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• W2105285232 abstract "A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-β1 (TGF-β1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-β1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-β1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-β1-induced phosphorylation of Src, ERK1/2, and Akt by 40–60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-β1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-β1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src. A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-β1 (TGF-β1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-β1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-β1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-β1-induced phosphorylation of Src, ERK1/2, and Akt by 40–60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-β1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-β1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src. We have previously reported in a series of reports that a Kunitz-type protease inhibitor, bikunin derived from human urine (also known as urinary trypsin inhibitor), suppresses expression of uPA, phosphorylation of ERK1/2, and cancer cell invasion in vitro and peritoneal disseminated metastasis and lung metastasis in vivo (1Kobayashi H. Shinohara H. Gotoh J. Fujie M. Fujishiro S. Terao T. Br. J. Cancer. 1995; 72: 1131-1137Crossref PubMed Scopus (52) Google Scholar, 2Kobayashi H. Suzuki M. Tanaka Y. Kanayama N. Terao T. J. Biol. Chem. 2003; 278: 7790-7799Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Suzuki M. Kobayashi H. Tanaka Y. Hirashima Y. Kanayama N. Takei Y. Saga Y. Suzuki M. Itoh H. Terao T. Int. J. Cancer. 2003; 104: 289-302Crossref PubMed Scopus (57) Google Scholar, 4Suzuki M. Kobayashi H. Tanaka Y. Hirashima Y. Kanayama N. Takei Y. Saga Y. Suzuki M. Itoh H. Terao T. J. Biol. Chem. 2003; 278: 14640-14646Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Kobayashi H. Suzuki M. Hirashima Y. Terao T. Biol. Chem. 2003; 384: 749-754Crossref PubMed Scopus (71) Google Scholar). More recently, we investigated the effects of soybean trypsin inhibitor on the uPA expression, signal transduction involved in the expression of uPA, and invasion in human ovarian cancer HRA cells (6Kobayashi H. Suzuki M. Kanayama N. Terao T. Clin. Exp. Metastasis. 2004; 21: 159-166Crossref PubMed Scopus (83) Google Scholar, 7Kobayashi H. Fukuda Y. Yoshida R. Kanada Y. Nishiyama S. Suzuki M. Kanayama N. Terao T. Int. J. Cancer. 2004; 112: 519-524Crossref PubMed Scopus (27) Google Scholar). Soybean trypsin inhibitor contains a Kunitz trypsin inhibitor (KTI) 1The abbreviations used are: KTI, Kunitz trypsin inhibitor; TGF, transforming growth factor; uPA, urokinase-type plasminogen activator; MAPK, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol 3-kinase; ODN, oligodeoxynucleotide; BBI, Bowman-Birk inhibitor; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; iAS, inverted antisense; CA, constitutively active; KD, kinase-dead; WT, wild-type. and a Bowman-Birk inhibitor (BBI) (8Friedman M. Brandon D.L. J. Agric. Food Chem. 2001; 49: 1069-1086Crossref PubMed Scopus (652) Google Scholar). We previously showed (6Kobayashi H. Suzuki M. Kanayama N. Terao T. Clin. Exp. Metastasis. 2004; 21: 159-166Crossref PubMed Scopus (83) Google Scholar, 7Kobayashi H. Fukuda Y. Yoshida R. Kanada Y. Nishiyama S. Suzuki M. Kanayama N. Terao T. Int. J. Cancer. 2004; 112: 519-524Crossref PubMed Scopus (27) Google Scholar) that 1) uPA expression observed in HRA cells is inhibited by preincubation of the cells with KTI with an IC50 of ∼2 μm, whereas BBI failed to repress uPA expression; 2) cell invasiveness is inhibited by treatment of the cells with KTI with an IC50 of ∼3 μm, whereas BBI failed to suppress cell invasion; 3) KTI suppresses HRA cell invasion by blocking uPA up-regulation, which may be mediated by one or more binding proteins other than a bikunin-binding protein and/or its receptor; and 4) TGF-β1-mediated activation of ERK1/2 is significantly reduced by preincubation of the cells with KTI. We conclude that KTI, but not BBI, could inhibit cell invasiveness at least through suppression of the MAPK-dependent uPA signaling cascade (6Kobayashi H. Suzuki M. Kanayama N. Terao T. Clin. Exp. Metastasis. 