Fatal factors of clinical manifestations and laboratory testing in patients with amniotic fluid embolism.

AIMS: To identify factors leading to fatality of patients with amniotic fluid embolism (AFE). METHODS: Patients who had fatal or nonfatal AFE were registered at the Hamamatsu University School of Medicine in the Department of Obstetrics and Gynecology from 1992 to 2006. Data collected included information about demographics and clinical characteristics. The fatal factors among these data were identified using chi(2) analysis and the Mann-Whitney test. RESULTS: One hundred and thirty-five patients met the criteria, which included fatal (n = 65) and nonfatal AFE (n = 70). Maternal full-term gestational weeks, multiparous and noncesarean sections were the risk factors for death found in this study (p < 0.01). Sialyl Tn levels (mean +/- SD) in the serum of patients with fatal AFE (69.7+/- 126.4 U/ml) were higher compared to those with nonfatal AFE (48.3+/- 161.8 U/ml; p = 0.003). Each of three items (cardiac arrest, dyspnea or loss of consciousness) was more common in fatal AFE (p < 0.01). Maternal pregnancy and labor complications were not associated with the distinction between fatal and nonfatal AFE. CONCLUSION: Factors associated with patients with fatal AFE were identified. These included multiparity, noncesarean section at full-term and the three symptoms mentioned above. Sialyl Tn levels could be a possible prognostic fatality factor.

Ovarian endometrioma--risks factors of ovarian cancer development.

OBJECTIVE: Our prospective studies in Japan have found an increased ovarian cancer incidence in women with ovarian endometrioma (standardized incidence ratio, 8.95; 95% confidence intervals, 4.12-5.3). The risk increased with increasing age at ovarian endometrioma diagnosis. The goal of this study was to define the risk factor(s) of ovarian cancer development in a Japanese population with ovarian endometrioma. We also analyzed whether the predisposition toward ovarian cancer is limited to endometrioid and clear cell carcinoma. STUDY DESIGN: A total of 6398 participants at 212 participating hospitals in Shizuoka, Japan, were enrolled in the Shizuoka Cohort Study on Endometriosis and Ovarian Cancer (SCSEOC) Trial, which had prospective and retrospective components. The follow-up period was up to 17 years (median, 12.8 years). The risks of development of ovarian cancer were assessed in 6398 women with ultrasonographically diagnosed ovarian endometriomas. Cox proportional-hazards regression function was used to estimate impact in terms of risk factors and possible development of ovarian cancer. RESULTS: The prospective study demonstrated that 46 (0.72%) of 6398 women developed histologically proven ovarian cancer and were operated upon during follow-up. Clear cell carcinoma (39%) and endometrioid adenocarcinoma (35%) were commonly observed among women with ovarian cancer. By multivariate analysis, tumor size > or =9 cm in diameter and postmenopausal women were independent predictive factors of patients with development of ovarian cancer. CONCLUSIONS: Some endometriosis lesions may predispose to clear cell and endometrioid ovarian cancers. Advancing age and the size of endometriomas were independent predictors of development of ovarian cancer among women with ovarian endometrioma.

Suppression of urokinase receptor expression by thalidomide is associated with inhibition of nuclear factor kappaB activation and subsequently suppressed ovarian cancer dissemination.

Thalidomide has been used to treat a variety of diseases ranging from alleviation of autoimmune disorders to prevention of metastasis of cancers. It has been shown previously that increased levels of urokinase-type plasminogen activator receptor (uPAR) correlate well with higher invasive phenotype. We examined whether thalidomide is able to suppress the expression of uPAR mRNA and protein in human ovarian cancer cell line HRA and human chondrosarcoma cell line HCS-2/8. Here, we show that: (a) thalidomide suppresses the expression of constitutive and transforming growth factor-beta1 (TGF-beta1)-induced uPAR mRNA and protein; (b) a nuclear factor kappaB (NF-kappaB) activation system (phosphorylation of IkappaB-alpha and degradation of IkappaB-alpha) is necessary for the TGF-beta1-induced increase in uPAR expression, because L-1-tosylamido-2-phenylethyl chloromethyl ketone, a NF-kappaB inhibitor, reduced the uPAR production as well as mRNA expression; (c) thalidomide failed to further strengthen L-1-tosylamido-2-phenylethyl chloromethyl ketone's action; (d) the once-daily i.p. administration of thalidomide (400 microg/g body weight/d) decreased progressive growth of HRA tumors and ascites formation in an in vivo animal model; and (e) the once-daily i.p. administration of thalidomide in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. We conclude that thalidomide down-regulates constitutive and TGF-beta1-stimulated uPAR mRNA and protein expression possibly through suppression of NF-kappaB activation. Furthermore, combination therapy with thalidomide plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian cancer dissemination.

