Biomedical subjects

Avraham Raz

Publications and source records attributed to Avraham Raz.

53 records · Page 3Linked to original sources

PubMed2003

Malignant transformation of thyroid follicular cells by galectin-3.

Galectin-3, a beta-galactoside binding lectin, is highly expressed in thyroid carcinomas of follicular cell origin, whereas neither benign thyroid adenomas nor normal thyroid tissues express galectin-3. We previously showed that antisense inhibition of galectin-3 expression markedly reduced the malignant phenotype of thyroid papillary carcinoma cells. In the present study we transfected galectin-3 cDNA into TAD-2 normal thyroid follicular cells. Stable transfectants expressing galectin-3 acquired the phenotype of serum-independent growth, clonogenicity in soft agar, as well as loss of contact inhibition. We also compared the gene expression profile of the galectin-3 transfectants to that of the vehicle control, which revealed that a series of genes were differentially expressed between the two. They include proliferating cell nuclear antigen, replication factor C, and retinoblastoma genes that participate in G1-S transition. These results indicate the transformation of thyroid follicular cells by galectin-3 and possible involvement of galectin-3 in cell cycle.

Antigens, Polyomavirus Transforming↗

PubMed2003

Differential expression of galectin-3 in pituitary tumors.

Galectin-3 (Gal-3), a beta-galactoside-binding protein, has been implicated in a variety of biological functions, including cell proliferation and differentiation, tumor cell adhesion, angiogenesis, apoptosis, tumor progression, and metastasis. We investigated the role of Gal-3 in the development and progression of pituitary tumors. Immunohistochemical and Western blot analysis of normal and neoplastic human pituitaries showed that only lactotroph (PRL) and corticotroph (ACTH) hormone-producing cells and tumors expressed Gal-3. Gal-3 was present in 24 of 38 (63.2%) PRL adenomas, 5 of 6 (83.3%) PRL carcinomas, 19 of 41 (46.3) ACTH adenomas, and 7 of 8 (87.5%) ACTH carcinomas, but not in 112 other pituitary adenomas and carcinomas. Pituitary folliculo-stellate cells, which have macrophage-type functions in the anterior pituitary, also expressed Gal-3. Hyperplastic and neoplastic pituitaries from p27(Kip1) (p27)-null mice, which produce mainly ACTH, showed increased Gal-3 expression levels compared with control mice. Treatment with transforming growth factor beta1, which regulates pituitary cell proliferation, reduced Gal-3 as well as p27 expression levels in cultured HP75 pituitary cells and Gal-3 in cultured pituitary cells from p27-null mice, suggesting that p27 is not necessary for the inhibitory effects of transforming growth factor beta1 on the cell cycle in the pituitary. The role of Gal-3 in pituitary cell function was examined by RNA interference experiments. Inhibition of Gal-3 gene expression by RNA interference decreased HP75 cell proliferation and increased apoptosis. These results indicate that Gal-3 has an important role in pituitary cell proliferation and tumor progression.

Adenoma↗

PubMed2003

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function.

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven-transmembrane domain receptor (gp78/AMF-R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site-directed mutagenesis, single- and double-deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins' binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.

Amino Acid Motifs↗

PubMed2003

Overexpression of the autocrine motility factor/phosphoglucose isomerase induces transformation and survival of NIH-3T3 fibroblasts.

Autocrine motility factor (AMF)/phosphoglucose isomerase (PGI) is a ubiquitous cytosolic enzyme and is produced as a leaderless secretory protein, released from cells via a nonclassical pathway. AMF/PGI acts extracellularly as a potent mitogen/cytokine (CXXC, chemokine). Increased expression of AMF/PGI and its receptor/CXXC-R has been found in a wide spectrum of malignancies, and is associated with cancer progression and metastasis. To directly elucidate the functional role of AMF/PGI on cell motility and neoplastic transformation, we stably transfected AMF/PGI cDNA into NIH-3T3 cells. Ectopic overexpression of AMF/PGI results in its secretion and activation via a constitutive autocrine activation loop that renders the cells highly motile, acquiring a transformed phenotype in vitro and tumorigenicity in vivo. The transformed phenotype of AMF/PGI-transfected cells leads in part resistance to induction of apoptosis induced by serum starvation, through the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway and down-regulation of caveolin-1 expression. Overexpression of this housekeeping gene induces resistance to apoptosis and neoplastic transformation, and, thus, AMF/PGI represents a novel class of oncogenic protein.