2004; 21: 159-166Crossref PubMed Scopus (83) Google Scholar, 7Kobayashi H. Fukuda Y. Yoshida R. Kanada Y. Nishiyama S. Suzuki M. Kanayama N. Terao T. Int. J. Cancer. 2004; 112: 519-524Crossref PubMed Scopus (27) Google Scholar). It has been reported that there appeared to be a specific interaction between bikunin and the tumor cell surface (9Kobayashi H. Gotoh J. Fujie M. Terao T. J. Biol. Chem. 1994; 269: 20642-20647Abstract Full Text PDF PubMed Google Scholar, 10Kobayashi H. Hirashima Y. Terao T. Mol. Hum. Reprod. 2000; 6: 735-742Crossref PubMed Scopus (12) Google Scholar, 11Kobayashi H. Hirashima Y. Sun G.W. Fujie M. Nishida T. Takigawa M. Terao T. J. Biol. Chem. 2000; 275: 21185-21191Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Hirashima Y. Kobayashi H. Suzuki M. Tanaka Y. Kanayama N. Fujie M. Nishida T. Takigawa M. Terao T. J. Biol. Chem. 2001; 276: 13650-136506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Tumor cells express two types of bikunin-binding proteins; a 40-kDa bikunin-binding protein, which is identical to cartilage link protein, and a 45-kDa bikunin-binding protein, a putative bikunin receptor (12Hirashima Y. Kobayashi H. Suzuki M. Tanaka Y. Kanayama N. Fujie M. Nishida T. Takigawa M. Terao T. J. Biol. Chem. 2001; 276: 13650-136506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13Suzuki M. Kobayashi H. Tanaka Y. Hirashima Y. Terao T. Biochim. Biophys. Acta. 2001; 1547: 26-36Crossref PubMed Scopus (32) Google Scholar). Bikunin binds link protein and bikunin receptor on tumor cell surface possibly via the N-terminal Kunitz domain I and the chondroitin sulfate side chain, respectively (13Suzuki M. Kobayashi H. Tanaka Y. Hirashima Y. Terao T. Biochim. Biophys. Acta. 2001; 1547: 26-36Crossref PubMed Scopus (32) Google Scholar). Bikunin must bind directly to both of the cell-associated bikunin-binding proteins to suppress expression of uPA and uPA receptor genes (14Kobayashi H. Suzuki M. Kanayama N. Nishida T. Takigawa M. Terao T. Eur. J. Biochem. 2002; 269: 3945-3957Crossref PubMed Scopus (38) Google Scholar). However, KTI has no chondroitin 4-sulfate glycosaminoglycan side chain. Our recent data (6Kobayashi H. Suzuki M. Kanayama N. Terao T. Clin. Exp. Metastasis. 2004; 21: 159-166Crossref PubMed Scopus (83) Google Scholar) showed that neither KTI nor BBI inhibits binding of radiolabeled-bikunin to HRA cells or bikunin does not inhibit KTI binding to the cells, suggesting that their binding molecule(s) in the plasma membranes is(are) different from those of bikunin. These data allow us to hypothesize that the mechanisms of KTI will be different from those of bikunin. These findings lead to many questions about the difference in the mechanisms between KTI and bikunin. We therefore investigated the effects of KTI on the TGF-β-induced uPA up-regulation and invasiveness of wild-type and transfected cells in relation to the status of Src, ERK, and PI3K/Akt in HRA cells, because TGF-β1 can stimulate uPA expression through the Src-dependent, ERK-specific signaling cascade (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We show for the first time that KTI suppresses uPA up-regulation and promotion of invasion possibly through one or more upstream targets of Src. Materials—A soybean KTI was obtained from Fuji Oil Co. Ltd., Osaka, Japan (6Kobayashi H. Suzuki M. Kanayama N. Terao T. Clin. Exp. Metastasis. 2004; 21: 159-166Crossref PubMed Scopus (83) Google Scholar, 7Kobayashi H. Fukuda Y. Yoshida R. Kanada Y. Nishiyama S. Suzuki M. Kanayama N. Terao T. Int. J. Cancer. 