Matrix metalloproteinase­1 and microRNA­486­5p in urinary exosomes can be used to detect early lung cancer: A preliminary report.

The present study describes a novel molecular-genetic method suitable for lung cancer (LC) screening in the work-place and at community health centers. Using urinary-isolated exosomes from 35 patients with LC and 40 healthy volunteers, the expression ratio of MMP-1/CD63, and the relative expression levels of both microRNA (miRNA)-21 and miRNA-486-5p were measured. MMP-1/CD63 expression ratio was significantly higher in patients with LC than in the healthy controls {1.342 [95% confidence interval (CI): 0.890-1.974] vs. 0.600 (0.490-0.900); P<0.0001}. The relative expression of miRNA-486-5p in male healthy controls was significantly different from that in female healthy controls, whereas there was no significant difference in miRNA-21. Receiver operating characteristic curve (ROC) analysis of MMP-1/CD63 showed 92.5% sensitivity and 54.3% specificity, whereas miRNA-486-5p showed 85% sensitivity and 70.8% specificity for men, and 70.0% sensitivity and 72.7% specificity for women. The logistic regression model used to evaluate the association of LC with the combination of MMP-1/CD63 and miRNA-486-5p revealed that the area under the ROC curve was 0.954 (95% CI: 0.908-1.000), and the model had 89% sensitivity and 88% specificity after adjusting for age, sex and smoking status. These data suggested that the combined analysis of MMP-1/CD63 and miRNA-486-5p in urinary exosomes may be used to detect patients with early-stage LC in the work-place and at community health centers, although confirmational studies are warranted.

ALDH1 and SALL4 Expression in Cell Block Samples from Patients with Lung Adenocarcinoma and Malignant Pleural Effusion.

Malignant pleural effusion (MPE) can accompany advanced lung adenocarcinoma. Recent studies suggest that MPE could contain a heterogeneous subpopulation of cells with stem-like properties, such as tumorigenicity and self-renewal, indicating that they could be the source of metastasis. Although previous studies analyzed the correlation between cancer stem cell (CSC) marker expression and clinical outcomes using lung cancer tissues, investigations regarding the association of MPE with CSC marker expression are limited. We performed immunohistochemistry to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and Sal-like 4 (SALL4) in 46 cell block samples of MPE from patients with lung adenocarcinoma. ALDH1-positive and SALL4-positive cancer cells in MPE were detected in 30 (65.2%) and 21 samples (45.7%), respectively. Cluster formation was detected in 26 samples (56.5%). The number of clusters was significantly higher in ALDH1-positive/SALL4-negative samples. SALL4 expression was inversely correlated with the cluster ratio (r = -0.356) and positively associated with the Ki-67 index (r = 0.326), suggesting that MPE cells with high SALL4 expression comprised the proliferative subpopulation. In conclusion, we demonstrated that MPE contains an ALDH1-positive/SALL4-negative subpopulation exhibiting cluster formation and a SALL4-positive proliferative subpopulation.

Novel breast cancer screening: combined expression of miR-21 and MMP-1 in urinary exosomes detects 95% of breast cancer without metastasis.

Serum and tissue miR-21 expression in patients with breast cancer (BC) is a useful biomarker for cancer diagnosis, progression, and treatment. Matrix metalloproteinase-1 (MMP-1) is also important in breast cancer carcinogenesis. However, miR-21 and MMP-1/CD63 in urine exosomes in these patients have not been examined. Urine samples were collected from patients with BC and 26 healthy females. Urinary exosomes were isolated and confirmed by western blotting with anti-CD63 antibody and electron microscopy observation. MiR-21 and MMP-1/CD63 expression was examined by quantitative RT-PCR and western blotting, respectively. Patients with very early stage breast cancer were evaluated. MiR-21 expression in the patients was 0.26 [95% CI: 0.20-0.78], which was significant lower than in the 26 controls (1.00 [95% CI: 1.01-3.37], p = 0.0947). MMP-1/CD63 expression in patients was significantly higher than in controls (1.74 [95% CI: 0.86-5.08] vs 0.535 [95% CI: -0.01-2.81], p = 0.0001). Sensitivity and specificity were 0.708 and 0.783 for miR-21 and 0.792 and 0.840 for MMP-1/CD63, respectively. Sensitivity and specificity of combined expression were 95% and 79%, respectively. The sensitivity of MMP-1/CD63 expression in urinary exosomes was better than that of miR-21 expression. Thus, miR-21 and MMP/CD63 may be useful markers for BC screening.

