Yasuyuki Hirashima

The protease inhibitor bikunin, a novel anti-metastatic agent

Abstract Bikunin is a Kunitz-type protease inhibitor predominantly found in human amniotic fluid. In cancers, administration of bikunin may block tumor cell invasion by a direct inhibition of tumor cell-associated plasmin activity as well as by inhibiting urokinase-type plasminogen activator (uPA) expression at the gene and protein levels, possibly through suppression of CD44 dimerization and/or the MAP kinase signaling cascade. Treatment of cancer patients with bikunin may be beneficial in the adjuvant setting to delay the onset of metastasis development and/or in combination with cytotoxic agents to improve treatment efficacy in patients with advanced ovarian cancer.

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

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.

Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.

Diagnosis, clinicopathologic features, treatment, and prognosis of small cell carcinoma of the uterine cervix; Kansai Clinical Oncology Group/Intergroup study in Japan

OBJECTIVES This is a multicenter, collaborative study to accumulate cases of small cell carcinoma of the uterine cervix (SmCC), to clarify its clinical and clinicopathologic features and prognosis, and to obtain findings to establish future individualized treatment. METHODS At medical centers participating in the Kansai Clinical Oncology Group/Intergroup, patients diagnosed with SmCC between 1997 and 2007 were enrolled. Clinicopathologic features and prognosis were retrospectively evaluated in patients with SmCC diagnosed at a central pathologic review. RESULTS A total of 71 patients were registered at 25 medical centers in Japan. Of these, 52 patients (73%) were diagnosed with SmCC based on a pathological review. These 52 patients diagnosed with SmCC were analyzed. The median follow-up period was 57 months. The 4-year progression-free survival (PFS) was: IB1, 59%; IB2, 68%; IIB, 13%; and IIIB, 17%. The 4-year overall survival (OS) was: IB1, 63%; IB2, 67%; IIB, 30%; IIIB, 29%; and IVB, 25%. For postoperative adjuvant therapy, postoperative chemotherapy (a platinum drug in all cases) was compared to non-chemotherapy. The 4-year PFS was 65% and 14%, and the 4-year OS was 65% and 29%. PFS was significantly better (p=0.002), and the OS tended to be better (p=0.073) in the group with postoperative chemotherapy. CONCLUSION Even in patients with early stage SmCC, the prognosis is poor. However, in early stage patients, by adding postoperative chemotherapy, the prognosis may improve. Currently, various treatment protocols are used at each medical center, but in the future, a standardized treatment protocol for SmCC will hopefully be established.

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Characterization of binding properties of urinary trypsin inhibitor to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8.

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP40), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP45). Here we characterize binding properties of UTI-BPs·UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP40 and UTI-BP45 bind125I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP40. Additional experiments, using various reagents to block binding of 125I-UTI and NG-UTI to the UTI-BP40 and UTI-BP45 confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP45. Analysis of binding of125I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP40 (which can bind NG-UTI), and the high affinity sites are the UTI-BP45. In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

Suppression of urokinase-type plasminogen activator expression from human ovarian cancer cells by urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, efficiently inhibits tumor cell invasion and metastasis. We examined the effect of UTI on urokinase-type plasminogen activator (uPA) expression in ovarian cancer cell lines, HOC-I and HRA. By Northern blot, Western blot, ELISA, and zymographic analyses, we demonstrated that UTI inhibited the expression of uPA mRNA and protein in these cells in a time- and dose-dependent manner, independent of whether induction was triggered by phorbol ester. Monoclonal antibody 4G12, which inhibits UTI binding to the cells, produced a dose-dependent abrogation in UTI-mediated down-regulation of uPA expression. These data suggest that UTI significantly down-regulates tumor cell uPA mRNA expression and protein secretion, and that UTI binding to the cells is necessary to exert the UTIs action.

Genetic downregulation of pregnancy-associated plasma protein-A (PAPP-A) by bikunin reduces IGF-I-dependent Akt and ERK1/2 activation and subsequently reduces ovarian cancer cell growth, invasion and metastasis.