Suppression of lipopolysaccharide-induced cytokine production of gingival fibroblasts by a soybean, Kunitz trypsin inhibitor.

BACKGROUND: Human bikunin, a Kunitz-type trypsin inhibitor, inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in tumor cells and inflammatory cells. OBJECTIVES: We analyzed the effect of a soybean-derived Kunitz trypsin inhibitor (KTI) on TNF-alpha production in human gingival fibroblasts stimulated by lipopolysaccharide (LPS), an inflammatory inducer. MATERIAL AND METHODS: Mitogen-activated protein kinase (MAPK) activation and cytokine levels were monitored using western blot and a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: Here, we show (i) a soybean KTI abrogates LPS-induced up-regulation of TNF-alpha mRNA and protein expression in a dose-dependent manner in gingival fibroblasts, (ii) KTI also blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins, (iii) inhibition by KTI of TNF-alpha induction correlates with the suppressive capacity of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 signaling pathways, implicating repressed ERK1/2 and p38 signalings in the inhibition, and (iv) pretreatment of cells with KTI blocked LPS-induced nuclear factor kappaB (NFkappaB) activation. CONCLUSION: Our results indicate that KTI inhibits LPS-induced up-regulation of cytokine expression possibly through suppression of ERK1/2 and p38 kinase-mediated NFkappaB activation. These findings may identify anti-inflammatory properties of KTI at the level of gingival fibroblasts and may be relevant to the use of KTI in modulating inflammation, including periodontal disease.

A soybean Kunitz trypsin inhibitor reduces tumor necrosis factor-alpha production in ultraviolet-exposed primary human keratinocytes.

BACKGROUND: Cytokines are produced as a consequence of photo-damaged DNA and oxidative stress in ultraviolet (UV)-exposed keratinocytes. A soybean Kunitz trypsin inhibitor (KTI) down-regulates the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in tumor cells and inflammatory cells. AIM: The effect of KTI on TNF-alpha production in UV-exposed primary human keratinocytes was analyzed. RESULTS: We show (i) UV induced up-regulation of TNF-alpha mRNA and protein expression in keratinocytes; (ii) cells treated with KTI before UV irradiation showed a significantly lower accumulation of TNF-alpha protein in a dose-dependent manner and a reduced UV-induced up-regulation of TNF-alpha mRNA expression; (iii) KTI inhibited the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins; (iv) UV irradiation transiently activated c-Jun N-terminal kinase (JNK) and Akt signaling but only weakly activated extracellular signal-regulated kinase (ERK) and p38; (v) KTI specifically inhibited UV-induced activation of ERK, JNK, and p38, but not Akt; (vi) treatment of cells with SP600125, a pharmacological inhibitor of JNK, predominantly suppressed UV-induced up-regulation of TNF-alpha expression; and (vii) KTI did not enhance suppression of UV-induced JNK phosphorylation by SP600125. CONCLUSIONS: KTI specifically inhibited UV-induced up-regulation of cytokine expression predominantly through suppression of JNK signaling pathway.

Reduced syndecan-1 expression stimulates heparin-binding growth factor-mediated invasion in ovarian cancer cells in a urokinase-independent mechanism.