Serum matrix metalloproteinase-1 level represents disease activity as opposed to fibrosis in patients with histologically proven nonalcoholic steatohepatitis.

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is prevalent in both economically developed and developing countries. Twenty percent of NASH progresses to cirrhosis with/without hepatocellular carcinoma, and there is an urgent need to find biomarkers for early diagnosis and monitoring progression of the disease. Using immunohistochemical and immunoelectron microscopic examination we previously reported that expression of matrix metalloproteinase-1 (MMP-1) increased in monocytes, Kupffer cells and hepatic stellate cells in early stage NASH. The present study investigated whether serum MMP-1 levels reflect disease activity and pharmaceutical effects in NASH patients. METHODS: We measured the serum levels of MMPs, tissue inhibitors of metalloproteinases (TIMPs), and several cytokines/chemokines in patients with histologically proven early and advanced stages of NASH and compared them with those in healthy controls. RESULTS: Serum MMP-1 levels in stage 1 fibrosis, but not in the more advanced fibrosis stages, were significantly higher than in healthy controls (P=0.019). There was no correlation between serum MMP-1 level and fibrosis stage. Serum MMP- 1 levels in NASH patients represented disease activity estimated by serum aminotransferase values during the follow-up period. In contrast, MMP-2, MMP-9 and TIMPs did not change with disease activity. Consistent with the finding that MMP-1 is expressed predominantly in monocytes and Kupffer cells, serum levels of monocyte chemotactic protein-1 and granulocyte-colony stimulating factor were significantly increased in NASH with stage 1 fibrosis. CONCLUSIONS: These results suggest that serum MMP-1 levels represent disease activity and may serve as a potential biomarker for monitoring the progression of NASH.

Lung Adenocarcinoma in Never Smokers: Problems of Primary Prevention from Aspects of Susceptible Genes and Carcinogens.

Global statistics estimate that approximately 25% of patients with lung cancer are never smokers. We suggest that genes related to susceptibility to metabolic syndrome were present among those related to susceptibility to lung adenocarcinoma (AC) in never smokers. There are many questions concerning lung AC in never smokers, which is increasing in incidence, with female predominance, good prognosis, unique genes related to susceptibility and good response to treatment with specific agents. The purpose of this review was to investigate the carcinogenesis of lung AC in never smokers focusing on genes related to susceptibility to lung AC and carcinogens, including environmental factors. In order to clarify the carcinogenesis of lung AC in never smokers, the definition of never smokers, survey of environmental tobacco smoke, the presence of the physical characteristics of metabolic syndrome, and other carcinogens should be investigated for primary prevention of lung AC.

Genes associated with succeptibility to lung adenocarcinoma among never smokers suggest the mechanism of disease.

Global statistics estimate that 15% of all cases of lung cancer in men and 53% in women are not attributable to smoking, and these data indicate that worldwide, approximately 25% of patients with lung cancer are never smokers. The etiology of lung cancer is disputed. The present study reviews the genes associated with susceptibility to lung cancer among never smokers and suggests possibilities for the involvement of metabolic syndrome. The environment appears to have changed the genes susceptible to lung cancer. Classical genes associated with lung cancer are decreasing and novel emerging genes may reflect changes in lifestyle. We provide evidence that the genes associated with susceptibility to lung cancer in never smokers are very similar to those reported in patients with metabolic syndrome, and that simply quitting smoking is not sufficient as the primary means of preventing lung cancer.

