A Kunitz-type protease inhibitor, bikunin, inhibits ovarian cancer cell invasion by blocking the calcium-dependent transforming growth factor-beta 1 signaling cascade.
Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.
Kobayashi
Hiroshi
H
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Shizuoka, 431-3192, Japan. hirokoba@hama-med.ac.jp
Suzuki
Mika
M
Tanaka
Yoshiko
Y
Kanayama
Naohiro
N
Terao
Toshihiko
T
D002118:Calcium / Q000378:metabolism*; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D009361:Neoplasm Invasiveness; D010051:Ovarian Neoplasms / Q000473:pathology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism
D008562:Membrane Glycoproteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium