Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin

Abstract

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik ؉ clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik ؉ clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik ؉ clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik ؉ clones. © 2003 Wiley-Liss, Inc. Key words: bikunin; invasion; transfection; urinary trypsin inhibitor; urokinase-type plasminogen activatorInvasion and metastasis by tumor cells have multifactorial steps, may require the action of proteolytic enzymes at each step and are complex processes in which many gene activities are involved. 1 Various proteins on the surfaces of tumor cells mediate attachment to and degradation of the basement membrane and components of the extracellular matrix. 2 Increased activity, membrane association and secretion of urokinase-type plasminogen activator (uPA), plasmin, certain cathepsins and matrix metalloproteinases (MMPs) have been shown to correlate positively with invasiveness and the metastatic properties of many tumor entities. [1][2][3][4] The penetrating tumor cell focuses proteolytic activity at its cell surface through receptors for uPA, plasmin and MMPs. 3,4 uPA and its receptor are instrumental in cell invasion and metastasis. 5 uPA has a pleiotropic effect on cell migration and spreading in vivo and in vitro through the activation of plasminogen or other protein factors at the cell surface or in the extracellular matrix. Thus, uPA is at the center of a complex system affecting cell invasion and metastasis. 5 Bikunin (bik), also known as urinary trypsin inhibitor, is a Kunitz-type protease inhibitor which presents in human amniotic fluid, urine and in...

Gene expression profiling of human ovarian tumours

“…Devoogdt et al (2003) demonstrated that transfection of the human SLP1 cDNA into Lewis lung carcinoma cells resulted in increased tumourigenicity and lung-colonising potential. In contrast, however, the protease inhibitor SPINT2 was shown to reduce the metastatic potential of ovarian cancer cells (Suzuki et al, 2003). This result highlights the complexity of defining the The score derived from this analysis using a significance threshold of 3.1 is shown.…”

Adenovirus-Mediated Expression of Tissue Factor Pathway Inhibitor-2 Inhibits Endothelial Cell Migration and Angiogenesis

“…Interestingly, bikunin, another Kunitz-type protease inhibitor, also inhibits cell invasion, possibly through direct inhibition of cell-associated plasmin activity. 32 Finding that overexpression of TFPI-2 increased apoptosis, both for ECs in vitro and for cells in Matrigel implants in vivo, indicates that TFPI-2 may also protect against aberrant neovessel formation through proteolysis-independent mechanisms. It remains to be established whether the process may also involve regulation of gene expression of proteins like VEGF-C, VEGFR1, or ECM components.…”

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

“…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 g/ml) in a 5% CO 2 atmosphere with constant humidity (18). SKOv-3 and OVCAR-3 ovarian cancer cells were obtained from the American Type Culture Collection (Manassas, VA).…”