Daily consumption of seaweed has been proposed as a factor in explaining lower postmenopausal breast cancer (BC) incidence and mortality rates in Japan. This clinical trial assessed the impact of introducing seaweed- to non-seaweed-consuming American postmenopausal women. Fifteen healthy postmenopausal women were recruited for a 3-month single-blinded placebo controlled clinical trial; five had no history of BC (controls) and ten were BC survivors. Participants ingested ten capsules daily (5 g day(-1)) of placebo for 4 weeks, seaweed (Undaria) for 4 weeks, then placebo for another 4 weeks. Blood and urine samples were collected after each treatment period. Urinary human urokinase-type plasminogen activator receptor concentrations (uPAR) were analyzed by ELISA, and urine and serum were analyzed for protein expression using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF-MS). Urinary creatinine standardized uPAR (in pg mL μg(-1) creatinine) changed significantly between groups, decreasing by about half following seaweed supplementation (placebo 1, 1.5 (95 % CI, 0.9-2.1) and seaweed, 0.9 (95 % CI, 0.6-1.1) while placebo 2 returned to pre-seaweed concentration (1.7 (95 % CI, 1.2-2.2); p = 0.01, ANOVA). One SELDI-TOF-MS-identified urinary protein (m/z 9,776) showed a similar reversible decrease with seaweed and is reported to be associated with cell attachment. One serum protein (m/z 8,928) reversibly increased with seaweed and may be the immunostimulatory complement activation C3a des-arginine. uPAR is higher among postmenopausal women generally, and for BC patients, it is associated with unfavorable BC prognosis. By lowering uPAR, dietary seaweed may help explain lower BC incidence and mortality among postmenopausal women in Japan.

Recurrent joint bleeding is the most common manifestation of haemophilia resulting in haemophilic arthropathy (HA). The exact pathophysiology is unknown, but it is suggested that arthropathy is stimulated by liberation of fibrinolytic activators from the synovium during haemarthrosis. The aim of this study was to test the hypothesis that haemarthrosis activates the local synovial fibrinolytic system in a murine haemophilia model. The right knees of haemophilic and control mice were punctured to induce haemarthrosis. The left knees served as internal control joints. Synovial levels of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), plasmin, and alpha-2-antiplasmin (A2AP) were compared between the punctured and control knees. In haemophilic mice, an increase in synovial cells expressing urokinase-type plasminogen activator (uPA) in the right punctured knee versus the left unaffected knee was observed: (47% vs 43%) (p=0.03). Additionally, in haemophilic mice, haemarthrosis induced an increase in uPA (0.016 ng/ml vs 0.01 ng/ml) (p=0.03) and plasmin (0.53 μg/ml vs 0.46 μg/ml) (p=0.01) as promoters of fibrinolysis. Synovial levels of PAI-1 (0.32 ng/ml vs 0.17 ng/ml) (p<0.01) was also increased, whereas synovial levels of A2AP were unchanged: (0.021 μg/ml vs 0.021 μg/ml) (p=0.15). Enhanced uPA production was confirmed in human stimulated synovial fibroblast cultures and elevated levels of plasmin were confirmed harmful to human cartilage tissue explants. In this study we demonstrate that haemarthrosis in haemophilic mice induces synovial uPA expression and results in an increase in synovial plasmin levels, making the joint more vulnerable to prolonged and subsequent bleedings, and adding directly to cartilage damage.

BACKGROUND & AIMS:

Hepatitis C virus (HCV) infection is a major cause of liver cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to confirm the potential of HCVcc as an HCV particle vaccine.

METHODS:

HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naïve Huh7.5.1 cells and urokinase-type plasminogen activator-severe combined immunodeficiency mice (uPA(+/+)-SCID mice) given transplants of human hepatocytes (humanized livers).

RESULTS:

Antibodies against HCV envelope proteins were detected in the sera of immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of iHCV-IgG into uPA(+/+)-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV.

CONCLUSIONS:

Inactivated HCV particles derived from cultured cells protect chimeric liver uPA(+/+)-SCID mice against HCV infection, and might be used in development of a prophylactic vaccine.

