Elevated cyclic AMP suppresses ConA-induced MT1-MMP expression in MDA- MB-231 human breast cancer cells

We have previously reported that induction of MMP-2 activation by Concanavalin A (ConA) in MDA-MB-231 human breast cancer cells involves both transcriptional and post-transcriptional mechanisms, and that the continuous presence of ConA is required for MMP-2 activation (Yu et al. Cancer Res, 55, 3272-7, 1995). In an effort to identify signal transduction pathways which may either contribute to or modulate this mechanism, we found that three different cAMP-inducing agents, cholera toxin (CT), forskolin (FSK), and 3- isobutyl-1-methylxanthine (IBMX) partially inhibited ConA-induced MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. Combinations of CT or FSK with IBMX exhibited additive effects on reduction of MT1-MMP mRNA expression and MMP-2 activation. Agents which increase cAMP levels appeared to target transcriptional aspects of ConA induction, reducing MT1-MMP mRNA and protein in parallel with the reduced MMP-2 activation. In the absence of ConA, down-regulation of constitutive production of MT1-MMP mRNA and protein was observed, indicating that cAMP acts independently of ConA. These observations may help to elucidate factors regulating MT1-MMP expression, which may be pivotal to the elaboration of invasive machinery on the cell surface.

Effects of short-duration and long-duration exercise on lipoprotein(a)

Purpose Most studies that use either a single exercise session, exercise training, or a cross-sectional design have failed to find a relationship between exercise and plasma lipoprotein(a) [Lp(a)] concentrations. However, a few studies investigating the effects of longer and/or more strenuous exercise have shown elevated Lp(a) concentrations, possibly as an acute-phase reactant to muscle damage. Based on the assumption that greater muscle damage would occur with exercise of longer duration, the purpose of the present study was to determine whether exercise of longer duration would increase Lp(a) concentration and creatine kinase. (CK) activity more than exercise of shorter duration. Methods Ten endurance-trained men (mean +/- SD: age, 27 +/- 6 yr; maximal oxygen consumption [(V)over dotO(2max)], 57 +/- 7 mL(.)kg(-1) min(-1)) completed two separate exercise sessions at 70% (V)over dotO(2max). One session required 900 kcal of energy expenditure (60 +/- 6 min), and the other required 1500 kcal (112 +/- 12 min). Fasted blood samples were taken immediately before (0-pre), immediately after (0-post), 1 d after (1-post), and 2 d after (2-post) each exercise session. Results CK activity increased after both exercise sessions (mean +/- SE; 800 kcal: 0-pre 55 +/- 11, 1-post 168 +/- 64 U(.)L(-1.)min(-1); 1500 kcal: 0-pre 51 +/- 5, 1-post 187 +/- 30, 2-post 123 +/- 19 U(.)L(-1.)min(-1); P < 0.05). However, median Lp(a) concentrations were not altered by either exercise session (800 kcal: 0-pre 5.0 mg(.)dL(-1), 0-post 3.2 mg(.)dL(-1), 1-post 4.0 mg(.)dL(-1), 2-post 3.4 mg(.)dL(-1); 1500 kcal: 0-pre 5.8 mg(.)dL(-1), 0-post 4.3 mg(.)dL(-1), 1-post 3.2 mg(.)dL(-1), 2-post 5.3 mg(.)dL(-1)). In addition, no relationship existed between exercise-induced changes in CK activity and Lp(a) concentration (800 kcal: r = -0.26; 1500 kcal: r = -0.02). Conclusion These results suggest that plasma Lp(a) concentration will not increase in response to minor exercise-induced muscle damage in endurance-trained runners.