3T3 Cells↗

PubMed2003

Overexpression of autocrine motility factor receptor (AMFR) in NIH3T3 fibroblasts induces cell transformation.

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of 78000 molecular weight (gp78), regulating cell motility signaling in vitro and metastasis in vivo. To test whether AMFR could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 fibroblast cells with expression vectors carrying the full-length cDNA for mouse AMFR and evaluated the effects of increased AMFR on transforming potential. The cells stably expressing high levels of AMFR as a result of transfection displayed a complete morphological change and acquired the ability to grow even in low serum. Furthermore, they were anchorage-independent for growth in soft agar and more motile in phagokinetic track assay. Interestingly, the enhanced expression of AMFR produced tumors in nude mice. Our findings provide a direct evidence that overexpression of the AMFR is associated with the acquisition of a transformation phenotype.

3T3 Cells↗

PubMed2002

Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin.

BACKGROUND: The role of dietary components in cancer progression and metastasis is an emerging field of clinical importance. Many stages of cancer progression involve carbohydrate-mediated recognition processes. We therefore studied the effects of high pH- and temperature-modified citrus pectin (MCP), a nondigestible, water-soluble polysaccharide fiber derived from citrus fruit that specifically inhibits the carbohydrate-binding protein galectin-3, on tumor growth and metastasis in vivo and on galectin-3-mediated functions in vitro. METHODS: In vivo tumor growth, angiogenesis, and metastasis were studied in athymic mice that had been fed with MCP in their drinking water and then injected orthotopically with human breast carcinoma cells (MDA-MB-435) into the mammary fat pad region or with human colon carcinoma cells (LSLiM6) into the cecum. Galectin-3-mediated functions during tumor angiogenesis in vitro were studied by assessing the effect of MCP on capillary tube formation by human umbilical vein endothelial cells (HUVECs) in Matrigel. The effects of MCP on galectin-3-induced HUVEC chemotaxis and on HUVEC binding to MDA-MB-435 cells in vitro were studied using Boyden chamber and labeling assays, respectively. The data were analyzed by two-sided Student's t test or Fisher's protected least-significant-difference test. RESULTS: Tumor growth, angiogenesis, and spontaneous metastasis in vivo were statistically significantly reduced in mice fed MCP. In vitro, MCP inhibited HUVEC morphogenesis (capillary tube formation) in a dose-dependent manner. In vitro, MCP inhibited the binding of galectin-3 to HUVECs: At concentrations of 0.1% and 0.25%, MCP inhibited the binding of galectin-3 (10 micro g/mL) to HUVECs by 72.1% (P =.038) and 95.8% (P =.025), respectively, and at a concentration of 0.25% it inhibited the binding of galectin-3 (1 micro g/mL) to HUVECs by 100% (P =.032). MCP blocked chemotaxis of HUVECs toward galectin-3 in a dose-dependent manner, reducing it by 68% at 0.005% (P<.001) and inhibiting it completely at 0.1% (P<.001). Finally, MCP also inhibited adhesion of MDA-MB-435 cells, which express galectin-3, to HUVECs in a dose-dependent manner. CONCLUSIONS: MCP, given orally, inhibits carbohydrate-mediated tumor growth, angiogenesis, and metastasis in vivo, presumably via its effects on galectin-3 function. These data stress the importance of dietary carbohydrate compounds as agents for the prevention and/or treatment of cancer.

Adenocarcinoma↗

PubMed2002

Galectin-3 enhances cyclin D(1) promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells.

Galectin-3 is a multifunctional carbohydrate-binding protein found in the nucleus, cytoplasm and the extracellular milieu. Nuclear galectin-3 expression is associated with cell proliferation, and its role in pre-mRNA splicing has been suggested. In this report, we investigated the role of galectin-3 on cyclin D(1) gene expression, a critical inducer of the cell cycle and a potential oncogene in human cancer. We found that galectin-3 induces cyclin D(1) promoter activity in human breast epithelial cells independent of cell adhesion through multiple cis-elements, including the SP1 and CRE sites. We present evidence that galectin-3 induction of the cyclin D(1) promoter may result from enhancement/stabilization of nuclear protein-DNA complex formation at the CRE site of the cyclin D(1) promoter. We also show that galectin-3 co-operates with, but does not depend on, pRb for cyclin D(1) promoter activation. The present study reveals a growth promoting activity of galectin-3 through cyclin D(1) induction, and suggests a novel function of nuclear galectin-3 in the regulation of gene transcription.

Base Sequence↗

PubMed2002

Autocrine motility factor secreted by tumor cells upregulates vascular endothelial growth factor receptor (Flt-1) expression in endothelial cells.

The autocrine motility factor (AMF) is known as a cytokine regulating tumor cells motility via AMF receptor (AMFR) and promotes their metastasis. Recently, AMFRs have been found on the surface of host cells and it was showed that AMF possibly affects them. The signaling of AMF-AMFR in the host endothelial cells induces expression of a vascular endothelial growth factor receptor (VEGFR) Flt-1 and AMFR feedback that is regulated at the transcriptional level. AMF-exposure stimulated the Flt-1 expression on human umbilical vein endothelial cells (HUVECs) surface and this AMF-treated cells exhibited high-responsibility against VEGF. The protein kinase C (PKC) and phosphatidylinositol 3 kinase (PI3K) play an important role in this signal transduction. The findings of our study suggest the possibility of "tumor AMF-->host AMFR-->PKC, PI3K-->-->VEGFR or AMFR-->angiogenesis, metastasis" as a new signal cross talk between the tumor and the host.

Blotting, Western↗

PubMed2002

Activation of small GTPase Rho is required for autocrine motility factor signaling.

The hallmark of tumor metastasis is the dissemination of cells from the primary growth site to distant organs. Autocrine motility factor (AMF), a tumor-associated C-X-X-C cytokine, the ligand for a unique 78 kDa seven transmembrane receptor, is a potent simulator of cell motility, a process that is a prerequisite for tumor progression and metastasis. Because little is known about AMF-dependent signaling, we sought to study whether AMF signaling involves family members of the Rho-like GTPases. AMF stimulation of human melanoma cells resulted in stress-fiber formation, concomitant with up-regulation and activation of both RhoA and Rac1 expression with no apparent changes in the expression level or activation state of Cdc42. Treatment of the cells with C3 exoenzyme before AMF stimulation inhibited both the formation of stress-fiber-like structures and the activation of RhoA. In addition, both c-Jun NH(2)-terminal kinase 1 and c-Jun NH(2)-terminal kinase 2 were simultaneously activated by AMF, supporting the notion that they are involved in the signaling pathway of RhoA. We thus conclude that AMF signaling shares a similar pathway to previously established paracrine factors signaling involving cytoskeletal rearrangement and morphological alterations mediated by the small RhoA-like GTPases.

ADP Ribose Transferases↗

PubMed2002

Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies.

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical with the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, the intracellular enzyme phosphohexose isomerase (PHI). The crystal structures of the inhibitor-free open form and the inhibitor (erythrose 4-phosphate, E4P, a strong inhibitor of AMF's cytokine activity)-bound closed form of human AMF have been determined at 1.9 A and 2.4 A resolution, respectively. Upon E4P binding, local conformation changes (open to closed) occur around the inhibitor-binding site. The E4P-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PHIs. The present study shows clearly that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF and PHI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate group. One likely scenario is that the compound could compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven-transmembrane helix protein. The other scenario is that the local conformation changes upon inhibitor binding may affect the AMF-AMFR interactions. To examine roles of the residues in the inhibitor-binding site, two mutant AMFs were prepared. Replacements of His389, which is hydrogen-bonded to the hydroxyl group of E4P by Phe, and Thr215, which is hydrogen-bonded to the phosphate group of E4P by Asp, result in mutant AMFs that are impaired in cytokine activity. These results suggest a role for these amino acids in recognition of a carbohydrate moiety of the AMFR. Since the E4P is one of the smallest compounds having AMF inhibitor activity, knowledge of the present crystal structure would provide an insight into the lead compound design of more effective AMF inhibitors.

