Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress

Malignant mesothelioma cells differentiate into sarcomatoid or epithelioid phenotypes. The sarcomatoid cell type is more resistant to chemotherapy and yields a worse prognosis. We have investigated whether selenite alone and in combination with doxorubicin induced apoptosis in variously differentiated mesothelioma cells. Selenite in concentrations that could potentially be administered to patients strongly inhibited the growth of the sarcomatoid mesothelioma cells (IC 50 = 7.5 µM), whereas epithelioid cells were more sensitive to doxorubicin. Benign mesothelial cells remained largely unaffected. Selenite potentiates doxorubicin treatment.. Apoptosis was the dominating mode of cell death. The toxicity of selenite was mediated by oxidative stress. Furthermore the activity of the thioredoxin system was directly dependent on the concentration of selenite. This offers a possible mechanism of action of selenite treatment. Our findings suggest that selenite is a promising new drug for the treatment of malignant mesothelioma.Cancer- och allergifondenSvenska läkaresällskapetHjärt-lungfondenAccepte

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1

Specific Syndecan-1 Domains Regulate Mesenchymal Tumor Cell Adhesion, Motility and Migration

Malignant mesothelioma is an asbestos induced cancer that is difficult to diagnose. Several studies have combined biomarkers to improve mesothelioma diagnosis, but with moderate success, and there is a need for new mesothelioma biomarkers. The tumour is often resistant to treatment and most patients will survive less than a year. An indicator of patient survival is the tumours growth pattern, which in turn is influenced by expressed proteoglycans. In this thesis work, we aim to improve the possibilities to diagnose malignant mesothelioma by combining biomarkers and by identifying new ones. We also investigate tumour driving mechanisms with focus on one of these suggested biomarkers, the cell-bound proteoglycan syndecan-1. We were able to construct a diagnostic two-step model based on biomarkers in patient material. By implementing a cut-off level and thereafter focusing on unresolved patients we combined hyaluronan and N-ERC/mesothelin (paper I), which significantly increased the diagnostic accuracy for malignant mesothelioma. To further improve diagnosis, we used mass spectrometry to find new biomarkers. We identified and validated galectin-1, which was excellent in discriminating mesotheliomas from adenocarcinomas (paper II). In the same study, we were also the first to describe aldo-keto reductase 1B10 as a novel prognostic mesothelioma biomarker. Syndecan-1 has been indicated as a marker for carcinomas. In paper I we describe how higher levels of syndecan-1 indicate the presence of a carcinoma over a mesothelioma. This was verified in paper II when syndecan-1 was identified as downregulated in fluids from mesothelioma patients compared to lung cancer patients. Paper III and paper IV focus on this proteoglycan. Malignant cell lines transfected with syndecan-1 and various truncated forms of syndecan-1 affected adhesion and migration, which are key features of cancer invasion (paper III). The results showed a domain- and cell type specific effect on the cells’ motility. Regulating syndecan-1 levels and analysing the global gene expression of mesothelioma cells made it evident that this proteoglycan has a strong influence on transforming growth factor β signalling and several growth factor pathways (paper IV). Links to cell migration and proliferation were furthermore identified, along with glycosaminoglycan modifying enzymes. These results can shed light on the complex role of syndecan-1 in invasion and growth of malignant mesenchymal cells. Taken together, this thesis work describes a complement to conventional mesothelioma diagnosis and identifies novel biomarkers. Furthermore, the potential biomarker syndecan-1 was shown to have an effect on cell motility and proliferation. These results increase our understanding of this aggressive malignancy

Phenotype-dependent apoptosis signalling in mesothelioma cells after selenite exposure

<p>Abstract</p> <p>Background</p> <p>Selenite is a promising anticancer agent which has been shown to induce apoptosis in malignant mesothelioma cells in a phenotype-dependent manner, where cells of the chemoresistant sarcomatoid phenotype are more sensitive.</p> <p>Methods</p> <p>In this paper, we investigate the apoptosis signalling mechanisms in sarcomatoid and epithelioid mesothelioma cells after selenite treatment. Apoptosis was measured with the Annexin-PI assay. The mitochondrial membrane potential, the expression of Bax, Bcl-XL, and the activation of caspase-3 were assayed with flow cytometry and a cytokeratin 18 cleavage assay. Signalling through JNK, p38, p53, and cathepsins B, D, and E was investigated with chemical inhibitors. Furthermore, the expression, nuclear translocation and DNA-binding activity of p53 was investigated using ICC, EMSA and the monitoring of p21 expression as a downstream event. Levels of thioredoxin (Trx) were measured by ELISA.</p> <p>Results</p> <p>In both cell lines, 10 μM selenite caused apoptosis and a marked loss of mitochondrial membrane potential. Bax was up-regulated only in the sarcomatoid cell line, while the epithelioid cell line down-regulated Bcl-XL and showed greater caspase-3 activation. Nuclear translocation of p53 was seen in both cell lines, but very little p21 expression was induced. Chemical inhibition of p53 did not protect the cells from apoptosis. p53 lost its DNA binding ability after selenite treatment and was enriched in an inactive form. Levels of thioredoxin decreased after selenite treatment. Chemical inhibition of MAP kinases and cathepsins showed that p38 and cathepsin B had some mediatory effect while JNK had an anti-apoptotic role.</p> <p>Conclusion</p> <p>We delineate pathways of apoptosis signalling in response to selenite, showing differences between epithelioid and sarcomatoid mesothelioma cells. These differences may partly explain why sarcomatoid cells are more sensitive to selenite.</p

