Toshihiko Terao

Immunologic studies in presumed amniotic fluid embolism

Objective To evaluate the potential role of immunologic mechanisms that involve mast cell degranulation (anaphylaxis) or complement activation in the mechanism of amniotic fluid embolism. Methods This study was a case series of nine women with presumed amniotic fluid embolism and a control group of 22 women who had normal labor. Women were from community and tertiary referral hospitals in Japan and the United States. Main outcome measures were maternal peripartum complement levels (C3 and C4), serum levels of tryptase, urinary histamine concentrations, and serum levels of a fetal antigen (sialyl Tn). Results Serum tryptase and urinary histamine measurements were negative in women with amniotic fluid embolism; seven of nine had elevated levels of fetal antigen. All eight who had serum available for testing had abnormally low levels of complement. Mean C3 level of 44.0 mg/dL and C4 level of 10.7 mg/dL were significantly lower than corresponding postpartum control values of 117.3 mg/dL and 29.4 mg/dL (P = .018 for C3, P = .012 for C4). Postpartum C3 and C4 levels decreased by 8% and 5%, respectively, compared with intrapartum values (P = .003 for C3, P = .021 for C4) but were still within normal range. Conclusion Serologic findings suggest a role for complement activation in the mechanism of amniotic fluid embolism. Laboratory data from this series did not implicate mast cell degranulation (anaphylaxis) in the pathophysiology of the disease.

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.

A soybean Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation.

We have previously reported in a series of papers that a Kunitz-type protease inhibitor, bikunin, suppresses up-regulation of urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and cancer cell invasion in vitro and peritoneal disseminated metastasis in vivo. In the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted, extracellular uPA, signal transduction involved in the expression of uPA and invasion in HRA human ovarian cancer cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman–Birk inhibitor (BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans; 2) that neither KTI nor BBI effectively inhibits enzymatic activity of uPA; 3) that uPA upregulation observed in HRA cells was inhibited by preincubation of the cells with KTI with an IC50 of ∼xa02xa0μM, whereas BBI failed to repress uPA upregulation, as measured by enzyme-linked immunosorbent assay; 4) that cell invasiveness was inhibited by treatment of the cells with KTI with an IC50 of ∼xa03xa0μM, whereas BBI failed to suppress cell invasion, as measured by an inxa0vitro invasion assay; 5) KTI suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated by a binding protein(s) other than a bikunin binding protein and/or its receptor; and 6) that transforming growth factor-beta 1 (TGF-β1)-mediated activation of ERK1/2 was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI, but not BBI, could inhibit cell invasiveness at least through suppression of uPA signaling cascade, although the mechanisms of KTI may be different from those of bikunin.

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.

Anti-metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent.

We have demonstrated that urinary trypsin inhibitor (UTI) purified from human urine is able to inhibit lung metastasis of mouse Lewis lung carcinoma (3LL) cells in experimental and spontaneous metastasis models. In this study, we have investigated whether UTI in combination with an anti-cancer drug, etoposide, can prevent tumour metastasis and show an enhanced therapeutic effect. Subcutaneous (s.c.) implantation of 3LL cells (1 x 10(6) cells) in the abdominal wall of C57BL/6 female mice resulted in macroscopic lung metastasis within 21 days. Microscopic lung metastasis was established by day 14 after tumour cell inoculation, and surgical treatment alone after this time resulted in no inhibition of lung metastasis. The number of lung tumour colonies in the group of mice which received surgery at day 21 was greater than in mice which had tumours left in situ (P = 0.0017). Surgical treatment on day 7, followed by UTI administration (s.c.) for 7 days, led to a decrease in lung metastasis compared with untreated animals. A significant inhibition of the formation of pulmonary metastasis was obtained with daily s.c. injections of UTI for 7 days immediately after tumour cell inoculation. UTI administration did not affect the primary tumour size at the time of operation. In addition, etoposide treatment alone led to a smaller primary tumours and yielded reduction of the formation of lung metastasis in the group of mice which received surgery at day 14 (P = 0.0026). Even in mice which received surgical treatment on day 14, followed by the combination of UTI (500 micrograms per mouse, days 14, 15, 16, 17, 18, 19 and 20) with etoposide (40 mg kg-1, days 14, 18 and 22), there was significant reduction of the formation of lung metastasis (P = 0.0001). Thus, the combination of an anti-metastatic agent with an anti-cancer drug, etoposide, might provide a therapeutically promising basis for anti-metastatic therapy.

CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase‐type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside‐in signal transduction pathways responsible for fragmented HA‐mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)‐2 and ‐9 as well as urokinase‐type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI‐1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS‐2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI‐1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c‐Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti‐CD44 mAb, MAP kinase inhibitors, neutralizing anti‐uPAR pAb, anti‐catalytic anti‐uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.

Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion

We investigated the effects of purified human urinary trypsin inhibitor (UTI) and fragments derived from UTI by proteolysis on the invasive potential of ovarian cancer cells (HOC-I) and gestational choriocarcinoma cells (SMT-ccl) using anin vitro reconstituted basement membrane invasion assay. These cells express cell-associated plasmin and functional uPA receptors that are partially occupied by ligands. SMT-ccl cells, which express threefold higher levels of cell-associated plasmin activity than HOC-I cells, showed approximately twofold increase in their invasive potential. For the invasion assay, HOC-I cells were primed with exogenous plasminogen, but SMT-ccl cells were not. Human leukocyte elastase (HLE)-digested UTI (22 kDa fragment; UTI-22) inhibited plasmin practically with the same strength as native UTI. Trypsindigested UTI (20 kDa fragment; UTI-20), however, did not inhibit plasmin significantly. Treatment of cells with UTI or UTI-22 reduced the incidence of tumor cell invasive capacity, whereas the inhibitory effect of UTI-20 was not remarkable. The inhibitory effect on tumor cell invasion was dose-dependent and non-toxic; moreover, it was not mediated by inhibition of the tumor cell chemotactic response or of cell attachment to matrigel. These results indicate that inhibition of the proteolytic enzyme plasmin specifically reduced the invasive capacity of tumor cellsin vitro.

Therapeutic efficacy of once‐daily oral administration of a Kunitz‐type protease inhibitor, bikunin, in a mouse model and in human cancer

Bikunin, a Kunitz‐type protease inhibitor, specifically inhibits tumor invasion and metastasis.

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.

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.