Binding Sites↗

PubMed2002

Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid.

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.

Animals↗

PubMed2002

Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation.

Galectin-3 is a multifunctional oncogenic protein found in the nucleus and cytoplasm and also the extracellular milieu. Although recent studies demonstrated an anti-apoptotic activity of galectin-3, neither the functional site nor the mechanism of how galectin-3 regulates apoptosis is known. In this study, we examined the subcellular localization of galectin-3 during apoptosis and investigated its anti-apoptotic actions. We report that galectin-3 translocates to the perinuclear membrane following a variety of apoptotic stimuli. Confocal microscopy and biochemical analysis revealed that galectin-3 is enriched in the mitochondria and prevents mitochondrial damage and cytochrome c release. Using a yeast two-hybrid system, we screened for galectin-3-interacting proteins that regulate galectin-3 localization and anti-apoptotic activity. Synexin, a Ca(2+)- and phospholipid-binding protein, was one of the proteins identified. We confirmed direct interaction between galectin-3 and synexin by glutathione S-transferase pull-down assay in vitro. We showed that galectin-3 failed to translocate to the perinuclear membranes when expression of synexin was down-regulated using an oligodeoxyribonucleotide complementary to the synexin mRNA, suggesting a role for synexin in galectin-3 trafficking. Furthermore, synexin down-regulation abolished anti-apoptotic activity of galectin-3. Taken together, these results suggest that synexin mediates galectin-3 translocation to the perinuclear mitochondrial membranes, where it regulates mitochondrial integrity critical for apoptosis regulation.

Annexin A7↗

PubMed2002

Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents.

Experimental evidence directly implicates complex carbohydrates in recognition processes, including adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells by one another. In addition, carbohydrates are recognized as differentiation markers and as antigenic determinants. Lectins are nonenzymatic proteins present in plants and animals, which preferentially bind to specific carbohydrate structures and play an important role in cell recognition. Modified carbohydrates and oligosaccharides have the ability to interfere with carbohydrate-protein interactions and therefore, inhibit the cell-cell recognition and adhesion processes, which play an important role in cancer growth and progression. Carbohydrate ligands therefore, are candidates to play important roles in cancer therapeutics.

Carbohydrate Metabolism↗

PubMed2002

Galectin-3 and metastasis.

Galectin-3, a 31 kDa member of the beta-galactoside-binding proteins, is an intracellular and extracellular lectin which interacts with intracellular glycoproteins, cell surface molecules and extracellular matrix proteins. Galectin-3 is expressed widely in epithelial and immune cells and its expression is correlated with cancer aggressiveness and metastasis. Galectin-3 is involved in various biological phenomena including cell growth, adhesion, differentiation, angiogenesis and apoptosis. Recent research revealed that galectin-3 is associated with several steps of invasion and metastasis, like angiogenesis, cell-matrix interaction, dissemination through blood flow and extravasation. Recently, we and others have shown that galectin-3 can be a reliable diagnostic marker in certain cancers and one of the target proteins of cancer treatment. In this review, we describe the involvement of galectin-3 in each steps of metastasis and clinical significance of galectin-3.

Animals↗

PubMed2002

A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78.

