竹井 裕二

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikunin-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunin-treated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by approximately 9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

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.

The aim of this study was clarification of the feasibility, effects, and adverse events of combination chemotherapy with paclitaxel and carboplatin in Japanese women with epithelial ovarian cancer. In patients with stage Ic to IV epithelial ovarian cancer, primary cytoreductive surgery was performed. Paclitaxel (175 mg/m(2)) was intravenously infused for 3 h. Subsequently, carboplatin was infused, with an area under the plasma concentration-time curve of 5. Ninety-one patients were enrolled in this study. The mean dose of paclitaxel at the sixth course was 161 mg/m(2). Of 51 patients, a complete response was observed in 19 patients (37%), and a partial response in 23 patients (45%). The response rate for clear cell adenocarcinoma was 25%. With regard to adverse events, grade 4 granulopenia was observed in 80% of patients, suggesting dose-limiting toxicity. However, none of the patients developed serious infection. With regard to non-hematological toxicity, grade 2 or 3 alopecia, arythralgia/myalgia, and peripheral neurotoxicity were noted in 97, 29, and 20% of the patients, respectively. Although hematological toxicity was relatively marked, this regimen can be applied in Japanese women.

We evaluated microsatellite instability (MSI) in primary lesions and lymph node metastatic lesions in 66 patients with endometrial carcinoma (FIGO stage IIIC) accompanied by lymph node metastasis. DNA was extracted from paraffin-embedded tissue of both the primary and lymph node metastatic lesions of endometrial carcinoma, and MSI was evaluated using microsatellite markers at five loci. Microsatellite instability was positive in the primary lesion in 27 patients (41%). All patients with MSI-positive primary lesions also showed MSI-positive in lymph node metastatic lesions. Of the other 39 patients with MSI-negative primary lesions, 4 showed MSI-positive in lymph node metastatic lesions. As the result of individual identification by polymerase chain reaction (PCR) using short tandem repeat loci in these 4 patients, PCR profiles of primary and metastatic lesions matched with those of normal controls in all 4 patients. Therefore, it was confirmed that both primary and metastatic lesions developed from the same individual. These results suggest that MSI is also involved in lymph node metastasis in the development and/or progression of endometrial carcinoma in some patients.