Kunitz-type Protease Inhibitor Bikunin Disrupts Phorbol Ester-induced Oligomerization of CD44 Variant Isoforms Containing Epitope v9 and Subsequently Suppresses Expression of Urokinase-type Plasminogen Activator in Human Chondrosarcoma Cells

Suzuki, Mika; Kobayashi, Hiroshi; Fujie, Manabu; Nishida, Takashi; Takigawa, Masaharu; Kanayama, Naohiro; Terao, Toshihiko

doi:10.1074/jbc.m108545200

Cited by 29 publications

(35 citation statements)

References 41 publications

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“…Northern Blot Hybridization with cDNA Probes-Northern blot hybridization was carried out as described previously (19,20). Samples of total RNA (10 g) were separated by electrophoresis through denaturing 1.2% agarose gels containing 1% formaldehyde and transferred onto nylon or nitrocellulose membranes using standard molecular biological techniques.…”

Section: Methodsmentioning

confidence: 99%

“…Samples of total RNA (10 g) were separated by electrophoresis through denaturing 1.2% agarose gels containing 1% formaldehyde and transferred onto nylon or nitrocellulose membranes using standard molecular biological techniques. Hybridization was carried out with [␣-32 P]dCTP by random oligonucleotide priming to specific activities of 0.4 -0.9 ϫ 10 9 cpm/ g. uPA cDNA was prepared as described (19,20). Filters were reprobed with the cDNA for glyceraldehyde-3-phosphate dehydrogenase to correct for the amount of RNA loaded onto the filters (19,20).…”

Section: Methodsmentioning

confidence: 99%

“…Hybridization was carried out with [␣-32 P]dCTP by random oligonucleotide priming to specific activities of 0.4 -0.9 ϫ 10 9 cpm/ g. uPA cDNA was prepared as described (19,20). Filters were reprobed with the cDNA for glyceraldehyde-3-phosphate dehydrogenase to correct for the amount of RNA loaded onto the filters (19,20). After hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at Ϫ70°C.…”

Section: Methodsmentioning

confidence: 99%

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Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Tanaka

Kobayashi

Suzuki

et al. 2004

Journal of Biological Chemistry

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Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-␤1 induces a dose-and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-␤1, we investigated which signaling pathway transduced by TGF-␤1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-␤1 activated Src kinase; 2) TGF-␤1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-␤1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-␤1-mediated Akt stimulation, whereas TGF-␤1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-␤1 was almost totally blocked by PP2 and PD98059 and partially (ϳ55%) by LY294002; 6) TGF-␤1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-B by pyrrolidinedithiocarbamate reduced the TGF-␤1-induced activation of the uPA gene by ϳ65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-␤1 activation of two distinct pathways (Src-MAPK-PI3K-NF-B-dependent and Src-MAPK-AP-1-dependent) for TGF-␤1-dependent uPA up-regulation and promotion of invasion.The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) 1 could be involved in the pathogenesis of peritoneal dissemination (1). uPA is a serine protease associated with various pathological conditions including tumor invasion and metastasis (2). One of the factors regulating the metastatic process is considered to be transforming growth factor-␤ (TGF-␤), which is a multifunctional cytokine that elicits numerous cellular effects pertinent to the metastatic process (1). TGF-␤ regulates a wide range of physiological and pathological cellular processes, including cell growth, differentiation, invasion, migration, mesenchymal transition, extracellular matrix synthesis, and cell death in many cell types including ovarian cancer cells (3). Recent data demonstrated that TGF-␤ specifically stimulates up-regulation of uPA mRNA and protein in certain types of neoplastic cells (1). The cellular mechanism(s) of the TGF-␤-induced uPA-dependent tumor invasion and metastasis has been extensively studied. The identity of the signaling pathway(s) involved in the TGF-␤-induced uPA up-regulation (4) remains less known. In a well...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Tanaka

Kobayashi

Suzuki

et al. 2004

Journal of Biological Chemistry

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“…The gene is also found to be selectively overexpressed in acute myelocytic leukemia [32]. The serine protease inhibitor, Kuniz type 2 [33], and the protein tyrosine phosphase, receptor type, F [34], take part in distinct cellular processes. The relevance of the up-regulation of these proteins is yet to be determined.…”

Section: Differential Expressions Of Genes In Bt-549 and Mda-mb-468 Cmentioning

confidence: 99%

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells

Tripathi

Misra

Chaudhuri

2005

Biochemical and Biophysical Research Communications

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Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process. KeywordsInvasiveness; Breast tumor; SLUG; Cytokeratin 8; Cytokeratin 19; Metallothionein 1G; Metallothionein 1L; siRNA; Microarray A serious gap in our understanding of cancer concerns malignancy and metastasis. We have yet to clearly identify gene defects that specifically permit cells to invade surrounding tissues, spread through the body, and form metastases. Local invasiveness requires a break down of the mechanisms that normally hold epithelial cells together. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. One of the known genotypic differences between these two cells is that BT-549 expresses the zinc finger repressor protein SLUG but the MDA-MB-468 cells do not [1]. Hence, we tested whether any gene overexpressed in the MDA-MB-468 cells is regulated by SLUG.We paid particular attention to SLUG because it is implicated as one of the determinants of tumor cell invasiveness [2][3][4][5][6]. This protein is a member of a superfamily of transcriptional regulators that are implicated in the formation of mesoderm and neural crest, as well as in We then overexpressed SLUG in MDA-MB-468 cells from a doxycycline-inducible promoter and compared the gene expression profile in the presence or absence of the inducer. We identified that cytokeratins 8 and 19 genes are repressed when SLUG expression is induced. We have verified the role of SLUG in regulating these cytokeratins by siRNA-mediated knock down of SLUG in BT-549 cells. We also report here our preliminary characterization of the promoters of these cytokeratin genes as it is regulated by SLUG exp...

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“…One study has shown that certain recombinant forms of bikunin can have an inhibitory effect on HIV-1 infectivity in vitro (42). In cancers, its expression by human glioblastoma cells can cause suppression of tumor invasion (43), whereas addition of bikunin to human chondrosarcoma cell cultures blocks cell spreading (44).…”

Section: Table II N-terminal Sequences Of M-bik and M-ptgf-␤mentioning

confidence: 99%

Identification of Placental Transforming Growth Factor-β and Bikunin Metabolites as Contaminants of Pharmaceutical Human Chorionic Gonadotrophin Preparations by Proteomic Techniques

Malatos

Neubert

Kicman

et al. 2005

Molecular & Cellular Proteomics

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A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization timeof-flight mass spectrometry of this complex showed three polypeptides at ϳ6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-␤ (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-␤ in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-␤, which may have transforming growth factor-␤ agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonad-

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Kunitz-type Protease Inhibitor Bikunin Disrupts Phorbol Ester-induced Oligomerization of CD44 Variant Isoforms Containing Epitope v9 and Subsequently Suppresses Expression of Urokinase-type Plasminogen Activator in Human Chondrosarcoma Cells

Cited by 29 publications

References 41 publications

Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells

Identification of Placental Transforming Growth Factor-β and Bikunin Metabolites as Contaminants of Pharmaceutical Human Chorionic Gonadotrophin Preparations by Proteomic Techniques

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