Publications
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Kugathas S, Audouze K, Ermler S, et al., 2016, Effects of Common Pesticides on Prostaglandin D2 (PGD2) Inhibition in SC5 Mouse Sertoli Cells, Evidence of Binding at the COX-2 Active Site, and Implications for Endocrine Disruption, ENVIRONMENTAL HEALTH PERSPECTIVES, Vol: 124, Pages: 452-459, ISSN: 0091-6765
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- Citations: 28
Orton F, Ermler S, Kugathas S, et al., 2013, Mixture effects at very low doses with combinations of anti-androgenic pesticides, antioxidants, industrial pollutant and chemicals used in personal care products, Toxicol.Appl.Pharmacol.
Many xenobiotics have been identified as in vitro androgen receptor (AR) antagonists, but information about their ability to produce combined effects at low concentrations is missing. Such data can reveal whether joint effects at the receptor are induced at low levels and may support the prioritisation of in vivo evaluations and provide orientations for the grouping of anti-androgens in cumulative risk assessment. Combinations of 30 AR antagonists from a wide range of sources and exposure routes (pesticides, antioxidants, parabens, UV-filters, synthetic musks, bisphenol-A, benzo(a)pyrene, perfluorooctane sulfonate and pentabromodiphenyl ether) were tested using a reporter gene assay (MDA-kb2). Chemicals were combined at three mixture ratios, equivalent to single components' effect concentrations that inhibit the action of dihydrotesterone by 1%, 10% or 20%. Concentration addition (CA) and independent action were used to calculate additivity expectations. We observed complete suppression of dihydrotestosterone effects when chemicals were combined at individual concentrations eliciting 1%, 10% or 20% AR antagonistic effect. Due to the large number of mixture components, the combined AR antagonistic effects occurred at very low concentrations of individual mixture components. CA slightly underestimated the combined effects at all mixture ratios. In conclusion, large numbers of AR antagonists from a wide variety of sources and exposure routes have the ability of acting together at the receptor to produce joint effects at very low concentrations. Significant mixture effects are observed when chemicals are combined at concentrations that individually do not induce observable AR antagonistic effects. Cumulative risk assessment for AR antagonists should apply grouping criteria based on effects where data are available, rather than on criteria of chemical similarity
Orton F, Rosivatz E, Scholze M, et al., 2012, Competitive androgen receptor antagonism as a factor determining the predictability of cumulative antiandrogenic effects of widely used pesticides, Environ.Health Perspect., Vol: 120, Pages: 1578-1584
BACKGROUND: Many pesticides in current use have recently been revealed as in vitro androgen receptor (AR) antagonists, but information about their combined effects is lacking. OBJECTIVE: We investigated the combined effects and the competitive AR antagonism of pesticide mixtures. METHODS: We used the MDA-kb2 assay to test a combination of eight AR antagonists that did not also possess AR agonist properties ("pure" antagonists; 8 mix: fludioxonil, fenhexamid, ortho-phenylphenol, imazalil, tebuconazole, dimethomorph, methiocarb, pirimiphos-methyl), a combination of five AR antagonists that also showed agonist activity (5 mix: cyprodinil, pyrimethanil, vinclozolin, chlorpropham, linuron), and all pesticides combined (13 mix). We used concentration addition (CA) and independent action (IA) to formulate additivity expectations, and Schild plot analyses to investigate competitive AR antagonism. RESULTS: A good agreement between the effects of the mixture of eight "pure" AR antagonists and the responses predicted by CA was observed. Schild plot analysis revealed that the 8 mix acted by competitive AR antagonism. However, the observed responses of the 5 mix and the 13 mix fell within the "prediction window" boundaries defined by the predicted regression curves of CA and IA. Schild plot analysis with these mixtures yielded anomalous responses incompatible with competitive receptor antagonism. CONCLUSIONS: A mixture of widely used pesticides can, in a predictable manner, produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Inasmuch as large populations are regularly exposed to mixtures of antiandrogenic pesticides, our results underline the need for considering combination effects for these substances in regulatory practice
Kortenkamp A, Martin O, Evans R, et al., 2012, Response to A critique of the European Commission Document, "State of the Art Assessment of Endocrine Disrupters" by Rhomberg and colleagues--letter to the editor, Crit Rev.Toxicol., Vol: 42, Pages: 787-789
Orton F, Rosivatz E, Scholze M, et al., 2011, Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens, Environ.Health Perspect., Vol: 119, Pages: 794-800