Versican but not decorin accumulation is related to malignancy in mammographically detected high density and malignant-appearing microcalcifications in non-palpable breast carcinomas

<p>Abstract</p> <p>Background</p> <p>Mammographic density (MD) and malignant-appearing microcalcifications (MAMCs) represent the earliest mammographic findings of non-palpable breast carcinomas. Matrix proteoglycans versican and decorin are frequently over-expressed in various malignancies and are differently involved in the progression of cancer. In the present study, we have evaluated the expression of versican and decorin in non-palpable breast carcinomas and their association with high risk mammographic findings and tumor characteristics.</p> <p>Methods</p> <p>Three hundred and ten patients with non-palpable suspicious breast lesions, detected during screening mammography, were studied. Histological examination was carried out and the expression of decorin, versican, estrogen receptor α (ERα), progesterone receptor (PR) and c-erbB2 (HER-2/neu) was assessed by immunohistochemistry.</p> <p>Results</p> <p>Histological examination showed 83 out of 310 (26.8%) carcinomas of various subtypes. Immunohistochemistry was carried out in 62/83 carcinomas. Decorin was accumulated in breast tissues with MD and MAMCs independently of the presence of malignancy. In contrast, versican was significantly increased only in carcinomas with MAMCs (median ± SE: 42.0 ± 9.1) and MD (22.5 ± 10.1) as compared to normal breast tissue with MAMCs (14.0 ± 5.8), MD (11.0 ± 4.4) and normal breast tissue without mammographic findings (10.0 ± 2.0). Elevated levels of versican were correlated with higher tumor grade and invasiveness in carcinomas with MD and MAMCs, whereas increased amounts of decorin were associated with <it>in situ </it>carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs.</p> <p>Conclusions</p> <p>The specific accumulation of versican in breast tissue with high MD and MAMCs only in the presence of malignant transformation and its association with the aggressiveness of the tumor suggests its possible use as molecular marker in non-palpable breast carcinomas.</p

Malignant mesothelioma : An experimental study with emphasis on proteoglycans in mesothelial cell growth and differentiation

Malignant mesothelioma is a highly aggressive tumor with median survival ranging from 4 to 12 months and, despite intense therapeutical efforts, it is invariably fatal. Mesothelioma cells are unique in the sense that they possess a biphasic growth potential and can be stimulated by serum growth factors to differentiate into stable epithelial or fibrous phenotypes. The prognosis of this tumor varies greatly depending on the differences in growth pattern, the most important predictor of poor prognosis being a fibrous phenotype. To study the molecular basis of mesothelial differentiation, we used benign and malignant mesothelial cells in various stages of phenotypic differentiation. In order to evaluate the impact of proteoglycans (PG) on this process, a series of PGs were analyzed by semi quantitative reversed transcriptase polymerase chain reaction. The cells with epithelial phenotype showed increased expression of syndecan-2, syndecan-4 and hyaluronan synthase, and fibroblast-like cells expressed more matrix PGs: versican, decorin and biglycan. The PG profile may serve as a "fingerprint", and reflect the maturation of mesothelial cells. The functional importance of syndecans in mesothelial differentiation was further shown by antisense targeting; down-regulation of each particular syndecan caused a loss of epithelial morphology, syndecans - 1 and -4 being also essential for cell adhesion. The differentiation of mesothelioma cells was influenced by treatment with various growth factors (TGF-beta2, EGF, FGF-2, IGF-I and PDGF-BB). These factors affected the proliferation and morphology of mesothelioma cells to various extents, and the PG profile changed, in parallel, with an induced epithelial-mesenchymal transition. Exposure to EG17 and IGF-I caused a fibroblast-like morphology simultaneously with a reduction in the syndecan expression levels. At the same time, the levels of shed syndecan-1 increased in the culture medium. The involvement of other regulatory molecules in mesothelioma differentiation was assessed by subtractive hybridization, which has revealed a limited number of genes being differentially expressed between cells of epithelial or fibrous phenotypes. Most of these genes were recovered from the epithelial cells, which may indicate a more mature phenotype. The expression level of thioredoxin reductase, a small redox-active protein involved in drug resistance, was extremely high in both cell sub-lines, and may reflect the generic insensitivity of mesotheliomas to chemotherapy. Although syndecans play a major role in regulating cell morphology, little is known about their subcellular distribution. Using confocal laser microscopy we found a substantial proportion of syndecans at intracellular locations, and syndecan-1 accumulated in the nucleus in a time-dependent manner. There was a close spatial relation of syndecans to tubulin in both interphase and mitotic cells. Vinblastine treatment interfered with the nuclear transport, and syndecan-1 and tubulin co- polymerize in paracrystalline occlusion bodies, in parallel with impaired nuclear transport. These findings suggest a tubulin-mediated transport mechanism. TGF-beta2 reduced the proliferation rate of mesothelioma cells, concomitantly with a delay in nuclear transport of syndecan-1. These data show that all syndecans are involved in maintaining the epithelial morphology, and that various amounts and translocation of syndecans may participate in molecular switches that regulate cell differentiation and proliferation. The above mechanisms may represent crucial steps, and possible future targets for therapy, that can be used to improve the management of patients with malignant mesothelioma