Neuroleukin (NLK) is a multifunctional protein involved in neuronal growth and survival, cell motility and differentiation, and glucose metabolism. We report herein that hippocampal expression of NLK and its receptor gp78 is associated with maze learning in rats. First, mRNA levels of NLK and gp78 were significantly increased in hippocampi of male Fischer-344 rats following training in the Stone T-maze and the Morris water maze. Second, a parallel increase was found in hippocampal NLK and gp78 proteins after maze learning. Third, NLK and gp78 mRNA and protein expression in hippocampus was reduced in a group of aged rats that showed more errors during the acquisition of the Stone maze task as compared with young rats. Finally, application of recombinant NLK to hippocampal neurons significantly enhanced glutamate-induced ion currents, functional molecular changes that have been correlated with learning in vivo. Taken together, our results identify a novel association of hippocampal expression of NLK and its receptor gp78 with rat maze learning. Interaction of NLK with gp78 and subsequent signaling may strengthen synaptic mechanisms underlying learning and memory formation.

Aging↗

PubMed2002

Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors.

Overexpression of the human epidermal growth factor receptor (HER) 2 has been linked to the development and maintenance of malignant phenotypes in breast tumors. In addition, the growth and dissemination of human cancers are regulated in part by the autocrine motility factor (AMF)/phosphoglucose isomerase shown to be up-regulated by heregulin (HRG) in breast cancer cells. This study was undertaken to explore the effect of anti-HER2 monoclonal antibody 4D5 [Herceptin (HCT)] on AMF expression and the potential of its augmentation by specific simple sugar AMF inhibitors. Here we show that HCT treatment of high HER2-expressing breast cancer SK-BR3, BT-474, and ZR-75R cells resulted in down-regulation of AMF mRNA and protein. HCT inhibited the ability of HRG to induce AMF expression in cells with a normal HER2 level, and HCT-mediated down-regulation could be reversed by HRG treatment in breast cancer cells with a high HER2 level. HCT also inhibited transcription from a chimeric pGL3-Luc vector-based reporter system containing the 1.8-kb promoter region of human AMF. Treatment of breast cancer cells with the combination of HCT and specific AMF inhibitors, erythrose 4-phosphate or D-mannose 6-phosphate, resulted in an additive inhibitory effect on both the growth rate and invasiveness of cells as compared with treatment with each agent alone. Results presented here suggest that HCT can effectively block both ligand-induced and constitutive expression of AMF associated with high HER2 overexpression, implying a role of the AMF pathway in the action of HCT. Accordingly, the combination of AMF inhibitor with HCT can potentiate the growth-inhibitory and anti-invasive action of HCT in breast cancer cells.

Antibodies, Monoclonal↗

PubMed2001

Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest.

Galectin-3, a beta-galactoside-binding protein, is implicated in cell growth, adhesion, differentiation, and tumor progression by interactions with its ligands. Recent studies have revealed that galectin-3 suppresses apoptosis and anoikis that contribute to cell survival during metastatic cascades. Previously, it has been shown that human galectin-3 undergoes post-translational signaling modification of Ser(6) phosphorylation that acts as an "on/off" switch for its sugar-binding capability. We questioned whether galectin-3 phosphorylation is required for its anti-apoptotic function. Serine to alanine (S6A) and serine to glutamic acid (S6E) mutations were produced at the casein kinase I phosphorylation site in galectin-3. The cDNAs were transfected into a breast carcinoma cell line BT-549 that innately expresses no galectin-3. Metabolic labeling revealed that only wild type galectin-3 undergoes phosphorylation in vivo. Expression of Ser(6) mutants of galectin-3 failed to protect cells from cisplatin-induced cell death and poly(ADP-ribose) polymerase from degradation when compared with wild type galectin-3. The non-phosphorylated galectin-3 mutants failed to protect cells from anoikis with G(1) arrest when cells were cultured in suspension. In response to a loss of cell-substrate interactions, only cells expressing wild type galectin-3 down-regulated cyclin A expression and up-regulated cyclin D(1) and cyclin-dependent kinase inhibitors, i.e. p21(WAF1/CIP1) and p27(KIP1) expression levels. These results demonstrate that galectin-3 phosphorylation regulates its anti-apoptotic signaling activity.

Anoikis↗