2004; 112: 519-524Crossref PubMed Scopus (27) Google Scholar, 15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Lipofectamine Plus reagent was purchased from Invitrogen. High molecular weight recombinant uPA and Glu-type plasminogen were obtained from American Diagnostics (Greenwich, CT). Boyden-type cell invasion chambers (BioCoat Matrigel™ invasion chambers) were obtained from Collaborative Biomedical (Franklin Lakes, NJ). Ultrapure natural human TGF-β1 was from Genzyme (Cambridge, MA) and R&D Systems (Minneapolis, MN). Genistein and PP2 were obtained from Calbiochem. Culture media, penicillin, streptomycin, and fetal bovine serum were purchased from Invitrogen. Tissue culture plastics were purchased from Costar/Corning (Cambridge, MA) and Falcon (BD Biosciences). Bovine serum albumin, Tris-base, dithiothreitol, phenylmethylsulfonyl fluoride, and ammonium persulfate were commercially obtained from Sigma. Acrylamide, bisacrylamide, and polyvinylidene difluoride membrane were from Bio-Rad. X-ray film was purchased from Eastman Kodak Co. ECL was purchased from Amersham Biosciences. Protein estimation reagents (BCA kit) were from Pierce. All other chemicals were of analytical grade. Pharmacological Inhibitors—The inhibitors were dissolved in Me2SO (cell culture grade, Sigma) and used in the following concentrations: wortmannin (100 nm, specific inhibitor of PI3K), LY294002 (10 μm, specific inhibitor of the p110 catalytic subunit of PI3K), herbimycin A (35 nm, tyrosine kinase inhibitor), SB202190 (30 μm, p38 kinase inhibitor), and PD98059 (50 μm, specific inhibitor of MEK). All of the inhibitors except wortmannin (Sigma) were obtained form Calbiochem. The inhibitors diluted in normal growth medium were added to wells containing confluent cells and incubated for 30 min to 1 h. TGF-β1 (10 ng/ml) was added to serum-free medium containing the respective inhibitors and incubated for the indicated periods of time, after which time the conditioned medium and cells were separately collected, and the cells were counted. The samples were stored at –80 °C until measured. Me2SO (0.05%, v/v) diluted in medium was used as a negative control. Antibodies—The antibodies against uPA (number 3689 (recognizes uPA B-chain) and number 3471 (reacts with uPA A-chain; interferes with binding of uPA to its receptor)) were gifts from Dr. R. Hart (American Diagnostics). Antibodies to human phospho-ERK1/2, phos-pho-p38 MAPK (Thr180/Tyr82), phospho-Akt (Ser473), and total Akt were from New England Biolabs (Beverly, MA). Anti-pan-ERK antibody and anti-phosphotyrosine antibody (clone PY20) were from BD Transduction Laboratories (Lexington, KY). Anti-p38 MAPK antibodies were from Santa Cruz Biotechnology, Inc. Anti-human phospho-Src antibody (Tyr418) was from BIOSOURCE International (Camarillo, CA). Anti-Src (GD11), anti-PI3K p85 subunit, and anti-phosphotyrosine antibody (clone 4G10) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat anti-rabbit/mouse IgG horseradish peroxidase-conjugated antibody was from Dako (Copenhagen, Denmark). Preparation of Oligodeoxynucleotides and Lipofection of HRA Cells— Antisense ODNs were selected for sequence target to PI3K p85 (antisense PI3K, 5′-GTA CTG GTA CCC CTC AGC ACT CAT-3′; sense PI3K, 5′-ATG AGT GCT GAG GGG TAC CAG TAC-3′) (16Morel J.C. Park C.C. Zhu K. Kumar P. Ruth J.H. Koch A.E. J. Biol. Chem. 2002; 277: 34679-34691Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The corresponding sense ODN was used as control for each antisense ODN. Furthermore, each inverted antisense (iAS) oligonucleotide (iAS c-Src ODN or iAS PI3K ODN) provided additional controls for the vehicle and transfection. The ODNs were synthesized, purified, and modified with phosphorothioate (17Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7413-7417Crossref PubMed Scopus (4370) Google Scholar). The constitutively active (CA)-Src plasmid (pUSEamp-Src (Y529F)), wild-type Src plasmid, Myc-tagged CA-Akt plasmid (pUSE-myc-active Akt) (18Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (840) Google Scholar), kinase-dead (KD)-Akt plasmid (pUSE-myc-dominant negative Akt), CA-MEK1 plasmid (pUSE-MEK1) (19Saxena M. Williams S. Brockdorff J. Gilman J. Mustelin T. J. Biol. Chem. 1999; 274: 11693-11700Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), wild-type (WT)-MEK1, or the control vector (pUSE) were obtained from Upstate Biotechnology (www.upstate.com/browse/Search.asp?query=Akt and www.upstate.com/browse/Search.asp?query=Mek) Confluent HRA monolayers were transfected with each plasmid in a pUSE vector according to the manufacturer's instructions. Each well of a 24-well plate was transfected with 250 ng to 2 μg of total DNA for 24 h as described previously (20Derrien A. Zheng B. Osterhout J.L. Ma Y.C. Milligan G. Farquhar M.G. Druey K.M. J. Biol. Chem. 2003; 278: 16107-16116Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Cell Culture—The ovarian cancer cell line, HRA, was grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a 5% CO2 atmosphere with constant humidity (3Suzuki M. Kobayashi H. Tanaka Y. Hirashima Y. Kanayama N. Takei Y. Saga Y. Suzuki M. Itoh H. Terao T. Int. J. Cancer. 