The expression of syndecan-1 generally appears down-regulated in human cancers and experimental models, whereas transfectional expression of syndecan-1 in cancer cells has been shown to inhibit aspects of their malignant behavior. To clarify how reduced levels of syndecan-1 may confer enhanced invasiveness, we transfected human ovarian cancer cell line HRA with antisense (AS) syndecan-1 oligodeoxynucleotide (ODN) and compared the properties of transfected cells to those of parental cells or sense (S) syndecan-1 cells. Here, we show: 1) there was lower proliferation in the AS syndecan-1 cells compared to controls (parental HRA cells and S syndecan-1 cells) when cells were incubated with HB-GFs (HB-EGF, HGF, or FGF2); 2) transfection of HRA cells with a syndecan-1 AS ODN enhanced the increase in HB-GF-dependent invasiveness; 3) in contrast, IGF-I stimulated cell proliferation and invasion, irrespective of whether cells were transfected with the AS syndecan-1 gene; 4) IGF-I stimulated ERK1/2 activation and uPA expression in both the control and AS cells, whereas the net effect of the reduction of syndecan-1 is to shift the HB-GF dose-response curve to the right; 5) the AS cells reduced activation and up-regulation of ERK1/2 phosphorylation and uPA expression, respectively, in response to HB-GFs; and 6) in comparison with early stage ovarian cancer tissues, there was a 3-fold decrease in syndecan-1 mRNA levels in advanced stage tissues. Taken together, these data suggest that decreased syndecan-1 expression may be associated with enhanced cell invasion possibly through the uPA-independent mechanism.

Suppression of urokinase expression and invasion by a soybean Kunitz trypsin inhibitor are mediated through inhibition of Src-dependent signaling pathways.

A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.

Suppressing effects of daily oral supplementation of beta-glucan extracted from Agaricus blazei Murill on spontaneous and peritoneal disseminated metastasis in mouse model.

PURPOSE: The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer. METHODS: We examined whether beta-(1-6)-D: -glucan extracted from A. blazei is a potential anticancer agent in an in vitro and in vivo animal model. RESULTS: Here we show that (1) beta-glucan had cytotoxic effect against human ovarian cancer HRA cells, but not against murine Lewis lung cancer 3LL cells, in vitro; (2) beta-glucan promotes p38 MAPK activity for suppressing HRA cell proliferation and amplifying the apoptosis cascade; (3) beta-glucan stimulates translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation; (4) treatment with SB203580, a p38 MAPK-specific inhibitor, suppresses beta-glucan-induced effects, indicating that activation of p38 MAPK is involved in the suppression of cell proliferation and mitochondrial activation-mediated cell death pathway; (5) in mice, oral supplementation with beta-glucan reduces pulmonary metastasis of 3LL cells and peritoneal disseminated metastasis of HRA cells and inhibits the growth of these metastatic tumors in lung or peritoneal cavity, in part, by suppressing uPA expression; and (6) in an in vivo experimental metastasis assay, however, the oral supplementation with beta-glucan after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies. CONCLUSION: Treatment with beta-glucan may be beneficial for cancer patients with or at risk for metastasis. The beta-glucan-dependent signaling pathways are critical for our understanding of anticancer events and development of cancer therapeutic agents.

Dietary supplementation of soybean kunitz trypsin inhibitor reduces lipopolysaccharide-induced lethality in mouse model.

We examined the modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean, as intraperitoneal (i.p.) injection and dietary supplements on bacterial lipopolysaccharide (LPS)-induced lethality in mice. We initially examined the suppressing effects of i.p. injection of KTI (50 mg/kg) and BBI (50 mg/kg) on LPS-induced lethality after i.p. injection of LPS. Furthermore, groups of female C57BL/6 were fed a basal diet (control group) or the basal diet supplemented with KTI (50 g/kg) or BBI (50 g/kg). Here, we show that i.p. and daily oral administration of KTI, but not BBI, caused a significant reduction of the LPS-induced lethality; that LPS significantly induced plasma TNF-alpha, IL-1beta, and IL-6 levels in mice after LPS challenge; that concomitant administration of KTI, but not BBI, inhibits the LPS-induced plasma levels of these cytokines; and that KTI, but not BBI, suppressed LPS-induced upregulation of cytokine expression through suppression of phosphorylation of three mitogen-activated protein (MAP) kinase pathways, ERK1/2, JNK, and p38, in peritoneal macrophages. These data allow us to speculate that i.p. injection and dietary supplementation of a soybean KTI may play a role as a potent anti-inflammatory agent by inhibiting activation of MAP kinases, leading to the suppression of cytokine expression.

