed2006

DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: the lack of this suppression in p53MUT cells contributes to apoptosis.

DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53(WT) colorectal carcinoma cells a long term cell cycle arrest and in p53MUT cells a transient arrest followed by apoptosis (Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherübl, H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Int. J. Cancer 101, 23-31; Bhonde, M. R., Hanski, M. L., Notter, M., Gillissen, B. F., Daniel, P. T., Zeitz, M., and Hanski, C. (2006) Oncogene 25, 165-175). The mechanism of the p53-independent apoptosis still remains largely unclear. Here we used five p53WT and five p53MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53MUT cells than in the cell cycle-arrested p53WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53MUT cells after SN-38 treatment contributes to DNA damage-induced apoptosis, whereas their suppression in p53WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53MUT cells activates the checkpoint and contributes to apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Vangl1 protein acts as a downstream effector of intestinal trefoil factor (ITF)/TFF3 signaling and regulates wound healing of intestinal epithelium.

The intestinal trefoil factor (ITF/TFF3) protects intestinal epithelia from a range of insults and contributes to mucosal repair. However, the signaling events that mediate healing responses are only partially understood. To identify ITF signaling pathways, proteins that were Ser/Thr phosphorylated in response to ITF stimulation were immunoprecipitated from human colon carcinoma cell lines and identified by mass spectrometry. We demonstrated that Van Gogh-like protein 1 (also designated Vang-like 1 or Vangl1), a protein with four transmembrane domains, was Ser/Thr phosphorylated in response to ITF stimulation. Vangl1 was present in normal human colon and all intestinal epithelial cell lines (IEC) tested. In transfected IEC, FLAG-Vangl1 was mostly present in the Nonidet P-40 soluble fraction as detected by Western blotting, corresponding to the localization of endogenous protein in cytoplasmic vesicular structures by confocal microscopy with rabbit polyclonal anti-human Vangl1 antibody (alpha-Vangl1). Vangl1 cell membrane association increased with differentiation, as demonstrated by co-localization with E-cadherin in differentiated IEC. Increased Vangl1 phosphorylation after stimulation with ITF corresponded to decreased cell membrane association with E-cadherin. Functionally, Vangl1 overexpression enhanced ITF unstimulated and stimulated wound closure of IEC, whereas siRNA directed against Vangl1 inhibited the migratory response to ITF. Vangl1 protein may serve as an effector mediating the ITF healing response of the intestinal mucosa.

Animals↗
PubMed2006

Characterization of mechanisms involved in secretion of active heparanase.

Heparanase is an endo-beta-D-glucuronidase involved in extracellular matrix remodeling and degradation and implicated in tumor metastasis, angiogenesis, inflammation, and autoimmunity. The enzyme is synthesized as a latent 65-kDa protein and is processed in the lysosomal compartment to an active 58-kDa heterodimer, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion. Here we show that secretion of the active enzyme is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was observed upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, as well as by the noncleavable ATP analogue adenosine 5'-O-(thiotriphosphate). Indeed, heparanase secretion was noted upon cell stimulation with a specific P2Y1 receptor agonist and was inhibited by P2Y receptor antagonists. The kinetics of heparanase secretion resembled the secretion of cathepsin D, a lysosomal enzyme, indicating that the secreted heparanase is of lysosomal origin. We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways. The secreted enzyme(s) then facilitate cell invasion associated with cancer metastasis, angiogenesis, and inflammation.

Adenosine Diphosphate↗
PubMed2002

Evidence for consistent patterns between flavonoid structures and cellular activities.

A wide variety of plant-derived compounds, including the polyphenolic flavonoids, is present in the human diet or is consumed for medicinal reasons. Epidemiological and animal studies tend to suggest a protective effect of flavonoids against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological activity are still largely unknown. Antioxidant properties of the flavonoids have been postulated as a mechanism for putative protective effect against cardiovascular disease. Nevertheless, these properties alone are not sufficient to explain the anti-carcinogenic potential of these polyphenols. The mechanisms by which the molecules interact with cells or are absorbed by them are very important for determining the intracellular concentration and distribution of the metabolites to internal organs. With the exception of the cells lining the gastrointestinal tract, all other cells in the body are only exposed to flavonoid metabolites and degradation products. No previous studies have addressed this aspect of cellular exposure, except for some methylated metabolites. Within the last decade, reports on flavonoid activities have been largely associated with enzyme inhibition and anti-proliferative activity. From our recent work on the human colon cancer cell line HT29 and comparison with published studies, structure-function relationships demonstrate that antioxidant, enzyme inhibitor or anti-proliferative activities are dependent on particular structure motifs. The present review also presents a summary of mechanistic data on a few elected compounds.

