RESUMEN
OBJECTIVE: We performed a detailed molecular analysis of bikunin-mediated anti-inflammation (suppressive effect of cytokine release, MAP kinase activation, and nuclear translocation of NF-kB) using a truncated form of bikunin. MATERIALS AND METHODS: We obtained bikunin derivatives that contained O-glycoside-linked N-terminal glycopeptide (Bik-m1), N-glycoside-linked C-terminal tandem Kunitz domains (Bik-m2), bikunin lacking O-glycoside (Bik-c), asialo bikunin (Bik-a), bikunin lacking N-glycoside (Bik-n), and purified C-terminal Kunitz domain II (kII) of bikunin (HI-8). Enzyme-linked immunosorbent assay and Western blot were carried out to measure secreted TNF-alpha and MAP kinase activation. RESULTS: We examined the TNF-alpha secretion in control and lipopolysaccharide (LPS)-treated neutrophils and did not see any changes of its protein levels in the cells pretreated with Bik-m1, Bik-m2, Bik-c, or HI-8. In all of the derivatives tested, only the derivatives that lacked N-glycoside side chain showed a significant suppression of TNF-alpha secretion by LPS. Only a small (21 amino acids) deletion of the N-terminal portion of bikunin (which corresponds to Bik-m2) abolished its suppressing activity of TNF-alpha secretion, thus suggesting that the N-terminal 21 amino acids play a critical role in anti-inflammation. Bik-m1 alone failed to show anti-inflammatory response. Bikunin failed to inhibit ionomycin-induced phosphorylation of MAP kinases. CONCLUSION: These data allow us to conclude that the cytokine expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain. Our results also suggest a possible role of bikunin for receptor-dependent MAP kinase activation.
Asunto(s)
alfa-Globulinas/química , alfa-Globulinas/fisiología , Regulación hacia Abajo/fisiología , Activación Neutrófila/fisiología , Neutrófilos/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Células Cultivadas , Humanos , Mediadores de Inflamación/química , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estructura Molecular , Neutrófilos/enzimología , Neutrófilos/patología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Activated neutrophils contribute to the development of preterm delivery. Because of its ability to suppress inflammation, bikunin, a Kunitz-type protease inhibitor, is currently in clinical trials. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on pro-inflammatory cytokine production and nuclear factor-kappaB (NF-kappaB) activation in mouse neutrophils stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show that bikunin: (i) blocks LPS-induced secretion of pro-inflammatory cytokines, including TNF-alpha and IL-1beta, in a dose-dependent manner; (ii) has an inhibitory effect on cytokine production at a concentration of 0.2 microM, reaching 65% inhibition at the highest doses of bikunin tested (5 microM); (iii) has the suppressive capacity of ERK1/2 and p38 signaling pathways; and (iv) inhibited sequentially the LPS-induced phosphorylation of IkappaB-alpha, degradation of IkappaB-alpha, and nuclear translocation of NF-kappaB. When the MAPK data are analyzed, a significant decrease in phosphorylation is not seen at 0.2 microM bikunin but is at 1.0 microM dosing. Bikunin can inhibit LPS-induced neutrophil activation and cytokine release, although it is unlikely that it works primarily through the inhibition of MAPK phosphorylation. These data suggest that such effects are important in vivo and play a major contributory role in abrogation of neutrophil-mediated inflammatory responses, such as preterm delivery.