BACKGROUND: Evidence suggests that there is widespread decline in male reproductive health and that antiandrogenic pollutants may play a significant role. There is also a clear disparity between pesticide exposure and data on endocrine disruption, with most of the published literature focused on pesticides that are no longer registered for use in developed countries. OBJECTIVE: We used estimated human exposure data to select pesticides to test for antiandrogenic activity, focusing on highest use pesticides. METHODS: We used European databases to select 134 candidate pesticides based on highest exposure, followed by a filtering step according to known or predicted receptor-mediated antiandrogenic potency, based on a previously published quantitative structure-activity relationship (QSAR) model. In total, 37 pesticides were tested for in vitro androgen receptor (AR) antagonism. Of these, 14 were previously reported to be AR antagonists ("active"), 4 were predicted AR antagonists using the QSAR, 6 were predicted to not be AR antagonists ("inactive"), and 13 had unknown activity, which were "out of domain" and therefore could not be classified with the QSAR ("unknown"). RESULTS: All 14 pesticides with previous evidence of AR antagonism were confirmed as antiandrogenic in our assay, and 9 previously untested pesticides were identified as antiandrogenic (dimethomorph, fenhexamid, quinoxyfen, cyprodinil, lambda-cyhalothrin, pyrimethanil, fludioxonil, azinphos-methyl, pirimiphos-methyl). In addition, we classified 7 compounds as androgenic. CONCLUSIONS: Due to estimated antiandrogenic potency, current use, estimated exposure, and lack of previous data, we strongly recommend that dimethomorph, fludioxonil, fenhexamid, imazalil, ortho-phenylphenol, and pirimiphos-methyl be tested for antiandrogenic effects in vivo. The lack of human biomonitoring data for environmentally relevant pesticides presents a barrier to current risk assessment
Rosivatz E, Woscholski R, 2011, Removal or masking of phosphatidylinositol(4,5)bisphosphate from the outer mitochondrial membrane causes mitochondrial fragmentation, Cell Signal., Vol: 23, Pages: 478-486
Mitochondria are central players in programmed cell death and autophagy. While phosphoinositides are well established regulators of membrane traffic, cellular signalling and the destiny of certain organelles, their presence and role for mitochondria remain elusive. In this study we show that removal of PtdIns(4,5)P(2) by phosphatases or masking the lipid with PH domains leads to fission of mitochondria and increased autophagy. Induction of general autophagy by amino acid starvation also coincides with the loss of mitochondrial PtdIns(4,5)P(2), suggesting an important role for this lipid in the processes that govern mitophagy. Our findings reveal that PKCalpha can rescue the removal or masking of PtdIns(4,5)P(2), indicating that the inositol lipid is upstream of PKC
Alimonti A, Nardella C, Chen Z, et al., 2010, A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis, J.Clin.Invest, Vol: 120, Pages: 681-693
Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss-induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a "pro-senescence" approach for cancer prevention and therapy
Ho KK, Rosivatz E, Gunn RM, et al., 2009, The novel molecule 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate inhibits phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signalling through JNK activation in cancer cells, FEBS J., Vol: 276, Pages: 4037-4050
Screening a compound library of compound 48/80 analogues, we identified 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3-kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose-dependent and time-dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC-3 and MCF-7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c-Jun NH2-terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC-3 and MCF-7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild-type and p38-null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK-null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC-3 and MCF-7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK-dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research
Garczarczyk D, Toton E, Biedermann V, et al., 2009, Signal transduction of constitutively active protein kinase C epsilon, Cell Signal., Vol: 21, Pages: 745-752