Cells Tissues Organs : preface

This volume stems from the 1st International Conference on Epithelial-Mesenchymal Transitions (EMT), which was convened by the editors on October 5–8, 2003 in the beautiful setting of Port Douglas, Queensland, Australia. EMT, the name given to the transformation of cells arranged in a coherent layer – epithelial cells – to more individualistic and potential motile cells – mesenchymal cells – was recognized decades ago by Prof. Elisabeth (Betty) Hay (Harvard Medical School, Boston, Mass., USA) as a primary mechanism in embryogenesis for remodelling tissues. More recently EMT has been seen as crucial to the spread and invasion of carcinoma, and more recently still, EMT-like changes have been detected in various pathologies marked by fi brosis. Despite the basic and clinical importance of EMT, this extremely rapidly growing fi eld had never previously had a conference devoted to it, and indeed the disciplines of developmental biology, cancer and pathology rarely interact although they have much to share. The chapters assembled for this volume encompass these three major themes of the meeting, development, pathology and cancer, and further highlight the commonality in terms of mechanisms and outcomes among them...

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Isolation and characterization of tumor cells from the ascites of ovarian cancer patients : molecular phenotype of chemoresistant ovarian tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture

Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al. Biol Reprod 1990; 43:946-955, 956-964). We have now investigated the regulation of testicular cell type IV collagenase and other metalloprotemases in vitro. Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels. However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mENA levels. Zyniographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-Wa type IV collagenase protein levels. However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloprotemnases (83 kDa and 110 Wa and in an activation of the 72-Wa rat type IV collagenase. Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation, lmmunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells. Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern. Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 Wa, 83 Wa, and 110 Wa and a triton-insoluble gelatinase of 225 Wa, These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Myogel supports the ex-vivo amplification of corneal epithelial cells

Limbal stem cell deficiency leads to conjunctivalisation of the cornea and subsequent loss of vision. The recent development of transplantation of ex-vivo amplified corneal epithelium, derived from limbal stem cells, has shown promise in treating this challenging condition. The purpose of this research was to compare a variety of cell sheet carriers for their suitability in creating a confluent corneal epithelium from amplified limbal stem cells. Cadaveric donor limbal cells were cultured using an explant technique, free of 3T3 feeder cells, on a variety of cell sheet carriers, including denuded amniotic membrane, Matrigel, Myogel and stromal extract. Comparisons in rate of growth and degree of differentiation were made, using immunocytochemistry (CK3, CK19 and ABCG2). The most rapid growth was observed on Myogel and denuded amniotic membrane, these two cell carriers also provided the most reliable substrata for achieving confluence. The putative limbal stem cell marker, ABCG2, stained positively on cells grown over Myogel and Matrigel but not for those propagated on denuded amniotic membrane. In the clinical setting amniotic membrane has been demonstrated to provide a suitable carrier for limbal stem cells and the resultant epithelium has been shown to be successful in treating limbal stem cell deficiency. Myogel may provide an alternative cell carrier with a further reduction in risk as it is has the potential to be derived from an autologous muscle biopsy in the clinical setting.

Factors regulating basement membrane invasion by tumor cells

Basement membranes serve as significant barriers to the passage of tumor cells but ones which metastatic cells can pass. This involves the production of a cascade of proteases leading to the activation of a specific collagenase that degrades the unique collagen network in basement membrane. Breast cancer cells, when estrogen dependent, show a requirement for estrogen for invasive activity. However, when these cells progress to an estrogen independent state and increased malignancy, they express an invasive phenotype constitutively. Studies with various anti-estrogens suggest that these responses are mediated via the estrogen receptor. Anti-estrogens lacking agonist activity suppress invasiveness as well as growth of the breast cancer cells.

Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity

During invasion and metastasis, cancer cells interact closely with the extracellular matrix molecules by attachment, degradation, and migration. We demonstrated previously the local degradation of fluorescently labeled gelatin matrix by cancer cells at invasive membrane protrusions, called invadopodia. Using the newly developed quantitative fluorescence-activated cell sorting-phagocytosis assay and image analysis of localized degradation of fluorescently labeled matrix, we document here that degradation and site- specific removal of cross-linked gelatin matrix is correlated with the extent of phagocytosis in human breast cancer cells. A higher phagocytic capacity is generally associated with increasing invasiveness, documented in other invasion and motility assays as well. Gelatin phagocytosis is time and cell density dependent, and it is mediated by the actin cytoskeleton. Most of the intracellular gelatin is routed to actively acidified vesicles, as demonstrated by the fluorescent colocalization of gelatin with acidic vesicles, indicating the intracellular degradation of the phagocytosed matrix in lysosomes. We show here that normal intracellular routing is blocked after treatment with acidification inhibitors. In addition, the need for partial proteolytic degradation of the matrix prior to phagocytosis is demonstrated by the inhibition of gelatin phagocytosis with different serine and metalloproteinase inhibitors and its stimulation by conditioned medium containing the matrix metalloproteinases MMP-2 and MMP-9. Our results demonstrate that phagocytosis of extracellular matrix is an inherent feature of breast tumor cells that correlates with and may even directly contribute to their invasive capacity. This assay is useful for screening and evaluating potential anti-invasive agents because it is fast, reproducible, and versatile.

Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

Invasive activity and chemotactic response to growth factors by Kaposi's sarcoma cells

Kaposi's sarcoma (KS) is a relatively low grade neoplasm, classically occurring in the skin of elderly men. A more virulent and invasive form of Kaposi's sarcoma has been described in patients with acquired immune deficiency syndrome (AIDS). The origin and identification of the tumor cells in these lesions is controversial. Here we have studied the behavior of cells derived from KS lesions in an in vitro assay which measures the ability of cells to invade through a reconstituted basement membrane. In agreement with previous work, KS cells obtained under selective culture conditions were invasive showing activity comparable to that of malignant tumor cells. Normal fibroblasts, smooth muscle cells, and endothelial cells did not demonstrate invasive behavior under the same experimental conditions. To characterize further the nature of the KS cells we tested the chemotactic response of cells from the most invasive line to a variety of growth factors and compared their response to those of fibroblasts, smooth muscle, and endothelial cells. These studies suggest that normal cells respond to a unique repertoire of chemotactic factors. The chemotactic response of the KS cells most closely resembled that of smooth muscle cells and was quite distinct from endothelial cells. These results indicate that the KS-derived cultures contain invasive cells with a smooth muscle cell-like phenotype.

Plasma lipid and lipoprotein responses during exercise

The purpose of this study was to examine the effect of prolonged exercise oil plasma lipid and lipoprotein concentrations and to identify caloric time-points where changes occurred. Eleven active male Subjects ran oil a treadmill at 70%,, of maximal fitness (VO2max) and expended 6 278.7 kilojoules (Kj) energy (1500 kcal). Blood samples were obtained at the 4185.8 Kj (1000 kcal) time-point during exercise and at each additional 418.6 Kj (100 kcal) expenditure until 6278.7 Kj was expended. After correcting for plasma volume changes, decreases in low-density lipoprotein cholesterol (LDL-C) were observed during exercise at time-points corresponding to 4604.4 and 5441.5 Kj (1100 and 1300 kcal) of energy expenditure, and immediately after exercise. Total cholesterol concentrations decreased significantly at exercise kilojoule expenditures of 4604.4, 5441.5 and 5860.1 (1100, 1300 and 1400 kcal). There were also exercise induced increases in high-density lipoprotein cholesterol (HDL-C) and HDL2-C concentrations immediately after exercise. Although acute lipid and lipoprotein changes are typically reported in the days following exercise, the Current data indicate that some lipoprotein concentrations change during acute exercise. Our data suggest that a threshold of exercise may be necessary to change lipoproteins during exercise. Future work Should identify potential mechanisms (lipoprotein lipase, cholesterol ester transport protein, LDL uptake) that alter lipoprotein concentrations during prolonged exercise.

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and β-catenin, localized increased levels of vimentin and redistribution of α6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.