Animals↗
PubMed2001

Corticosteroids impair intestinal epithelial wound repair mechanisms in vitro.

BACKGROUND: Despite enormous progress in the medical treatment of inflammatory bowel diseases (IBD), corticosteroids still represent the most effective drugs in the management of acute IBD. Unfortunately, surgical intervention under concomitant therapy with corticosteroids is often complicated by impaired intestinal wound healing. Our aim was to assess the effects of the corticosteroids prednisolone and budesonide on different aspects of intestinal epithelial wound healing in vitro to identify potential causes for impaired intestinal wound healing under corticosteroid therapy. METHODS: The effects of both corticosteroids on intestinal epithelial cell function were studied in non-transformed small intestinal epithelial intestinal epithelial cell line IEC-6 cells and human colon cancer-derived HT-29 cells. Effects on epithelial migration were assessed using an in vitro wounding model. Effects on epithelial cell proliferation were assessed using colorimetric 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assays. Transforming Growth Factor beta (TGFbeta) mRNA and protein expression were determined by semi-quantitative RT-PCR and ELISA. RESULTS: Prednisolone and budesonide caused a significant dose-dependent inhibition of intestinal epithelial cell migration and proliferation in IEC-6 and HT-29 cells. Both corticosteroids induced apoptosis of intestinal epithelial cells in a dose-dependent fashion. Neither corticosteroid modulated the expression of TGFbeta mRNA and the synthesis of TGFbeta peptide. However, both corticosteroids stimulated the bioactivation of latent TGFbeta peptide. CONCLUSIONS: Prednisolone and budesonide inhibit intestinal epithelial cell restitution and proliferation in vitro. Both processes play a key role in the rapid resealing of the mucosal barrier following intestinal injury. Thus, impaired intestinal epithelial wound healing under corticosteroid therapy in vivo may be caused by inhibition of intestinal epithelial cell restitution and proliferation.

Anti-Inflammatory Agents↗
PubMed2006

The magnitude and time-dependence of the apoptotic response of normal and malignant cells subjected to ionizing radiation versus hyperthermia.

PURPOSE: The purpose of the study was to examine the optimal time of exposure and dose of heat and ionizing radiation that results in the killing of human cancer cells in vitro via apoptosis vs. necrosis. MATERIALS AND METHODS: Human mammary carcinoma, colorectal carcinoma and normal bovine capillary endothelial (BCE) cell lines were subjected to 20 Gy ionizing radiation and 6, 12, 24, and 72 h later assessed for apoptosis using detection of apoptotic bodies and caspase assays. Necrosis was detected by loss of cells from the surface and lactate dehydrogenase (LDH) release. The colorectal carcinoma cells were subjected to hyperthermia using temperatures ranging from 39 - 44 degrees C for 5, 15 or 45 min. exposures and at varying times post-treatment, apoptosis and necrosis were measured. RESULTS: In response to ionizing radiation, none of the cells underwent necrosis and some cell types apoptosed 24 and 72 h posttreatment. The colorectal cancer cells exhibited a steady increase of apoptosis at 6, 12, and 24 h. When these cells were exposed to 40 degrees C for 5 min, caspases increased within 6 h and a significant fraction (50%) of cells apoptosed. If the time of exposure to 40 degrees C was increased to 15 or 45 min, 80% and 100% of the dying cells apoptosed, respectively. A temperature of 39 degrees C did not cause cell death even after 45 min exposures. If heat was elevated to 42 or 44 degrees C, increased necrosis was observed with a corresponding decrease in apoptosis. CONCLUSIONS: These studies reveal time and temperature dependent in vitro cell responses to ionizing radiation and water-bath hyperthermia.

Animals↗
PubMed2000

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

Animals↗
PubMed2006

The pro-inflammatory cytokines, IL-1beta and TNF-alpha, inhibit intestinal alkaline phosphatase gene expression.