The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCepsilon isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCepsilon-signaling, we investigated the effects of constitutively active rat PKCepsilon (PKCepsilonA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCepsilonA/E expression by doxycycline, the major part of PKCepsilonA/E was localized to the Golgi. This led (i) to phosphorylations of PKCepsilon(S729), Elk-1(S383), PDK1(S241) and Rb(S807/S811), (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCepsilonA/E-expression by doxycycline for 24 h and additional short-term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), PKCepsilonA/E translocated to the plasma membrane and increased phosphorylation of MARCKS(S152/156). Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1(S383), PDK1(S241), Rb(S807/S811), PKCdelta(T505), p38MAPK(T180/Y182), MEK1/2(S217/S221) and ERK2(T185/T187). MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCepsilon localized to the plasma membrane but not by PKCalpha or delta, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCepsilon can induce distinctly different signaling from the Golgi and from the plasma membrane
Rosivatz E, Woscholski R, 2009, Measurement of PTEN activity in vivo by imaging phosphorylated Akt, Methods Mol.Biol., Vol: 462, Pages: 213-222
This chapter describes an indirect approach to measure PTEN's lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation and phosphorylation of the survival kinase Akt can be used as readout for cellular PTEN activity. Here we have outlined a detailed procedure employing a phosphoserine-specific anti-Akt antibody to examine the content of phosphorylated Akt by immunofluorescence and its dependence on PTEN activity
Mulet X, Rosivatz E, Ho KK, et al., 2009, Spatial localization of PtdInsP2 in phase-separated giant unilamellar vesicles with a fluorescent PLC-delta 1 PH domain, Methods Mol.Biol., Vol: 462, Pages: 135-144
This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis
Rosivatz E, 2008, Imaging the boundaries-innovative tools for microscopy of living cells and real-time imaging, J.Chem.Biol., Vol: 1, Pages: 3-15
Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes "at work" and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein-protein interaction designed for use in living cells
Busch GK, Tate EW, Gaffney PR, et al., 2008, Specific N-terminal protein labelling: use of FMDV 3C pro protease and native chemical ligation, Chem.Commun.(Camb.), Pages: 3369-3371
We report an effective strategy for generating N-terminal cysteinyl proteins by proteolytic cleavage using the enzyme 3C pro, suitable for a wide range of applications via native chemical ligation
Ho KK, Anderson AA, Rosivatz E, et al., 2008, Identification of cyclin A2 as the downstream effector of the nuclear phosphatidylinositol 4,5-bisphosphate signaling network, J.Biol.Chem., Vol: 283, Pages: 5477-5485
In addition to the well characterized phosphoinositide second messengers derived from the plasma membrane, increasing evidence supports the existence of a nuclear phosphoinositide signaling network. The aim of this investigation was to dissect the role played by nuclear phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in cell cycle progression and to determine the cell cycle regulatory component(s) that are involved. A number of cytosolic/nuclear PtdIns(4,5)P2-deficient Swiss 3T3 cell lines were established, and their G 0/G 1/S cell cycle phase transitions induced by defined mitogens were examined. Our results demonstrate that nuclear PtdIns(4,5)P2 down-regulation caused a delay in phorbol ester-induced S phase entry and that this was at least in part channeled through cyclin A2 at the transcriptional level. In summary, these data identify cyclin A2 as a downstream effector of the nuclear PtdIns(4,5)P2 signaling network and highlight the importance of nuclear PtdIns(4,5)P2 in the regulation of mammalian mitogenesis
Rosivatz E, 2007, Inhibiting PTEN., Biochem Soc Trans, Vol: 35, Pages: 257-259, ISSN: 0300-5127
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is well known as a tumour suppressor. In dephosphorylating the 3-position of the inositol ring of phosphoinositides such as PtdIns(3,4,5)P(3), PTEN's lipid phosphatase activity is an important counteracting mechanism in PI3K (phosphoinositide 3-kinase) signalling. This is essential for cell motility and migration due to the achievement of a PtdIns(3,4,5)P(3)/PtdIns(4,5)P(2) gradient that is also involved in metastasis. Furthermore, PTEN's tumour suppressor role is linked to the control of cell-cycle progression and cell proliferation by counteracting Akt (also called protein kinase B) signalling which is PtdIns(3,4,5)P(3)-dependent. Akt is upstream of several kinases involved in proliferation and apoptotic signalling which are often found to be deregulated or mutated in tumours. However, Akt is also the key enzyme in insulin signalling regulating glucose uptake and cell growth. Therefore PTEN has recently moved into the spotlight as a drug target in diabetes. This review summarizes studies undertaken on PTEN's role in glucose uptake, insulin resistance, diabetes and its controversial role in GLUT (glucose transporter)-mediated glucose uptake. Currently available techniques for inhibiting PTEN and the suitability of PTEN as a drug target will be discussed.