This study investigated the antimetastatic activity of four Hericium erinaceus edible mushroom extracts using CT-26 murine colon carcinoma cells as an indicator of inhibition of cell migration to the lung. Hot water (HWE) and microwaved 50% ethanol (MWE) extracts of H. erinaceus strongly elicited cancer cell death through apoptosis and inhibited metastasis of cancer cells to the lungs by 66% and 69%, respectively. HWE and MWE reduced the expression of matrix metalloproteinases MMP-2 and MMP-9 in cells and their activities in culture media. Urokinase-type plasminogen activator (u-PA), another extracellular matrix (ECM)-degrading proteinase, also showed decreased protein expression. In CT-26 cells, HWE and MWE down-regulated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylations. The reduced phosphorylations seem to cause reduction of activity of the MMPs, thereby blocking migration and invasion of cells. Dietary administration of HWE and MWE reduced the formation of tumor nodules in the lung by about 50% and 55%, respectively, and prevented increases in lung weight caused by cancer cell metastasis. These results demonstrate the effectiveness of HWE and MWE as beneficial antimetastatic agents, targeting their upstream signaling molecules for mediating the expression of the ECM-degrading proteinases. Acidic and alkaline extracts were not bioactive. Bioactivity seems to be related to composition. H. erinaceus edible mushrooms have the potential to serve as a health-promoting functional food.

Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53 binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.

A small subset of mammary tumor initiating cells (also known as breast cancer stem cells; bCSC), characterized by expression of different markers [CD44high/CD24low/epithelial specific antigen(ESA)+], aldehyde dehydrogenase-1 (ALDH1) activity, and ability to form mammospheres under ultra-low attachment culture conditions, are suspected to evade conventional therapies leading to disease recurrence. Elimination of both therapy-sensitive epithelial tumor cells and therapy-resistant bCSC is therefore necessary for prevention of breast cancer. We have shown previously that a nontoxic small-molecule constituent of edible cruciferous vegetables (benzyl isothiocyanate; BITC) inhibits mammary cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice by causing epithelial tumor cell apoptosis. The present study demonstrates efficacy of BITC against bCSC in vitro and in vivo. Mammosphere formation frequency and CD44high/CD24low/ESA+ and/or ALDH1+ populations in cultured MCF-7 (estrogen-receptor positive) and SUM159 (triple-negative) human breast cancer cells were decreased significantly in the presence of plasma achievable concentrations of BITC. BITC administration in the diet (3 μmol BITC/g diet for 29 weeks) resulted in a marked decrease in bCSC in the MMTV-neu mice tumors in vivo. Overexpression of full-length Ron as well as its truncated form (sfRon), but not urokinase-type plasminogen activator receptor, conferred near complete protection against BITC-mediated inhibition of bCSC in MCF-7 cells. The BITC treatment downregulated protein levels of Ron and sfRon in cultured breast cancer cells and in tumor xenografts. Ron overexpression resulted in up-regulation of bCSC-associated genes Oct-4, SOX-2, and Nanog. In conclusion, the present study indicates that BITC treatment eliminates bCSC in vitro and in vivo.

Resistance to cisplatin-based chemotherapy is responsible for the majority of deaths from head and neck squamous cell carcinoma (HNSCC). In this study, using genome-wide gene expression analysis to investigate potential molecular mediators of HNSCC chemoresistance, we identified SERPINB2, a known inhibitor of extracellular serine proteinase urokinase-type plasminogen activator (uPA), as an important candidate. Whereas SERPINB2 is known to function as a suppressor of uPA molecular cascades, many of which play important roles in tumor invasion and metastasis, a role for SERPINB2 in cancer drug resistance has not been examined. By using quantitative real-time PCR and Western blot analysis, we determined that SERPINB2 mRNA and protein levels correlated with chemoresistance in HNSCC cell lines, and significantly lower SERPINB2 expression levels were observed in two cisplatin resistant HNSCC subclones compared to their isogenic drug-sensitive parental lines. Immunohistochemical analysis of HNSCC tumor tissues from patients treated with neoadjuvant cisplatin-based chemotherapy (n = 67 cases) revealed a significant association between SERPINB2 protein levels, tumor differentiation and patient relapse. Moreover, SERPINB2 down-regulation was a strong predictor of reduced overall survival in patients with HNSCC who received cisplatin-based chemotherapy (P = 0.001, log rank test). Studies using either siRNA-mediated down-regulation or forced over-expression of SERPINB2 in HNSCC cell lines confirmed a functional role for SERPINB2 in drug resistance. The findings were further supported using chemical inhibitors of STAT3 activity (a downstream effecter of uPAR signaling pathway), showing that STAT3 suppression altered HNSCC cell line cisplatin sensitivity. This is the first report on a role for SERPINB2 in acquired resistance to cisplatin in patients with HNSCC. © 2013 Wiley Periodicals, Inc.

BACKGROUND:

Semaphorin 5A, a member of the semaphorin family, was originally identified as an axonal guidance factor functioning during neuronal development. Previously, we showed that the expression of semaphorin 5A might contribute to the metastasis of gastric cancer. However, less information is currently available as to the involvement of uPA in the semaphorin 5A-induced metastasis and invasion of gastric cancer cells.

