The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.
The RNA-binding protein Musashi-1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch-1 signaling pathway. Musashi-1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi-1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase-1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi-1 expression is 20.8-fold upregulated in SP1 compared to SP-and equally distributed between ALDH1 and ALDH-cell pools. siRNA-mediated knockdown of Musashi-1 mRNA expression lead to an altered expression of the signaling receptor Notch-1 and its downstream targets, the transcription factor Hes-1 and the cell cycle regulators p21and cyclin B1, as determined by Western blotting and quantitative real-time PCR. Flow cytometric and ELISA analyses revealed that Musashi-1-mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell-like properties. Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness-related factors Notch-1, Hes-1 and p21 WAF1/CIP1 , thus emerging as a novel future target for endometrial carcinoma therapy.Similar to stem cells, a subpopulation of cancer cells is characterized by a high proliferative capacity, self-renewal, expression of multidrug-resistance proteins, and a high degree of plasticity.
Klaus Schulze-Osthoff and Marek Los share senior authorship. ABSTRACTCryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase-3 is induced during the freeze-thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD-fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony-forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl-2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjuryassociated cell death is mediated by a Bcl-2-controlled mitochondrial pathway. Thus, our data suggest the use of zVAD-fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.Keywords: apoptosis • cryopreservation • stem cells • cathepsins • calpains W ith the recent advances in cell and tissue transplantation, tissue engineering, in vitro fertilization, and other genetic technologies, preservation of biological materials has become increasingly important in medical care and biotechnological industries. Cryopreservation is the only reliable form of long-term storage of viable cells and tissues. However, both the freeze and the thaw process result in considerable cell and tissue injury (1, 2). Various mechanisms, including oxidative stress, mechanical injury due to ice crystal formation, altered physical properties of cellular structures, osmotic injury, and disturbed ion homeostasis due to Na + /K + -ATPase inhibition are responsible for cell damage during hypothermia and freezethaw processes (3)(4)(5)(6)(7)(8). Traditional protocols rely on supplementation of the freeze medium with penetrating cryoprotectants such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, or hydroxyethyl starch (2). Their major role is the prevention of lethal ice formation and osmotic injury. Stabilization of cellular structures with small carbohydrate sugars such as trehalose has recently been shown to also markedly improve cell recovery (9). Despite significant improvements of cryopreservation protocols, clinical procedures are often not satisfactory, and up to 50% of the cryopreserved cells may die within the first 24 h after the thaw process (10). There are some indications that hypothermia is associated with apoptosis (9, 11), although the role of apoptosis in cryoinjury has not been studied in detail. Because inhibition of proteases of the caspase family protects against apoptosis in various experimental settings (12-15), we examined whether addition of the ...
ScopeIn this study, human exposure to the mycotoxin ochratoxin A (OTA) and its thermal degradation product 2’R‐ochratoxin A (2’R‐OTA, previously named as 14R‐Ochratoxin A [22]) through coffee consumption was assessed. LC‐MS/MS and the dried blood spot (DBS) technique were used for the analysis of blood samples from coffee and noncoffee drinkers (n = 50), and food frequency questionnaires were used to document coffee consumption.Methods and resultsFor the detection of OTA and 2’R‐OTA in blood, a new sensitive and efficient sample preparation method based on DBS was established and validated. Using this technique 2’R‐OTA was for the first time detected in biological samples. Comparison between coffee drinkers and noncoffee drinkers showed for the first time that 2’R‐OTA was only present in blood from the first group while OTA could be found in both groups in a mean concentration of 0.21 μg/L. 2’R‐OTA mean concentration was 0.11 μg/L with a maximum concentration of 0.414 μg/L. Thus, in average 2’R‐OTA was approx. half the concentration of OTA but in some cases even exceeded OTA levels. No correlation between the amounts of coffee consumption and OTA or 2’R‐OTA levels was observed.ConclusionThe results of this study revealed for the first time a high exposure of coffee consumers to 2’R‐OTA, a compound formed from OTA during coffee roasting. Since little information is available regarding toxicity and possible carcinogenicity of this compound, further OTA monitoring in blood including 2’R‐OTA is advisable.
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