Plasma bikunin as a favorable prognostic factor in ovarian cancer.

PURPOSE: Bikunin is a multifunctional glycoprotein, which mediates suppression of tumor cell invasion and metastasis. The measurement of bikunin levels in the tissue of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the evaluation of prognosis. The high bikunin expression in ovarian cancer tissue would enable the use of soluble bikunin protein present in the circulation of ovarian cancer patients as a biomarker of disease. PATIENTS AND METHODS: We developed a double-antibody immunoassay for bikunin and detected its presence in normal human circulation. We quantified, by enzyme-linked immunosorbent assay and/or immunoblot assay bikunin in sera from 200 healthy women (controls), 200 patients with benign gynecologic diseases, and 327 patients with ovarian cancer before surgical removal of the tumor. RESULTS: When the values of bikunin corresponding to the median were used as the cutoff value (11.5 microg/mL), low plasma bikunin was strongly associated with late-stage, suboptimal debulking with large residual tumor (> 2 cm) and low response to chemotherapy. The median survival time of the patients with a high bikunin level was more than 60 months as compared with 26 months among those with low bikunin level (P = .002). This difference corresponded to a 2.2-fold increased risk of dying for the lower plasma bikunin patients (hazard ratio, 0.45; P = .023) and remained significant in multivariate analysis (hazard ratio, 0.63; P = .041). CONCLUSION: Preoperative plasma bikunin concentration is a strong and independent favorable prognostic marker for ovarian cancer.

Thalidomide inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in endometriotic stromal cells, possibly through suppression of nuclear factor-kappaB activation.

OBJECTIVE: Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. TNF-alpha induces IL-8 production in endometriotic cells through nuclear factor-kappaB (NF-kappaB) activation. Thalidomide (Thal) inhibits inflammation by down-regulating the expression of proinflammatory cytokines in tumor cells and inflammatory cells. However, the mechanism of Thal action in human endometriotic stromal cells has not yet been elucidated. MAIN OUTCOME MEASURES: We examined whether Thal abrogates TNF-alpha-induced up-regulation of IL-8 expression in endometriotic stromal cells. RESULTS: Here, we show 1) that treatment of endometriotic stromal cells with TNF-alpha increased the expression of phosphorylated IkappaBalpha and degradation of total IkappaBalpha, which in turn activates NF-kappaB; 2) Thal significantly inhibits the TNF-alpha-induced expression of phosphorylated IkappaBalpha and degradation of IkappaBalpha; 3) TNF-alpha activation induced increased nuclear translocation of NF-kappaB, which was inhibited by pretreatment with either Thal or N-tosyl-L-phenylalanine chloromethyl ketone, an NF-kappaB inhibitor. Thal did not enhance the N-tosyl-L-phenylalanine chloromethyl ketone's action; and 4) Pretreatment with Thal reduced TNF-alpha-induced IL-8 protein production as well as mRNA expression. CONCLUSION: The current study showed for the first time that Thal treatment attenuated the expression of IL-8 by reducing TNF-alpha-induced NF-kappaB activation.

Bikunin suppresses lipopolysaccharide-induced lethality through down-regulation of tumor necrosis factor- alpha and interleukin-1 beta in macrophages.