Asunto(s)
alfa-Globulinas/farmacología , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células Cultivadas , Citoprotección/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Previously, we showed that bikunin, a Kunitz-type protease inhibitor, inhibits invasion and metastasis in several types of cancer cells possibly through suppression of upregulation of urokinase-type plasminogen activator (uPA) expression. Bikunin corresponds to a light chain of the inter-alpha inhibitor. To explore critical role of endogenous bikunin, we used bikunin knockout (Bik-/-) mice. Here, we show that 1) higher frequency of spontaneous 3LL lung metastasis was observed in Bik-/- mice compared to Bik+/+ mice, suggesting that bikunin deficiency increases the sensitivity of mice to lung metastasis; 2) administration of exogenous bikunin caused a significant reduction of lung metastasis in Bik-/- and Bik+/+ mice; 3) primary and metastatic tumors significantly upregulated uPA and PAI-1 expression in Bik-/- mice relative to Bik+/+ mice at least through phosphorylation of ERK1/2 and 4) exogenous bikunin suppressed phosphorylation of ERK1/2 and upregulation of uPA and PAI-1 expression in 3LL cells in response to G-CSF. These data allow us to conclude that the increased sensitivity of Bik-/- mice to lung metastasis in vivo is due to a lack of circulating proteins of the inter-alpha inhibitor family, especially bikunin.
Asunto(s)
Carcinoma Pulmonar de Lewis/patología , Neoplasias Pulmonares/fisiopatología , Neoplasias Pulmonares/secundario , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Inhibidor de la Tripsina de Soja de Kunitz/genética , Animales , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
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.
Asunto(s)
FN-kappa B/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Talidomida/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Condrosarcoma/tratamiento farmacológico , Condrosarcoma/metabolismo , Condrosarcoma/patología , Sinergismo Farmacológico , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , FN-kappa B/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Talidomida/administración & dosificación , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Mediadores de Inflamación/farmacología , Lipopolisacáridos/farmacología , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Inhibidores de Tripsina/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Células Cultivadas , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Encía/citología , Humanos , Interleucina-1/antagonistas & inhibidores , Interleucina-6/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Queratinocitos/efectos de la radiación , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Factor de Necrosis Tumoral alfa/genética , Rayos Ultravioleta , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , L-Lactato Deshidrogenasa/análisis , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Factores de Crecimiento de Fibroblastos/farmacología , Glicoproteínas de Membrana/genética , Neoplasias Ováricas/patología , Proteoglicanos/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Adulto , Anciano , Northern Blotting , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Factor 1 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Estadificación de Neoplasias , Oligonucleótidos Antisentido/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sindecano-1 , Sindecanos , TransfecciónRESUMEN
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.
Asunto(s)
Glycine max/enzimología , Transducción de Señal/fisiología , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/fisiología , Línea Celular Tumoral , Represión Enzimática/efectos de los fármacos , Represión Enzimática/fisiología , Femenino , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Glycine max/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Peritoneales/tratamiento farmacológico , beta-Glucanos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Agaricus , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Carcinoma Pulmonar de Lewis/prevención & control , Proliferación Celular/efectos de los fármacos , Esquema de Medicación , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Ratones , Neoplasias Ováricas/patología , Neoplasias Peritoneales/secundario , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , beta-Glucanos/administración & dosificaciónRESUMEN
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.
Asunto(s)
Suplementos Dietéticos , Lipopolisacáridos/farmacología , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Administración Oral , Animales , Antiinflamatorios/farmacología , Western Blotting , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Femenino , Inflamación , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de Señal , Glycine max/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/sangre , Glicoproteínas de Membrana/sangre , Neoplasias Ováricas/mortalidad , Inhibidor de la Tripsina de Soja de Kunitz/sangre , Western Blotting , Interacción de Doble Vínculo , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedades de los Genitales Femeninos/sangre , Humanos , Inmunoensayo , Análisis Multivariante , Neoplasias Ováricas/sangre , Neoplasias Ováricas/cirugía , Pronóstico , Curva ROCRESUMEN
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.
Asunto(s)
Endometriosis/metabolismo , Proteínas I-kappa B/antagonistas & inhibidores , Interleucina-8/biosíntesis , Talidomida/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Células Cultivadas , Femenino , Humanos , Interleucina-8/genética , Inhibidor NF-kappaB alfa , Células del Estroma/metabolismo , Clorometilcetona de Tosilfenilalanila/farmacologíaRESUMEN
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.