Alves CC, Rosivatz E, Schott C, et al., 2007, Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin., J Pathol, Vol: 211, Pages: 507-515, ISSN: 0022-3417
Epithelial-mesenchymal transition (EMT) involving down-regulation of E-cadherin is known to play an important role in tumour progression. The aim of our study was to investigate the mRNA expression of two EMT regulators-Slug and E12/E47-in primary human gastric carcinomas and to compare this with the expression of E-cadherin and other EMT regulators (Snail, Twist, and SIP1). We studied a series of 59 gastric carcinomas by real-time quantitative RT-PCR in formalin-fixed and paraffin-embedded tissues. Thirty-four cases (58%) showed Slug up-regulation in the tumour; reduced or negative expression of E-cadherin was present in 24 of these (71%, p<0.0001). Twenty-one cases (36%) showed E12/E47 up-regulation that was not significantly associated with E-cadherin down-regulation (p=0.5734). Slug up-regulation accompanied by E-cadherin down-regulation correlated with the presence of distant metastases (p=0.0029) and with advanced pTNM stages (p=0.0424). A statistically significant association was found between Slug up-regulation and the expression of SIP1 in intestinal (p=0.0014) and Snail in diffuse (p=0.0067) carcinomas. We present the first study integrating the analysis of several EMT regulators in primary gastric carcinomas and conclude that Slug up-regulation is associated with E-cadherin down-regulation in diffuse and intestinal-type gastric carcinoma, and that this effect could be complemented by the presence of other EMT regulators.
Byrne RD, Rosivatz E, Parsons M, et al., 2007, Differential activation of the PI 3-kinase effectors AKT/PKB and p70 S6 kinase by compound 48/80 is mediated by PKCalpha, Cell Signal., Vol: 19, Pages: 321-329
The secretagogue compound 48/80 (c48/80) is a well known activator of calcium mediated processes and PKCs, and is a potent inducer of mast cell degranulation. As the latter process is a phosphoinositide 3-kinase (PI 3-kinase) mediated event, we wished to address whether or not c48/80 was an activator of PI 3-kinases. The data presented here reveal that c48/80 is an effective activator of PI 3-kinases as judged by the increased phosphorylation of PKB and p70(S6K) in fibroblasts in a PI 3-kinase dependent fashion. Compound 48/80 effectively translocates PKB to the plasma membrane and induces phosphorylation at serine 473 (S473), detected by fluorescence imaging of fixed cells. At higher concentrations the secretagogue is inhibitory towards PKB phosphorylation on S473. Conversely, p70(S6K) phosphorylation on T389 is unaffected at high doses. We provide evidence that the differential effect on the two PI 3-kinase effectors is due to activation of PKCalpha by c48/80, itself a PI 3-kinase dependent process. We conclude that compound 48/80 is an effective activator of PI 3-kinase dependent pathways, leading to the activation of effectors including PKB/Akt, p70(S6K) and PKCalpha. The latter is only activated by higher doses of c48/80 resulting in an inhibition of the c48/80 induced PKB phosphorylation, thus explaining the observed biphasic activation profile for PKB in response to this secretagogue
Becker K-F, Rosivatz E, Blechschmidt K, et al., 2007, Analysis of the E-cadherin repressor Snail in primary human cancers., Cells Tissues Organs, Vol: 185, Pages: 204-212
Epithelial-mesenchymal transition (EMT), a normal developmental process, is known to play a crucial role in tumor progression. Molecules involved in this process, such as the E-cadherin repressor Snail, facilitate migration and invasion of carcinoma cells. A growing number of studies addressing the expression of Snail in clinical samples have been reported and are discussed in this review. A total of 2,112 cases from 9 different tumor types were evaluated. So far, a clear picture has emerged only in some cancer types analyzed with regard to overexpression of Snail and clinical-pathological parameters. Currently, it seems that Snail may play a role in hormone-dependent carcinomas but may be of minor importance in gastrointestinal cancers for tumor dedifferentiation and the maintenance of the invasive phenotype. It should be kept in mind, however, that the threshold for Snail activity does not have to be the same in every tumor type analyzed. The recent introduction of well-characterized novel monoclonal antibodies reacting with the short-lived nuclear Snail protein may help to establish a potential clinical usefulness for this master molecule of EMT, at least for certain types of cancer.