2003; 104: 289-302Crossref PubMed Scopus (57) Google Scholar). SKOv-3 ovarian cancer cells were obtained from the American Type Culture Collection (Manassas, VA). These cells were maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum and antibiotics. For all experiments in which TGF-β1 was added, cells were incubated in the serum-free medium. TGF-β1 (10 ng/ml) was added either alone or in combination in cancer cells preincubated for 30 min to 1 h with inhibitors. The protein concentrations in the supernatants of cell extracts were measured by the Bio-Rad protein assay. Total RNA isolations were done using the TRIzol reagent (Invitrogen). Preparation of Cell Lysate—The cell monolayers treated with or without various agents for the indicated times were washed with phosphate-buffered saline. 1 × 106 cells were lysed in 750 μl of lysis buffer containing 20 mm Tris-HCl (pH 7.5), 12.5 mm 2-glycerophosphate, 150 mm NaCl, 1.5 mm MgCl2, 2 mm EGTA, 10 mm NaF, 0.5% Triton X-100, 2 mm dithiothreitol, 1 mm sodium vanadate, and 1 mm phenylmethylsulfonyl fluoride at 4 °C for 15 min and scraped with a rubber policeman (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Cell extracts were then centrifuged at 3000 × g to remove cell debris. All samples were stored at –70 °C until use. In parallel, cells treated in the same condition in different dishes were harvested and counted using a hemocytometer. Northern Blot Hybridization with cDNA Probes—Northern blot hybridization was carried out as described previously (14Kobayashi H. Suzuki M. Kanayama N. Nishida T. Takigawa M. Terao T. Eur. J. Biochem. 2002; 269: 3945-3957Crossref PubMed Scopus (38) Google Scholar, 21Suzuki M. Kobayashi H. Fujie M. Nishida T. Takigawa M. Kanayama N. Terao T. J. Biol. Chem. 2002; 277: 8022-8032Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Samples of total RNA (10 μg) 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 [α-32P]dCTP by random oligonucleotide priming to specific activities of 0.4–0.9 × 109 cpm/μg. uPA cDNA was prepared as described (14Kobayashi H. Suzuki M. Kanayama N. Nishida T. Takigawa M. Terao T. Eur. J. Biochem. 2002; 269: 3945-3957Crossref PubMed Scopus (38) Google Scholar, 21Suzuki M. Kobayashi H. Fujie M. Nishida T. Takigawa M. Kanayama N. Terao T. J. Biol. Chem. 2002; 277: 8022-8032Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Filters were reprobed with the cDNA for glyceraldehyde-3-phosphate dehydrogenase to correct for the amount of RNA loaded onto the filters (14Kobayashi H. Suzuki M. Kanayama N. Nishida T. Takigawa M. Terao T. Eur. J. Biochem. 2002; 269: 3945-3957Crossref PubMed Scopus (38) Google Scholar, 21Suzuki M. Kobayashi H. Fujie M. Nishida T. Takigawa M. Kanayama N. Terao T. J. Biol. Chem. 2002; 277: 8022-8032Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). After hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at –70 °C. Filters were quantitated by scanning densitometry using a Bio-Rad model 620 video densitometer with a one-dimensional Analyst software package for Macintosh. Western Blot Analysis—Each cell was harvested, and cell pellets were lysed as described above. Centrifuged lysates (50 μg) from each cell were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane by semidry transfer. Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 2% bovine serum albumin. Blots were probed with the following primary antibodies overnight at 4 °C: monoclonal anti-uPA (antibody 3471 plus 3689), anti-β-actin, anti-phospho-Src, anti-Src, anti-phospho-Akt, ant-Akt, anti-phospho-ERK1/2, or anti-ERK1/2 antibodies. This was followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5000 for 1 h. Detection was achieved by enhanced chemiluminescence (Amersham Biosciences) and exposed to film. Densitometric analysis of Western blots was carried out using the Macintosh Image System (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Extracellular Matrix Invasion Assay—Chemoinvasion assays were carried out in a Boyden chamber as described (22Kobayashi H. Suzuki M. Tanaka Y. Hirashima Y. Terao T. J. Biol. Chem. 2001; 276: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The upper surface of chamber was precoated with a layer of artificial basement membrane, Matrigel. The cell suspension (1 × 105 cells/well) was added to the upper chamber. The lower chamber was filled with fibroblast-conditioned medium, which acted as a chemoattractant. To measure invasion, incubation was at 37 °C for 24 h. The invaded cells in the lower side of the filter were stained with hematoxylin. Triplicate filters were used for each cell type and assay condition, and 10 random fields were counted per filter under a microscope (×400). The experiments of inhibition of cell invasiveness were performed as follows. Because wortmannin is unstable at 37 °C in culture medium, it was added every 6 h to the upper chamber of the Matrigel invasion assay. Statistics—Data are expressed as mean ± S.D. of at least three independent triplicate experiments. All statistical analysis was performed using StatView for Macintosh. Statistical analysis was performed by one-way analysis of variance followed by Student's t test. p < 0.05 was considered statistically significant. KTI Suppressed TGF-β1-induced Src Phosphorylation—Previously, we have investigated signaling pathways involved in TGF-β1 activation in human ovarian cancer cell lines, HRA and SKOv-3 (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We showed that TGF-β1 induced a marked rise in the level of phosphorylated Src protein in a time-dependent manner (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In the present study, we examined whether a soybean KTI suppressed TGF-β1-induced phosphorylation of Src in HRA and SKOv-3 cells by Western blot. As compared with nonstimulated HRA cells, a 7-fold increase in phosphorylated Src was observed at 20 min in response to 10 ng/ml TGF-β1 (Fig. 1, lane 1 versus lane 3). The anti-Src antibodies immunoblotted similar amounts of Src protein in HRA cells, irrespective of whether cells were stimulated with TGF-β1. Here, we showed that KTI suppressed TGF-β1-stimulated Src phosphorylation in a dose-dependent manner (lanes 4–6). Furthermore, KTI also suppressed TGF-β1 (10 ng/ml, 20 min)-induced phosphorylation of Src in SKOv-3 cells in a dose-dependent manner (lanes 10–12). KTI Suppressed TGF-β1-induced Phosphorylation of Akt through PI3K—In the previous study (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), we observed concentration-dependent phosphorylation of ERK1/2 and Akt in response to TGF-β1 beginning with only 0.4–1 ng/ml TGF-β1. Src activation has been linked to the activation of MAPK and PI3K in HRA cells (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The facts that a MEK inhibitor, PD98059, inhibited the TGF-β1-induced Akt activation and that PI3K inhibitors, LY294002 and wortmannin, had no effect on the TGF-β1-induced ERK1/2 activity suggest a presence of cross-talk between the ERK and Akt cascades and that PI3K is a downstream target of MAPK (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Here, we show the ability of KTI to TGF-β1-induced phosphorylation of Akt and ERK1/2 by Western blot using antibodies to phosphorylated Akt (or phosphorylated ERK1/2) or total Akt (or total ERK1/2), respectively. As shown in Fig. 2A, the increase in Akt phosphorylation in response to TGF-β1 could be reduced when HRA cells were preincubated with KTI (10 μm, 1 h; lane 2 versus lane 3), wortmannin (100 nm, 1 h; lane 4), or LY294002 (10 μm, 30 min; lane 6) by 50%, 80%, or 80%, respectively. Pretreatment of HRA cells with 50 μm PD98059 (lane 8) also markedly abrogated the TGF-β1-stimulated Akt phosphorylation by 80%. In contrast, TGF-β1-dependent Akt phosphorylation was not suppressed by p38 MAPK inhibitor, SB202190 (lane 10). The combined treatment of KTI together with pharmacological inhibitors (lanes 4–9) did not provide a further suppression of TGF-β1-induced Akt phosphorylation. The role of PI3K in Akt activation was more specifically demonstrated through inhibition with PI3K antisense ODN. We used antisense