BACKGROUND: Lipopolysaccharide (LPS) is the primary mediator of gram-negative sepsis; it induces the production of macrophage-derived cytokines. It has been shown that bikunin, a Kunitz-type protease inhibitor, inhibits LPS-induced cytokine expression. METHODS: To explore the role of bikunin, bikunin knockout (Bik(-/-)) mice were used for in vitro cytokine experiments and in vivo animal models. RESULTS: We show that a higher level of LPS-mediated death was induced in Bik(-/-), compared with wild-type (wt), mice; the administration of bikunin caused a significant reduction in LPS-induced lethality; LPS significantly increased tumor necrosis factor (TNF)- alpha and interleukin-1 beta levels in Bik(-/-), relative to wt, mice after LPS challenge; concomitant administration of bikunin inhibited the LPS-induced plasma levels of these cytokines; bikunin suppressed the LPS-induced up-regulation of cytokine expression through the suppression of the phosphorylation of ERK1/2, JNK, and p38 in macrophages; and LPS-induced up-regulation of TNF- alpha expression was not enhanced in Bik(-/-) macrophages without endogenous bikunin. CONCLUSIONS: These data allow us to speculate that the increased sensitivity of Bik(-/-) mice to LPS-induced death in vivo is due to a lack of circulating bikunin in plasma. Bikunin may play a role as a potent anti-inflammatory agent.

Bikunin down-regulates heterodimerization between CD44 and growth factor receptors and subsequently suppresses agonist-mediated signaling.

We provided evidence previously that bikunin, a Kunitz-type protease inhibitor, can disrupt dimerization of CD44 proteins, which may result in suppression of receptor-mediated MAP kinase signaling. However, to what extent dimerization may alter ligand-induced signaling has not been documented. Given the recent recognition that some growth factor receptors can form heterodimers with CD44, the present study was undertaken to determine whether the CD44 and growth factor receptors (e.g., EGFR, FGFR, HGFR, VEGFR, TGF-betaRI, or TGF-betaRII) can form heterodimers in cancer cells and, if so, to investigate the potential functional consequences of such heterodimerization. We also examined whether bikunin can abrogate these heterodimerizations and inhibit CD44/growth factor-dependent signaling. Here, we show direct evidence for heterodimerization of CD44-FGFR and CD44-TGF-betaRI in human chondrosarcoma HCS-2/8 cells, CD44-EGFR complex in human glioma U87MG cells, and CD44-TGF-betaRI heterodimer in human ovarian cancer HRA cells. Coupling of CD44 and growth factor receptor may be selective, depending on a cell type. Bikunin does not alter the ligand binding, whereas functionally reduces heterodimerization between CD44 and growth factor receptors. The disruption of heterodimerization substantially reduces receptor-induced tyrosine phosphorylation and ERK1/2 activation. Taken together, our data suggest that bikunin-mediated suppression of heterodimerization between CD44 and growth factors may inhibit the agonist-promoted activation of the signaling pathway.

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages.

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.

Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI)--ITI target genes in mouse ovary identified by microarray analysis.

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik(-/-) female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.

Upregulation of bikunin in tumor-infiltrating macrophages as a factor of favorable prognosis in ovarian cancer.

OBJECTIVE: This study was carried out to clarify the localization of bikunin, a Kunitz-type protease inhibitor, and relation between expression of individual bikunin protein and ovarian cancer progression. METHODS: We performed a retrospective study on the immunohistochemical expression of bikunin, urokinase-type plasminogen activator (uPA) and macrophages (CD68) in surgical specimens derived from 89 ovarian cancer patients to investigate correlations between the expression of bikunin and the clinicopathologic features and the prognosis. Furthermore, bikunin and uPA levels were measured by immunoblot analysis. RESULTS: Immunohistochemical staining revealed that the localization of bikunin was similar to that of CD68 for macrophages. We identified high expression of bikunin in 40 (45%) of 89 ovarian cancers. The results of Western blot analysis showed a significant correlation with immunohistochemical data. There was a significant inverse correlation between bikunin levels and uPA levels in ovarian cancer tissues. High bikunin expression was an independent predictor for disease-free survival (P = 0.040) and overall survival (P = 0.042). The 5-year survival rate of the 49 patients with low bikunin expression in ovarian cancers was 39%, whereas that of the other 40 patients with high bikunin expression was 63%. In addition, macrophage-derived bikunin protein was induced by exogenous IL-6. CONCLUSION: Bikunin derived from tumor-infiltrating macrophages might be a prognostic indicator as an antiinvasive factor supplied from macrophages within and around the tumor possibly through down-regulation of tumor-associated uPA expression.