The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cell-cell adhesion and to suppress invasion.

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF-LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF-LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a beta-catenin-dependent activation of the WNT pathway.

Functional and molecular characterization of the epithelioid to round transition in human colorectal cancer LoVo cells.

In subclones of the human colon cancer LoVo cell line, there is a reproducible spontaneous transition from an epithelioid (E) to a round (R) morphotype. The E to R transition is associated with increased cell growth, absence of E-cadherin-dependent compaction in a slow aggregation assay, loss of contact inhibition of motility and directional migration in a wound filling motility assay. Furthermore, none of the E subclones from LoVo was invasive into chick heart fragments. This is in contrast to the R subclones that were either nonadherent or adherent and invasive. Macroarray analysis demonstrated transcriptional downregulation of plakoglobin in R type LoVo cells and this was confirmed at the level of the mRNA by quantitative RT-PCR. Western blotting showed lower expression of all components of the E-cadherin/catenin complex in R subclones. Interestingly, treatment of R subclones with the demethylating agent 5-aza-2'-deoxycytidine resulted in restoration of the E morphotype, higher expression of E-cadherin, but not plakoglobin mRNA, and higher expression of E-cadherin and plakoglobin at the protein level.

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Invasion of retinal pigment epithelial cells: N-cadherin, hepatocyte growth factor, and focal adhesion kinase.

PURPOSE: To investigate the role of N-cadherin and hepatocyte growth factor (HGF) in the invasion of collagen type I by human retinal pigment epithelial (RPE) cells. METHODS: RPE sheets from eight human eyes were used for characterization through Western blot analysis of the expression of cadherin in total lysates or after immunoprecipitation with anti beta-catenin antibody. First-passage primary cultures of RPE sheets were successfully established from 28 of 56 human eyes. First-passage primary RPE cell cultures on glass substrate, consisting of patches of cells, were used for immunocytochemistry. Fifteen first-passage primary RPE cell cultures in culture vessels were grown to confluence. Four of the 15 first-passage primary RPE cell cultures were investigated for cadherin expression by immunocytochemistry, and the other 11 were further subcultured for two to six passages. These 11 cultures were used for functional assays and investigated for expression of cadherin at regular time intervals. Cells from passage-3 to -4 primary RPE cell cultures were tested for invasion into collagen type I gels, with or without neutralizing antibodies for HGF and N-cadherin, respectively. Activation of the c-Met receptor for HGF and of focal adhesion kinase (FAK) was investigated by immunoprecipitation with anti-phosphotyrosine antibody, gel electrophoresis, and immunostaining on Western blot. Levels of HGF in conditioned medium (CM) of RPE cells were determined by ELISA. RESULTS: RPE cells in culture displayed two phenotypes: Both fibroblast-like and epithelioid cells were present in all 15 first-passage primary cultures and in further passaged cultures derived therefrom. When seeded on collagen, all RPE cells acquired a fibroblast-like phenotype and invaded the collagen type I gel. RPE cells also stimulated branching morphogenesis of MDCK/AZ epithelial canine kidney cell colonies inside collagen. HGF was found in RPE CM, suggesting an autocrine loop for invasion through its c-Met receptor. HGF-neutralizing antibody inhibited invasion of collagen. The major cadherin expressed by RPE cells in culture was N(euronal)-cadherin. Invasion of collagen by RPE cells was also inhibited by an N-cadherin-neutralizing antibody. N-cadherin and c-Met coimmunoprecipitated in RPE cells. FAK and c-Met were both phosphorylated in RPE cells in culture, and phosphorylation was inhibited by antibodies neutralizing either N-cadherin or HGF. CONCLUSIONS: The present investigation provides evidence for an autocrine HGF/c-Met loop that stimulates RPE cell invasion into collagen through FAK. The invasion-stimulatory molecule N-cadherin also activates FAK in invasive RPE cells.

Structure and function of the N-cadherin/catenin complex in retinoblastoma.

PURPOSE: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.

Alkylphospholipids reversibly open epithelial tight junctions.