Rosivatz E, Matthews JG, McDonald NQ, et al., 2006, A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN), ACS Chem.Biol., Vol: 1, Pages: 780-790
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulin-dependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a small- molecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the PtdIns(3,4,5)P3 substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular PtdIns(3,4,5)P3 levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development
Rosivatz E, Becker KF, Kremmer E, et al., 2006, Expression and nuclear localization of Snail, an E-cadherin repressor, in adenocarcinomas of the upper gastrointestinal tract, Virchows Arch., Vol: 448, Pages: 277-287
Transcriptional E-cadherin down-regulation can be mediated by Snail, a zinc finger transcription factor. To be able to examine nuclear Snail immunoreactivity in archival human cancers, we established a monoclonal antibody against the purified human Snail protein. The specificity of the selected rat antibody Sn9H2 was demonstrated by Western blot analysis using extracts from different cell lines and by immunofluorescence and immunohistochemistry of primary tissues. Subsequently, a series of 340 adenocarcinomas of the upper gastrointestinal tract, including tumours from the oesophagus (n=154), cardia (n=102) and stomach (n=84), arranged in tissue microarrays, were examined for Snail expression and were correlated to E-cadherin expression and clinico-pathological parameters. Nuclear Snail immunoreactivity was seen in 27 tumours (7.9%) and tended to be more frequent in oesophageal adenocarcinomas (11.1%) than in cardiac (6.9%) or gastric (3.6%) carcinomas (p=0.0428). In 35% of the Snail-positive cases, E-cadherin immunoreactivity was lost. No correlation was found for nuclear Snail expression and tumour grade, Lauren's classification, WHO classification, tumour stage and tumour size. The pattern of Snail expression observed with our new hybridoma, Sn9H2, which is currently the only antibody that reacts with endogenous nuclear (active) Snail, suggests only a minor role of Snail in tumours of the upper gastrointestinal tract
Fuchs M, Hermannstadter C, Specht K, et al., 2005, Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3, J.Cell Physiol, Vol: 202, Pages: 805-813
Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread
Rosivatz E, Becker I, Bamba M, et al., 2004, Neoexpression of N-cadherin in E-cadherin positive colon cancers, Int.J.Cancer, Vol: 111, Pages: 711-719
In our study, we aimed to investigate the expression of N-cadherin and E-cadherin and their dependency on epithelial-mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E-cadherin and N-cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N-cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real-time quantitative RT-PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane-associated immunoreactivity of N-cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse-type gastric cancers with abnormal N-cadherin expression. Reduced and/or cytoplasmic E-cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N-cadherin was almost exclusively expressed in those cases showing normal E-cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E-cadherin reduction and upregulation of N-cadherin. For the first time, we postulate a role for N-cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N-cadherin can exert a dominant function over E-cadherin's adhesive function and thus promote tumor invasiveness
Kremer M, Quintanilla-Martinez L, Fuchs M, et al., 2003, Influence of tumor-associated E-cadherin mutations on tumorigenicity and metastasis, Carcinogenesis, Vol: 24, Pages: 1879-1886
In this study, we investigated whether tumor-associated E-cadherin mutations impair the tumor-suppressive function of the cell adhesion molecule and influence metastasis formation in a severe combined immunodeficiency mouse model. The investigated E-cadherin mutations were in frame deletions of exons 8 (del 8) or 9 (del 9) and a point mutation in exon 8 (p8). Transfected human MDA-MB-435S carcinoma cells stably expressing wild-type (wt) or mutant E-cadherin were injected into the mouse mammary fat pad. Mice transplanted with wt E-cadherin transfectants developed significantly smaller tumors than animals transplanted with the E-cadherin-negative parental cell line. Animals transplanted with del 9 or p8 E-cadherin transfectants produced medium size tumors, indicating that these mutations impair the tumor-suppressive function of E-cadherin. In contrast, mice transplanted with del 8 E-cadherin transfectants developed tumors of approximately the same sizes as animals transplanted with wt E-cadherin expressing cells. Lung metastases were induced by all cell lines without significant differences. Immunohistochemical analysis of E-cadherin expression in the tumors revealed a heterogeneous staining pattern, indicating loss or down-regulation of E-cadherin in some tumor cells. Metastases were completely negative for E-cadherin. Our data suggest that the type of mutation determines whether the tumor-suppressive function of E-cadherin is impaired