AIM:

The present study was designed to test whether semaphorin 5A mediates the invasion and metastasis of gastric cancer via PI3K/Akt/uPA signaling.

METHODS:

The semaphorin 5A-overexpressing cell was established from the gastric cancer cell line AGS. The effect of semaphorin 5A on the expression of uPA was evaluated by ELISA and Western blotting as well as RT-PCR assays, respectively. Synthetic or natural inhibitors and dominant-negative mutants were used to determine the hierarchical relationship between semaphorin 5A, PI3K/Akt and uPA in the invasion and metastasis of gastric cancer.

RESULTS:

Overpression of semaphorin 5A enhanced the expression of uPA, and synthetic or natural inhibitors of uPA abolished semaphorin 5A-induced cell migration and invasion. Semaphorin 5A overexpression promoted the phosphorylation of Akt. Blocking effects of PI3K/Akt using pharmacologic inhibitors, dominant-negative mutants abolished the ability of semaphorin 5A to induce uPA expression and cell invasion and migration.

CONCLUSION:

Semaphorin 5A could promote invasion and metastasis of gastric cancer through the PI3K/Akt/uPA signal transduction pathway. Semaphorin 5A and its regulated molecules could be the potential targets for cancer therapy.

Abstract 1. Human chimeric mice (h-PXB mice) having humanized liver, constructed by transplantation of human hepatocytes, were evaluated as an experimental model for predicting human drug metabolism. Metabolism of zaleplon in h-PXB mice was compared with that in rat chimeric mice (r-PXB mice) constructed by transplantation of rat hepatocytes. 2. Zaleplon is metabolized to 5-oxo-zaleplon by aldehyde oxidase and to desethyl-zaleplon by cytochrome P450 (CYP3A4) in rat and human liver preparations. 3. Liver S9 fraction of h-PXB mice metabolized zaleplon to 5-oxo-zaleplon and desethyl-zaleplon in similar amounts. However, liver S9 fractions of r-PXB and control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice predominantly metabolized zaleplon to desethyl-zaleplon. 5-Oxo-zaleplon was detected as a minor metabolite. 4. Oxidase activity of h-PXB mouse liver cytosol toward zaleplon was about 10-fold higher than that of r-PXB or control mice. In contrast, activities for desethyl-zaleplon formation were similar in liver microsomes from these mice, as well as rat and human liver microsomes. 5. In vivo, the level of 5-oxo-zaleplon in plasma of h-PXB mice was about 7-fold higher than that in r-PXB or control mice, in agreement with the in vitro data. Thus, aldehyde oxidase in h-PXB mice functions as human aldehyde oxidase, both in vivo and in vitro. 6. In contrast, the plasma level of desethyl-zaleplon in r-PXB and control mice was higher than that in h-PXB mice. 7. These results suggest h-PXB mice with humanized liver could be a useful experimental model to predict aldehyde oxidase- and CYP3A4-mediated drug metabolism in humans.

Quercetin, a principal flavanoid compound in onions, has been shown to possess a wide spectrum of pharmacological properties, including anticancer activities. Our earlier study showed that quercetin induced cytotoxic effects on SAS human oral cancer cells. In this study, we found that quercetin significantly reduced wound closure of SAS cells in culture plates after 12- and 24-h treatments. Results indicated that quercetin inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, as measured by western blotting and gelatin zymography. The results from western blotting also showed that quercetin reduced the protein levels of MMP-2, -7, -9 and -10, vascular endothelial growth factor (VEGF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, inductible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), urokinase-type plasminogen activator (uPA), phosphatidylinositide-3 kinases (PI3K), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IKBα), IKB-α/β, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor kinase, alpha/beta (p-IKKα/β), focal adhesion kinase (FAK), son of sevenless homolog-1 (SOS1), growth factor receptor-bound protein-2 (GRB2), mitogen-activated protein kinase kinase kinase-3 (MEKK3), MEKK7, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, c-Jun N-terminal kinase 1/2 (JNK1/2), p38, p-p38, Jun proto-oncogene (c-JUN) and p-c-JUN but it did not affect Ras homolog gene family, member A (RhoA), Protein kinase C (PKC) and rat sarcoma viral oncogene homolog (RAS) in SAS cells. Confocal laser microscopy also showed that quercetin promoted the expressions of RhoA and Rho-associated, coiled-coil containing protein kinase-1 (ROCK1), but inhibited the expression of NF-κB p65 in SAS cells. It is concluded from these data that inhibition of migration and invasion of SAS cells by quercetin is associated with the down-regulation of PKC and RhoA by blocking MAPK and PI3K/AKT signaling pathways and NF-κB and uPA, resulting in inhibition of MMP-2 and MMP-9 signaling.