Alkylphospholipids, such as the antitumor ether lipid 1-O-octadecyl-2-O-methylglycero-3-phosphocholine, modulate cancer cell invasion through changes in the adherens junction E-cadherin complex, a major organizer of epithelia. We wanted to know whether alkylphospholipids would also change tight junctions, molecular complexes that seal cell-to-cell contacts in polarised epithelia. Therefore, human colorectal cancer cell layers T84 were established in two-compartment culture chambers and the functional integrity of tight junctions was evaluated through their transepithelial electrical resistance. Incorporation of alkylphospholipids causes a rapid and reversible decrease of transepithelial electrical resistance. This decrease is due to an increased paracellular permeability and is temperature-independent. Unlike methyl-beta-cyclodextrin, alkyl-phospholipids do not specifically displace lipids from raft-like membrane domains. Nevertheless, alkylphospholipids change the detergent-solubility of zonula occludens-protein and occludin. Our data, together with the literature, indicate that non-toxic doses of alkylphospholipids affect more than one cell-cell adhesion complex, probably through their incorporation into the plasma membrane lipid bilayer.

Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography.

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.

The citrus methoxyflavone tangeretin affects human cell-cell interactions.

Two effects of the citrus methoxyflavone tangeretin on cell-cell interactions are biologically relevant. Firstly, tangeretin upregulates the function of the E-cadherin/catenin complex in human MCF-7/6 breast carcinoma cells. This leads to firm cell-cell adhesion and inhibition of invasion in vitro. Secondly, tangeretin downregulates the interleukin-2 receptor on T-lymphocytes and natural killer cells. This leads to a decrease in the cytotoxic competence of these immunocytes against cancer cells. The second effect can become important when high doses of tangeretin are combined with adjuvant tamoxifen treatment for breast cancer. Experiments with nude mice bearing MCF-7/6 tumors showed that tangeretin given orally at high doses, abrogated the therapeutic suppression of tumor growth exerted by tamoxifen. No evidence for a tumor promoting effect of tangeretin by itself was found in these experiments. Tangeretin may be an interesting molecule for application in cases where immunosuppression could be clinically beneficial.

Chick heart invasion assay.

Tumors are microecosystems in which a continuous cross talk between cancer cells and host cells decides on the invasive behavior of the tumor cell population as a whole (Mareel et al., Encyclopedia of cancer, San Diego, CA, Academic Press, 1997). Both compartments secrete activating and inhibitory factors that modulate activities such as cell-extracellular matrix (ECM) interaction, cell-cell adhesion, remodeling of the ECM, and cell motility. For this reason, confrontations of cancer cells with a living normal host tissue in organ culture have been introduced by several groups: Wolff and Schneider in France (Wolff and Schneider, C R S Soc Biol (Paris) 151:1291-1292, 1957), Easty and Easty in the United Kingdom (Easty and Easty, Nature 199:1104-1105, 1963), and Schleich in Germany (Schleich et al., J Natl Cancer Inst 56:221-237, 1976). Embryonic chick heart fragments in organ culture maintain many histological features of their tissue of origin: They are composed of myocytes, fibroblasts, and endothelial cells, and their ECM contains fibronectin, laminin, and several collagen types. Moreover, the fragments remain contractile, and this activity allows the monitoring of their functional integrity during organ culture.

Opioids affect focal contact-mediated cell-substrate adhesion.

Earlier observations suggested that opioids modify cell-substrate adhesion on agar. In this study, we wanted to investigate whether opioids also interfere with cell adhesion to biologically relevant substrates, including interstitial matrix and basement membrane components. Human embryonic kidney 293 cells stably transfected with FLAG-tagged micro-opioid receptor were used as an experimental model. The cells were cultured on tissue culture plastic, collagen types I and IV, fibronectin, laminin and human amnion fragments in the absence or presence of morphine. Cultures were immunolabelled with antivinculin to visualize focal contacts. Morphine-treated cultures on tissue culture plastic, collagen types I and IV and fibronectin, but not on laminin, covered less substrate than untreated cultures; individual cells were difficult to distinguish and their nuclei piled up in contrast to untreated cultures. The same effects were observed on human amnion fragments: morphine changed the morphotype of cultures on the stromal side, but not on the basement membrane side. Cultures that were treated with morphine displayed fewer focal contacts than untreated cultures on collagen type I, whereas untreated and morphine-treated cultures were indistinguishable on laminin.

Synthesis, characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds.

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.