Fricke E, Keller G, Becker I, et al., 2003, Relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression and Ki-67 staining in diffuse-type gastric carcinoma, Int.J.Cancer, Vol: 104, Pages: 60-65
E-cadherin mutations are found in 50% of diffuse-type gastric carcinoma, but not in intestinal gastric carcinoma. Because cell-cell adhesion mediated by E-cadherin plays an important role in epithelial cell survival, E-cadherin mutations could alter the apoptotic behavior of tumor cells. p53 and Bcl-2 family members are also important regulators of cellular apoptosis. This is the first study that investigates the relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression, and Ki-67 expression in diffuse-type gastric carcinoma (24 cases, E-cadherin mutation status: wild-type in 8 patients and mutant in 16 patients). The mutation status of exons 5-8 of p53 was analyzed by denaturing high pressure liquid chromatography (DHPLC) in formalin-fixed, paraffin-embedded tumor sections, followed by direct sequencing of cases with aberrant chromatographic patterns. p53 mutations were found in 1 of 8 tumors without E-cadherin mutation (12.5%) and in 1 of 16 tumors with E-cadherin mutation (6.3%), a difference that was not statistically significant (p = 1.00). p53 accumulation was found in 8 of 24 tumors (33.3%) by immunohistochemical staining. p53 accumulation was significantly more frequent in tumors without E-cadherin mutations (5 of 8 tumors, 62.5%) than in gastric carcinoma tissues with E-cadherin mutations (3 of 16 tumors, 18.8%, p = 0.03). Bcl-2 staining was not observed in gastric carcinoma cells without E-cadherin mutations, but was detectable in 5 of 16 tumors with E-cadherin mutations (31.3%), a difference that was not statistically significant (p = 0.13). No relationship was observed between Ki-67 staining and the E-cadherin mutation status (p = 1.00). These data suggest that the presence of E-cadherin mutations can significantly alter the accumulation of the apoptosis-regulating p53 protein, whereas no correlation with the p53 mutation status or with Ki-67 staining was observed
Rosivatz E, Becker I, Specht K, et al., 2002, Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer, Am.J.Pathol., Vol: 161, Pages: 1881-1891
Epithelial-mesenchymal transition (EMT) involving down-regulation of E-cadherin is thought to play a fundamental role during early steps of invasion and metastasis of carcinoma cells. The aim of our study was to elucidate the role of EMT regulators Snail, SIP1 (both are direct repressors of E-cadherin), and Twist (an activator of N-cadherin during Drosophila embryogenesis), in primary human gastric cancers. Expression of Snail, SIP1, and Twist was analyzed in 48 gastric carcinomas by real-time quantitative RT-PCR in paraffin-embedded and formalin-fixed tissues. The changes of expression levels of these genes in malignant tissues compared to matched non-tumorous tissues were correlated with the expression of E- and N-cadherin. From 28 diffuse-type gastric carcinomas analyzed reduced E-cadherin expression was detected in 11 (39%) cases compared to non-tumorous tissues. Up-regulated Snail could be found in 6 cases with reduced or negative E-cadherin expression. However, there was no correlation to increased SIP1 expression. Interestingly, we could detect abnormal expression of N-cadherin mRNA in 6 cases, which was correlated with Twist overexpression in 4 cases. From 20 intestinal-type gastric cancer samples reduced E-cadherin expression was found in 12 (60%) cases, which was correlated to up-regulation of SIP1, since 10 of these 12 cases showed elevated mRNA levels, whereas Snail, Twist, and N-cadherin were not up-regulated. We present the first study investigating the role of EMT regulators in human gastric cancer and provide evidence that an increase in Snail mRNA expression is associated with down-regulation of E-cadherin in diffuse-type gastric cancer. We detected abnormally positive or increased N-cadherin mRNA levels in the same tumors, probably due to overexpression of Twist. SIP1 overexpression could not be linked to down-regulated E-cadherin in diffuse-type tumors, but was found to be involved in the pathogenesis of intestinal-type gastric carcinoma. We concl
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