A Kunitz‐type protease inhibitor, bikunin, downregulates expression of uPA and its receptor uPAR at the mRNA and protein levels in several types of tumor cells. Our recent work showed that, using a cDNA microarray analysis, pregnancy‐associated plasma protein‐A (PAPP‐A) is a candidate bikunin target gene. To clarify how reduced levels of PAPP‐A may confer repressed invasiveness, we transfected human ovarian cancer cell line HRA with antisense (AS)‐PAPP‐A cDNA and compared the properties of the transfected cells to those of parental HRA cells. Here, we show that regulation of uPA mRNA and protein by IGF‐I depends on the PI3K and MAPK signaling pathways and phosphorylation of Akt and ERK1/2 is required for IGF‐I‐mediated cell invasion; that IGFBP‐4 protease in HRA cells is identified as PAPP‐A; that reduced PAPP‐A expression is associated with the upregulation of IGFBP‐4 expression; that higher intact IGFBP‐4 levels were associated with low invasive potential and growth rate in AS‐PAPP‐A cells in response to IGF‐I; that IGF‐I stimulates Akt and ERK1/2 activation of both the control and antisense cells, but the relative potency and efficacy of IGF‐I were lower in the antisense cells compared to the control; and that genetic downregulation of PAPP‐A reduces the proliferation, invasion and metastasis of HRA cells. In conclusion, our data identify a novel role for PAPP‐A as a bikunin target gene. IGF‐I‐induced IGFBP‐4 proteolysis by PAPP‐A may enhance cell growth and invasion through IGF‐I‐dependent Akt and ERK1/2 activation and subsequently upregulation of uPA.

Inter-alpha-trypsin inhibitor is concentrated in the pericellular environment of mouse granulosa cells through hyaluronan-binding

OBJECTIVE The binary complex involving hyaluronan and inter-alpha-trypsin inhibitor (ITI) is an important component of the cumulus oocyte complex. The aim of this study is to investigate the physiological association between ITI and its derivatives and hyaluronan or its binding protein (HABP). STUDY DESIGN ITI and its derivatives (heavy chains of ITI and urinary trypsin inhibitor, UTI) were tested for their ability to interact with hyaluronan or HABP. HABP was used to locate the distribution of hyaluronan in mice ovaries. RESULT ITI and heavy chains of ITI, but not UTI, could specifically bind to immobilized hyaluronan. Furthermore HABP could specifically bind immobilized hyaluronan with high affinity, and also to immobilized ITI and its derivatives. 6 h after the injection of human chorionic gonadotropin, the hyaluronan staining in the preovulatory ovaries displayed a heterogenous appearance in which the most intense stainings were observed in cumulus oocyte complex. The distribution of ITI was found to be similar to that of hyaluronan. CONCLUSION The hyaluronan binding sites of ITI are located in the heavy chains of this molecule. ITI is concentrated in the pericellular environment of granulosa cells through hyaluronan-binding. The altered amount of hyaluronan and ITI in the preovulatory ovaries may contribute to their important clinical characteristics including cumulus oocyte complex expansion.

The relationship between positive peritoneal cytology and the prognosis of patients with FIGO stage I/II uterine cervical cancer

Objective The purpose of this study was to assess whether peritoneal cytology has prognostic significance in uterine cervical cancer. Methods Peritoneal cytology was obtained in 228 patients with carcinoma of the uterine cervix (International Federation of Gynecology and Obstetrics [FIGO] stages IB1-IIB) between October 2002 and August 2010. All patients were negative for intraperitoneal disease at the time of their radical hysterectomy. The pathological features and clinical prognosis of cases of positive peritoneal cytology were examined retrospectively. Results Peritoneal cytology was positive in 9 patients (3.9%). Of these patients, 3/139 (2.2%) had squamous cell carcinoma and 6/89 (6.7%) had adenocarcinoma or adenosquamous carcinoma. One of the 3 patients with squamous cell carcinoma who had positive cytology had a recurrence at the vaginal stump 21 months after radical hysterectomy. All of the 6 patients with adenocarcinoma or adenosquamous carcinoma had disease recurrence during the follow-up period: 3 with peritoneal dissemination and 2 with lymph node metastases. There were significant differences in recurrence-free survival and overall survival between the peritoneal cytology-negative and cytology-positive groups (log-rank p<0.001). Multivariate analysis of prognosis in cervical cancer revealed that peritoneal cytology (p=0.029) and histological type (p=0.004) were independent prognostic factors. Conclusion Positive peritoneal cytology may be associated with a poor prognosis in adenocarcinoma or adenosquamous carcinoma of the uterine cervix. Therefore, the results of peritoneal cytology must be considered in postoperative treatment planning.