Fibrogenesis and Carcinogenesis in Nonalcoholic Steatohepatitis (NASH): Involvement of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinase (TIMPs).

Nonalcoholic steatohepatitis (NASH) is emerging worldwide because life-styles have changed to include much over-eating and less physical activity. The clinical and pathophysiological features of NASH are very different from those of HBV- and HCV-chronic liver diseases. The prognosis of NASH is worse among those with nonalcoholic fatty liver diseases (NAFLD), and some NASH patients show HCC with or without cirrhosis. In the present review we discuss fibrogenesis and the relationship between fibrosis and HCC occurrence in NASH to clarify the role of MMPs and TIMPs in both mechanisms. Previously we proposed MMP and TIMP expression in the multi-step occurrence of HCC from the literature based on viral-derived HCC. We introduce again these expressions during hepatocarcinogenesis and compare them to those in NASH-derived HCC, although the relationship with hepatic stem/progenitor cells (HPCs) invasion remains unknown. Signal transduction of MMPs and TIMPs is also discussed because it is valuable for the prevention and treatment of NASH and NASH-derived HCC.

Novel strategies for hepatocellular carcinoma based on MMPs science.

Hepatocellular carcinoma (HCC) is a cancer with extremely poor prognosis. This review discusses the pathological characteristics of multi-step hepatocarcinogenesis, tumor growth, invasion and metastasis, the expression of matrix metalloproteinases (MMPs) and their inhibitors via signal transduction in relation to dedifferentiation of hepatoma cells. It introduces the reports on anti-cancer agents in the field of MMP science, and finally describes novel strategies for the early stages of HCC in relation to cancer stem cells.

Introduction: MMPs, ADAMs/ADAMTSs research products to achieve big dream.

Matrix metalloproteinases (MMPs) are known to participate in cancer invasion and metastasis. Copious research on MMPs and their inhibitors has promoted the understanding of the mechanisms of cancer invasion and metastasis as well as the development of effective drugs for cancer treatment. This review discusses the classification of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in relation to tumor growth and invasion mechanisms via signal transduction, followed by the possibilities for cancer treatment. The review focuses especially on the development of anti-cancer agents in the field of MMP science.

Matrix metalloproteinase-13 promotes recovery from experimental liver cirrhosis in rats.

OBJECTIVE: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. METHODS: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl(4)) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl(4) injection. The role of MMP-13 in stably expressing cell lines was also analyzed. RESULTS: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. CONCLUSION: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.

ELISA measurement for urinary 3-hydroxyproline-containing peptides and its preliminary application to healthy persons and cancer patients.

BACKGROUND/AIM: We reported that endogenous urinary 3-hydroxyproline (3-Hyp) is useful for cancer screening because cancer invasion involves the destruction of basement membrane. A simple and sensitive assay is desired. PATIENTS AND METHODS: An ELISA method using a specific antibody against a synthetic peptide of 10 amino acids including 3-Hyp corresponding to the amino acid sequences of collagen type IV alpha chain was applied to urine samples from 180 healthy controls and 22 cancer patients. RESULTS: The values in controls were 2.44+/-1.90 (SD) mg peptide/g creatinine for 52 men and 2.87+/-2.01 for 128 women, while the values in 22 cancer patients were very low at 0.110+/-0.137 (p<0.001). DISCUSSION: The discrepancy in the data between our previous and present studies is based on the difference of targets measured. 3-Hyp-containing peptides in cancer patients might be destroyed by the elevated peptidase levels found in these patients. CONCLUSION: This ELISA assay may be useful for cancer screening.

Additional candidates to conventional genes susceptible for lung cancer and changing trend in Japan.