Asunto(s)
Regulación hacia Abajo , Inflamación/mortalidad , Interleucina-1/metabolismo , Macrófagos Peritoneales/inmunología , Glicoproteínas de Membrana/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Inflamación/tratamiento farmacológico , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/farmacología , Inhibidor de la Tripsina de Soja de Kunitz/administración & dosificación , Inhibidor de la Tripsina de Soja de Kunitz/genética , Inhibidor de la Tripsina de Soja de Kunitz/farmacologíaRESUMEN
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.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Glicoproteínas de Membrana/farmacología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Línea Celular Tumoral , Dimerización , Activación Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunoprecipitación , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FosforilaciónRESUMEN
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.
Asunto(s)
Lipopolisacáridos/administración & dosificación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/administración & dosificación , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidor de la Tripsina de Soja de Kunitz/administración & dosificación , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Activación de Macrófagos/efectos de los fármacos , RatonesRESUMEN
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.
Asunto(s)
Glicoproteínas de Membrana/farmacología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Factor de Crecimiento Transformador beta/fisiología , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario/genética , Femenino , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/orina , Neoplasias Ováricas , Procolágeno/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Timidina/metabolismo , Transfección , Factor de Crecimiento Transformador beta/efectos de los fármacos , Inhibidor de la Tripsina de Soja de Kunitz/genética , Inhibidor de la Tripsina de Soja de Kunitz/aislamiento & purificación , Inhibidor de la Tripsina de Soja de Kunitz/orinaRESUMEN
The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Familia-src Quinasas/metabolismo , Femenino , Flavonoides/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias Ováricas/metabolismo , Proteínas Quinasas/metabolismo , Pirimidinas/farmacología , Transducción de Señal/fisiología , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta1 , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) 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. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ascitis/tratamiento farmacológico , Glicoproteínas de Membrana/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/administración & dosificación , Proteínas Serina-Treonina Quinasas , Inhibidor de la Tripsina de Soja de Kunitz/uso terapéutico , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/farmacocinética , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/patología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Distribución Tisular , Inhibidor de la Tripsina de Soja de Kunitz/administración & dosificación , Inhibidor de la Tripsina de Soja de Kunitz/farmacocinéticaRESUMEN
BACKGROUND: Bikunin, a Kunitz-type protease inhibitor, specifically inhibits tumor invasion and metastasis. METHODS: The authors initially evaluated the therapeutic efficacy of once-daily oral administration of different doses of bikunin against human ovarian carcinoma HRA cells growing in the peritonea of nude mice. For the in vivo studies, female 7-week-old nude mice were randomized to 1 of 4 groups: bikunin-treated groups (n = 9 in each group) received 3, 10, or 30 microg/g body weight per day bikunin for 7 days via gastrointestinal gavage, and a control group (n = 9) received the vehicle solution (phosphate-buffered saline) via gastrointestinal gavage. On Day 9, the abdominal cavity was examined by two observers who were blinded to treatment. RESULTS: After oral administration, intact bikunin was detectable in mouse serum specimens at 3 and 6 hours. This was followed by a decline at 12 hours. The mice given bikunin at the highest dose level had a 40% decrease in tumor load. The highest uptake in the tumor was obtained with [125I]bikunin 12 hours postadministration. No effect on either food intake or body weight was observed in the treated versus sham groups. The current study was the first to report the potent activity of once-daily oral administration of bikunin against ovarian carcinoma. Next, the authors performed a Phase I trial to determine the maximum-tolerated dose (MTD) and safety of a once-daily oral administration schedule. The indication was locally advanced uterine cervical carcinoma after definitive treatment. An escalating dose (3, 10, and 30 mg/kg per day) of bikunin was administered orally to nine patients for 7 days. There were no dose-limiting toxicities and the MTD of the bikunin schedule was not defined. The authors also obtained preliminary data on its effect on urokinase-type plasminogen activator expression at the highest dose level. CONCLUSIONS: Once-daily oral administration of bikunin was found to be safe in humans and exhibited signs of biologic activity.