Syndecan-1 in Cancer: Implications for Cell Signaling, Differentiation, and Prognostication

Syndecan-1, a cell surface heparan sulfate proteoglycan, is critically involved in the differentiation and prognosis of various tumors. In this review, we highlight the synthesis, cellular interactions, and the signalling pathways regulated by syndecan-1. The basal syndecan-1 level is also crucial for understanding the sequential changes involving malignant transformation, tumor progression, and advanced or disseminated cancer stages. Moreover, we focus on the cellular localization of this proteoglycan as cell membrane anchored and/or shed, soluble syndecan-1 with stromal or nuclear accumulation and how this may carry different, highly tissue specific prognostic information for individual tumor types

Utility of BerEp4/calretinin and desmin/epithelial membrane antigen (EMA) dual immunocytochemical staining in effusion cytology

Abstract Background Pleural mesothelioma (PM) is typically diagnosed late during the disease. Earlier detection can increase the chance of effective therapy. Recurrent pleural effusions are the earliest symptoms displaying an array of cytomorphological changes from reactive atypia to malignancy. Diagnosis is possible on effusion cytology by applying molecular and immunocytochemical markers, the main difficulty being when to suspect PM and to differentiate PM from metastatic adenocarcinoma and reactive mesothelial proliferations. Methods We evaluated the diagnostic performance of two immunocytochemical dual stains (BerEp4/Calretinin and Desmin/Epithelial Membrane Antigen (EMA)) on 149 ethanol‐fixed cytospin preparation as an initial step to solve the mentioned diagnostic difficulty. The immunocytochemical reactivity pattern was evaluated by two independent investigators. The final diagnosis corresponded to PM (n = 20), metastatic adenocarcinoma (n = 83), and mesotheliosis (n = 46) in these cases. Results Calretinin had 99% specificity and 98% sensitivity for indicating a mesothelial phenotype, while BerEp4 distinguished the adenocarcinoma cases with 98% specificity and 99% sensitivity. EMA displayed 96% specificity and 99% sensitivity in malignant cases, while Desmin without EMA present showed 99% specificity and 96% sensitivity for indicating benign mesothelial proliferation. Conclusions Interpretation of the four immunoreactions is improved when performed as dual stains. The dual staining is a useful tool in the initial handling of atypical effusions and guides the subsequent choice of antibody panels for more detailed subclassification of malignant effusions

Cytoskeletal Organization Correlates to Motility and Invasiveness of Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is a rare but highly aggressive cancer that primarily originates from the pleura, peritoneum or pericardium. There is a well-established link between asbestos exposure and progression of MM. Direct invasion of the surrounding tissues is the main feature of MM, which is dependent on dysregulated communication between the mesothelium and the microenvironment. This communication is dependent on the dynamic organization of the cytoskeleton. We have analyzed the organization and function of key cytoskeletal components in MM cell lines of increasing malignancies measured as migratory and invasive properties, and we show that highly malignant and invasive MM cells have an organization of the actin filament and vimentin systems that is distinct from the less malignant MM cell lines. In addition, the Hippo tumor suppressor pathway was inactivated in the invasive MM cells, which was seen as increased YAP nuclear localization