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Acetylation↗
PubMed2005

A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo.

Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bifidobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture.

Animals↗
PubMed2006

Decreased hephaestin activity in the intestine of copper-deficient mice causes systemic iron deficiency.

Copper and iron metabolism intersect in mammals. Copper deficiency simultaneously leads to decreased iron levels in some tissues and iron deficiency anemia, whereas it results in iron overload in other tissues such as the intestine and liver. The copper requirement of the multicopper ferroxidases hephaestin and ceruloplasmin likely explains this link between copper and iron homeostasis in mammals. We investigated the effect of in vivo and in vitro copper deficiency on hephaestin (Heph) expression and activity. C57BL/6J mice were separated into 2 groups on the day of parturition. One group was fed a copper-deficient diet and another was fed a control diet for 6 wk. Copper-deficient mice had significantly lower hephaestin and ceruloplasmin (approximately 50% of controls) ferroxidase activity. Liver hepcidin expression was significantly downregulated by copper deficiency (approximately 60% of controls), and enterocyte mRNA and protein levels of ferroportin1 were increased to 2.5 and 10 times, respectively, relative to controls, by copper deficiency, indicating a systemic iron deficiency in the copper-deficient mice. Interestingly, hephaestin protein levels were significantly decreased to approximately 40% of control, suggesting that decreased enterocyte copper content leads to decreased hephaestin synthesis and/or stability. We also examined the effect of copper deficiency on hephaestin in vitro in the HT29 cell line and found dramatically decreased hephaestin synthesis and activity. Both in vivo and in vitro studies indicate that copper is required for the proper processing and/or stability of hephaestin.

Anemia, Iron-Deficiency↗
PubMed2000

Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis.

Current opinion assumes epithelial integrity during spontaneous apoptotic cell death. We measured, for the first time, the local conductances associated with apoptoses and show leaks of up to 280 nS (mean 48 +/- 19 nS) in human intestinal epithelium. The results disprove the dogma that isolated cell apoptosis occurs without affecting the epithelial cell permeability barrier. After induction by tumor necrosis factor alpha (TNF-alpha) the apoptotic leaks were dramatically enhanced: not only was the frequency increased by threefold, but the mean conductance also increased by 12-fold (597+/-98 nS). Thus, apoptosis accounted for about half (56%) of the TNF-alpha-induced permeability increase whereas the other half was caused by degradation of tight junctions in nonapoptotic areas. Hence, spontaneous and induced apoptosis hollow out the intestinal barrier and may facilitate loss of solutes and uptake of noxious agents.

Apoptosis↗
PubMed2001

The homocamptothecin BN 80915 is a highly potent orally active topoisomerase I poison.

BN 80915, a lead compound of the homocamptothecin (hCPT) family, has entered clinical trials. BN 80915 is a difluoro-hCPT where the six-membered alpha-hydroxylactone ring of camptothecin (CPT) is replaced by a seven-membered beta-hydroxylactone ring. Preclinical data reported here show that in spite of the modification to the crucial E-ring of CPTs, BN 80915 retains topoisomerase I poisoning activity as shown in living HT29 cells as well as in cell-free assays, where BN 80915 always performs better than SN-38 or TPT. In antiproliferative assays BN 80915 is also very potent as evidenced by IC50s values consistently lower than those of SN38 in sensitive cell lines as well as in their related multidrug-resistant lines overexpressing P-glycoprotein or multidrug resistance-associated protein. Furthermore, in human plasma, in contrast to CPT analogs, the hydrolysis of BN 80915 is slow, leading to improved plasma stability, and irreversible, thus avoiding toxicity related to the accumulation of active principle during excretion in the urinary tract. These findings may account for the good in vivo efficacy observed in PC3 xenograft experiments where BN 80915 administered orally at very low doses doubled the tumor growth delay in comparison to CPT-11 administered i.p. Altogether, these results strongly support further development of BN 80915.

Adenocarcinoma↗
PubMed2006

NPI-2358 is a tubulin-depolymerizing agent: in-vitro evidence for activity as a tumor vascular-disrupting agent.