ODN targeting of the gene for PI3K (lanes 12 and 13) and corresponding control ODNs (S PI3K ODN (lanes 14 and 15) and inverted antisense (iAS) PI3K ODN (data not shown)). PI3K p85 protein expression was reduced by the antisense strategy (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Previously, we showed that antisense PI3K ODN transfection abrogated Akt phosphorylation in HRA cells, irrespective of whether cells were stimulated with TGF-β1 (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Note that TGF-β1-stimulated Akt phosphorylation was not impaired by S PI3K ODN or iAS ODN (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In this study, KTI inhibited the TGF-β1-induced phosphorylation of Akt in the S PI3K ODN cells (lanes 14 and 15). However, KTI did not affect phospho-Akt level in AS PI3K ODN transfected cells (lanes 12 and 13), because TGF-β1 could not induce Akt phosphorylation. To confirm that KTI-dependent suppression of TGF-β1-stimulated Akt phosphorylation is mediated by ERK, HRA cells expressing CA-MEK1 or control MEK1 were pretreated with KTI, and then stimulated with TGF-β1. We found that overexpression of CA-MEK1, but not control MEK1, increased basal ERK phosphorylation (Fig. 2C, lane 1 versus lane 4). KTI could not significantly suppress the TGF-β1-induced phosphorylation of ERK in HRA cells transfected with CA-MEK1 (Fig. 2C, lane 5 versus lane 6). On the other hand, KTI inhibited the ERK phosphorylation in the control MEK1 cells (Fig. 2C, lane 8 versus lane 9). In a parallel experiment, phospho-Akt band was strongly detected in cells expressing CA-MEK1 (Fig. 2E, lanes 4–6), but not in cells expressing control MEK1 (Fig. 2E, lanes 7–9), irrespective of whether cells were stimulated with TGF-β1. Furthermore, pretreatment for 1 h with 10 μm KTI could not significantly diminish the TGF-β1-induced phosphorylation of Akt band in the CA-MEK1 cells (Fig. 2E, lane 5 versus lane 6), supporting that KTI may be involved in one or more upstream targets of ERK. As shown in Fig. 2G, the increase in ERK1/2 phosphorylation in response to TGF-β1 could be blocked when HRA cells were preincubated with KTI (10 μm, 1 h, lane 3) or PD98059 (50 μm, lane 8) by 60 and 70%, respectively. In contrast, pretreatment of HRA cells with wortmannin (100 nm, 1 h, lane 4) or LY294002 (10 μm, 30 min, lane 6) did not abrogated the TGF-β1-stimulated ERK1/2 phosphorylation, because PI3K/Akt is a downstream target of ERK (15Tanaka Y. Kobayashi H. Suzuki M. Kanayama N. Terao T. J. Biol. Chem. 2004; 279: 8567-8576Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In addition, TGF-β1-dependent ERK1/2 phosphorylation was not suppressed by SB202190 (lane 10). KTI failed to strengthen the PD98059-induced suppression of TGF-β1-induced ERK1/2 phosphorylation (lane 9 versus lane 8). We also showed that antisense PI3K ODN transfection did not abrogate ERK1/2 phosphorylation in HRA cells in response to TGF-β1 (lane 12). As expected, KTI inhibited the TGF-β1-induced phosphorylation of ERK1/2 in the AS PI3K ODN cells (lanes 12 and 13) or S PI3K ODN cells (lanes 14 and 15). KTI Suppressed TGF-β1-stimulated Phosphorylation of ERK1/2 or Akt via an Src-dependent Mechanism—The pharmacological Src inhibitor PP2 inhibited the ERK1/2 (Fig. 3A, lane 4) and Akt (Fig. 3C, lane 4) activation by 65–75%. KTI did not enhance the suppression by PP2 of TGF-β1-induced phosphorylation of ERK1/2 (Fig. 3A, lane 5) and Akt (Fig. 3C, lane 5). Furthermore, general tyrosine kinase inhibition with genistein (lane 6) or herbimycin A (lane 7) also significantly attenuated TGF-β1-induced ERK1/2 or Akt phosphorylation. Suppression of TGF-β1-induced activation of Src by KTI supports that the critical position of KTI may not be a downstream target(s) of Src pathway. Effects of KTI on the Phosphorylation of ERK1/2 and Akt in Cells Expressing CA-c-Src—Our biochemical approach clearly showed that KTI may be correlated" @default.
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• W2105285232 title "Suppression of Urokinase Expression and Invasion by a Soybean Kunitz Trypsin Inhibitor Are Mediated through Inhibition of Src-dependent Signaling Pathways" @default.
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