Suzuki Mika, Kobayashi Hiroshi, Tanaka Yoshiko, Hirashima Yasuyuki, Kanayama Naohiro, Takei Yuji, Saga Yasushi, Suzuki Mitsuaki, Itoh Hiroshi, Terao Toshihiko
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
Int J Cancer. 2003 Apr 10;104(3):289-302. doi: 10.1002/ijc.10950.
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.
比库宁（bik）是一种库尼茨型蛋白酶抑制剂，也被称为尿胰蛋白酶抑制剂，被认为是抑制肿瘤细胞侵袭和转移的主要参与者，可能是通过直接抑制细胞相关纤溶酶活性以及抑制尿激酶型纤溶酶原激活剂（uPA）的mRNA表达来实现的。在本研究中，我们用携带编码人比库宁cDNA的表达载体转染了高侵袭性的人卵巢癌细胞系HRA。我们的研究旨在调查比库宁过表达的影响以及稳定转染克隆中肿瘤细胞表型和侵袭性的变化。HRA细胞的比库宁基因转染产生了以下结果：1）用比库宁cDNA转染HRA产生了5个稳定表达功能性比库宁的变体；2）与亲本细胞相比，比库宁阳性（bik(+)）克隆的uPA mRNA表达显著降低；3）比库宁负向调节ERK1/2活性；4）对比库宁阳性克隆进行分泌阻断处理消除了比库宁介导的ERK1/2激活和uPA表达的抑制；5）HRA细胞中观察到的侵袭调节主要由uPA-纤溶酶-MMP-2系统介导；6）用比库宁基因转染HRA相对于亲本细胞显著降低了侵袭，但不影响增殖、黏附或迁移；7）植入比库宁阳性克隆的动物腹膜播散减少且生存期延长。我们得出结论，用比库宁cDNA转染HRA细胞可组成性抑制ERK1/2激活，这导致uPA表达受到抑制，随后减少了比库宁阳性克隆的播散。