The polymorphism of CYP1A1*2A or CYP1A1*2B, and the linkage of CYP1A1*2A, CYP1A1*2B, GSTM1 and GSTT1 polymorphisms have been established as susceptible genes or gene-gene interactions of tobacco-related lung cancer. New candidate genes susceptible for lung cancer such as NQO1 (NAD(P)H:quinine oxidoreductase), NAT2 (N-acetyltransferase 2), and several others have been reported. In the present review we focus on new candidate genes susceptible for lung cancer, then examine all Japanese references by meta-analysis on susceptible genes over the past 20 years, and discuss whether new candidates and changing trend in Japan could be caused by environmental change.

Activation of two distinct MAPK pathways governs constitutive expression of matrix metalloproteinase-1 in human pancreatic cancer cell lines.

Elevated matrix metalloproteinase-1 (MMP-1) expression is known to correlate with poor prognosis of pancreatic cancer. We investigated the molecular mechanisms of constitutive expression of MMP-1 in pancreatic cancer cell lines. Expression of MMP-1 mRNA and protein as well as its enzymatic activity were observed in three pancreatic cancer cell lines. Transient transfection assays of two MMP-1 promoter/luciferase constructs (full-length 4.4-kb or proximal 0.6-kb region) showed high levels of transcription in pancreatic cancer cells compared with non-MMP-1 producing cells. The 0.6-kb promoter region of MMP-1 gene contained three activator protein-1 (AP-1) sites and the strong AP-1 activity was detected by electrophoretic mobility shift assays (EMSAs). In these cells, production and phosphorylation of c-Jun were commonly observed. Phosphorylated c-Jun NH2-terminal kinase (p-JNK) and activator transcription factor-2 (p-ATF-2) were also detected in two of the three cell lines. Phosphorylated extracellular signal-regulated kinase (p-ERK) was observed in one. The promoter activity, AP-1-binding activity and MMP-1 production were suppressed by a specific inhibitor of JNK or MEK. K-ras mutation, reported to be present in three cell lines used, is known to activate JNK and ERK pathways. Considering the facts together, our results revealed that activation of JNK/AP-1 or ERK/AP-1 pathway plays crucial roles in constitutive transactivation of MMP-1 in these cancer cells. This study contributes to provide new insights into strategies for inhibiting tumor cell invasion in pancreatic cancer.

Negligible contribution of bone marrow-derived cells to collagen production during hepatic fibrogenesis in mice.

BACKGROUND & AIMS: Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients. METHODS: BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of alpha2(I) collagen gene (COL1A2) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates. RESULTS: The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli. CONCLUSIONS: By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Glycyrrhizin and its metabolite inhibit Smad3-mediated type I collagen gene transcription and suppress experimental murine liver fibrosis.

AIMS: Glycyrrhizin has been widely used for the treatment of chronic hepatitis C. It decreases the serum levels of aminotransferases, and suppresses progression of liver fibrosis as well as subsequent occurrence of hepatocellular carcinoma. Although previous studies have shown that glycyrrhizin and its metabolite inhibit collagen gene expression, its underlying mechanisms are virtually unknown. This study was aimed to explore molecular mechanisms responsible for the inhibitory effect of glycyrrhizin on type I collagen gene transcription. MAIN METHODS: Effects of glycyrrhizin and its metabolite, glycyrrhetinic acid, on collagen promoter activity were examined by using transgenic reporter mice harboring alpha2(I) collagen gene (COL1A2) promoter. Their effects on the TGF-beta/Smad signaling pathway were studied by cell transfection assays and immunofluorescence studies using cultured hepatic stellate cells. KEY FINDINGS: Administration of glycyrrhizin or its metabolite, glycyrrhetinic acid, significantly suppressed COL1A2 promoter activation and progression of liver fibrosis induced by repeated carbon tetrachloride injections. In cultured hepatic stellate cells, glycyrrhetinic acid, but not glycyrrhizin, inhibited type I collagen synthesis mostly at the level of gene transcription. This inhibitory effect of glycyrrhetinic acid was abolished by a mutation introduced into a Smad3-binding region within the COL1A2 promoter. Glycyrrhetinic acid did not affect gene expression of TGF-beta receptors or Smad proteins, but inhibited nuclear accumulation of Smad3 in activated hepatic stellate cells. In addition to those direct inhibitory effects on COL1A2 transcription, glycyrrhetinic acid also suppressed activation of quiescent hepatic stellate cells in primary culture. SIGNIFICANCE: The results provide a molecular basis for the anti-fibrotic effect of glycyrrhizin treatment.

Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.

UNLABELLED: Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)-derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)-expressing BM cells. The distribution and characteristics of EGFP-positive (EGFP(+)) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP(+) cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP-negative (EGFP(-)) liver resident cells, expressed matrix metalloproteinase (MMP)-13 and MMP-9. Whereas MMP-13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP-9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony-stimulating factor (G-CSF) treatment significantly enhanced migration of BM-derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G-CSF and HGF synergistically increased MMP-9 expression along the fibrous septa. CONCLUSION: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis.

[A survey of subjective symptoms and life-style in junior high school students].

PURPOSE: A questionnaire survey was conducted to examine a relationship between indefinite complaints and life styles in junior high school students and to apply the results to life guidance. METHODS: Twenty nine public junior high schools were selected by random sampling of 13 cities and 3 towns in Kanagawa Prefecture. A self-reported questionnaire was supplied to all subjects, containing 100 life-style related items, including a simplified CMI (Cornel Medical Index) questionnaire. RESULTS: The number of students who were evaluated as "point medical examination" based on physical symptoms of CMI and "disorder" based on the mental and physical symptom increased with the grade for both males and females. The students who reported "the life rhythm was always irregular" tended to be in the group evaluated as "disorder" or "point medical examination". The life rhythm was related to bedtime, sleeping hours and eating habits. It was observed that the students whose daily habits were always irregular had late bedtime, short sleeping hours and an irregular eating habit. As a result of the quantification method for the second type of analysis, it was found that sleeping time was the most related factor to the life rhythm. Furthermore 90% and more of students reported they did not consider nutritional balance with regard to their eating habits. CONCLUSION: It was suggested that those students who had irregular life rhythm needed sufficient sleep and nutrition guidance to maintain their health.

All-trans retinoic acid down-regulates human albumin gene expression through the induction of C/EBPbeta-LIP.

ATRA (all-trans retinoic acid), which is a major bioactive metabolite of vitamin A and a potent regulator of development and differentiation, mediates down-regulation of the human albumin gene. However, the mechanism of ATRA-mediated down-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrate that ATRA causes a marked decrease in the activity of the albumin promoter, the region between nt -367 and -167 from the transcription start site, where C/EBP (CCAAT/enhancer-binding protein)-binding sites are tightly packed, is indispensable for ATRA-mediated down-regulation. ChIP (chromatin immunoprecipitation) assays revealed that in vivo binding of C/EBPalpha to the region markedly decreases upon incubation with ATRA, whereas ATRA treatment marginally increases the recruitment of C/EBPbeta. We found that ATRA has the ability to differentially and directly induce expression of a truncated isoform of C/EBPbeta, which is an LIP (liver-enriched transcriptional inhibitory protein) that lacks a transactivation domain, and to increase the binding activity of C/EBPbeta-LIP to its response element. Overexpression of C/EBPbeta-LIP negatively regulates the endogenous expression of albumin, as well as the activity of the albumin promoter induced by C/EBP transactivators such as C/EBPalpha and full-length C/EBPbeta. In conclusion, we propose a novel model for down-regulation of the albumin gene, in which ATRA triggers an increase in the translation of C/EBPbeta-LIP that antagonizes C/EBP transactivators by interacting with their binding sites in the albumin promoter.