The diketopiperazine NPI-2358 is a synthetic analog of NPI-2350, a natural product isolated from Aspergillus sp., which depolymerizes microtubules in A549 human lung carcinoma cells. Although structurally different from the colchicine-binding site agents reported to date, NPI-2358 binds to the colchicine-binding site of tubulin. NPI-2358 has potent in-vitro anti-tumor activity against various human tumor cell lines and maintains activity against tumor cell lines with various multidrug-resistant (MDR) profiles. In addition, when evaluated in proliferating human umbilical vein endothelial cells (HUVECs), concentrations as low as 10 nmol/l NPI-2358 induced tubulin depolymerization within 30 min. Furthermore, NPI-2358 dose dependently increases HUVEC monolayer permeability--an in-vitro model of tumor vascular collapse. NPI-2358 was compared with three tubulin-depolymerizing agents with vascular-disrupting activity: colchicine, vincristine and combretastatin A-4 (CA4). Results showed that the activity of NPI-2358 in HUVECs was more potent than either colchicine or vincristine; the profile of CA4 approached that of NPI-2358. Altogether, our data show that NPI-2358 is a potent anti-tumor agent which is active in MDR tumor cell lines, and is able to rapidly induce tubulin depolymerization and monolayer permeability in HUVECs. These data warrant further evaluation of NPI-2358 as a vascular-disrupting agent in vivo. Currently, NPI-2358 is in preclinical development for the treatment of cancer.

Angiogenesis Inhibitors↗
PubMed1997

Cell proliferation enhances entry of Listeria monocytogenes into intestinal epithelial cells by two proliferation-dependent entry pathways.

Bacterial entry into intestinal host cells is the result of a fairly sophisticated manipulation of host cell machinery by the pathogens. To study further the potential cell target of Listeria spp., the in-vitro entry of L. monocytogenes strains into intestinal cells was examined in relation to the metabolism, proliferation and differentiation of the cells by the alamarBlue assay, [3H] thymidine incorporation, and brush border-associated enzyme activities, respectively. The study showed that cell metabolism was not involved in the entry of L. monocytogenes in three cell models (two human and one porcine). On the other hand, entry was closely related to the proliferation process and poorly related to the differentiation state of the cells. The use of L. monocytogenes mutants lacking invasion proteins showed that InlA and InlB acted in synergy to mediate the entry of L. monocytogenes into proliferative cells, whereas InlA alone seemed to be involved in the entry into non-proliferative cells. These two entry pathways could correspond to the two cellular processes used by L. monocytogenes to enter proliferative and non-proliferative cells, as suggested by the use of cytochalasin D, nocodazole, chloroquine and monodansylcadaverine. Taken together, we propose a hypothesis in which the entry of L. monocytogenes is mediated by interaction between randomly distributed E-cadherin on the surface of proliferative cells. In contrast, the entry into non-proliferative cells may involve pp60c-src, a proto-oncogenic tyrosine kinase signal that modifies E-cadherin localisation. In conclusion, these results suggest that L. monocytogenes may preferentially enter crypt cells in vivo by a microfilament-dependent process, whereas the few bacteria that infect villus cells enter by an E-cadherin-internalin interaction that mediates microtubule-dependent endocytosis.

Animals↗
PubMed2006

Campylobacter jejuni-infected human epithelial cell lines vary in their ability to secrete interleukin-8 compared to in vitro-infected primary human intestinal tissue.

Campylobacter jejuni causes symptoms of acute inflammatory diarrhoea in man. C. jejuni interaction with epithelial cells elicits interleukin-8 (IL-8) production, and IL-8 recruits neutrophils to sites of infection. Cell culture models of bacterial interaction with epithelium are useful to define bacteria-host interaction and are used because it is thought they mimic the same bacteria-host cell interaction in the natural disease. This study looks at the ability of C. jejuni strains to elicit IL-8 production from a variety of cell lines previously used for investigating the interaction of C. jejuni with host cells. A spectrum of IL-8 responses was observed, with minimal IL-8 elicited from Caco-2 cells and more marked responses elicited from HeLa and T84 cells. These in vitro-infected cell line responses were compared to IL-8 production from in vitro C. jejuni-infected human colonic and ileal tissue. The in vitro-infected tissue elicited the highest IL-8 responses and the cytokine was manifested earlier compared to the infected cell lines.

Caco-2 Cells↗