Shigenari Hozawa

Side population of pancreatic cancer cells predominates in TGF-β-mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride

BACKGROUND/AIMS Liver fibrosis is a dynamic state between matrix production and degradation. Since our report in 1974, many studies have examined collagenase and liver fibrosis, but not the identification of cells responsible for collagenase production in vivo. The aim of this study was to investigate the gene expression of interstitial collagenase in the progressive and recovery phases of experimental rat liver fibrosis by in situ hybridization. METHODS We examined the gene expression of interstitial collagenase (MMP-13) in the progressive and recovery phase of experimental rat liver fibrosis induced by chronic CCl4 intoxication by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. In order to identify the cells expressing MMP-13 mRNA by in situ hybridization, immunohistochemistry was performed using serial sections. RESULTS In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR, but not by in situ hybridization. The livers of rats treated with CCl4 for 4 weeks showed fatty metamorphosis but no definite fibrosis. Positive signals for MMP-13 mRNA were observed in scattered mesenchymal cells, within lobules which seem to be stellate cells from immunohistochemical staining. Once the fibrosis became prominent, the faint band for MMP-13 mRNA was detected only by RT-PCR and very few signals, if any, by in situ hybridization. On the other hand, in the recovery phase of liver fibrosis, gene expression of MMP-13 was markedly enhanced. Strong positive cells by in situ hybridization were observed mainly at the interface between the resolving fibrous septa and the parenchyma. Overlapping both images of in situ hybridization and immunohistochemical staining with the help of a computer revealed that some positive cells, but not all cells, were stellate cells stained with a-smooth muscle actin antibody. CONCLUSIONS MMP-13 participates in the degradation of newly-formed matrix in the recovery from rat liver fibrosis more than in the remodeling of extracellular matrix for the formation of fibrosis. Hepatic stellate cells play a crucial role in MMP-13 production in the recovery from fibrosis, though not all stellate cells were positive for MMP-13 mRNA. Further investigation into gene expression of MMP-13 in recovery will lead to new strategies for the treatment of liver cirrhosis.

Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat

BACKGROUND/AIMS The aim of this study was to investigate whether both matrix metalloproteinase-2 (MMP-2) and membrane type 1 MMP (MT1-MMP) participate in the spontaneous resolution of liver fibrosis. METHODS Transcription of both genes was examined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Gelatinase activity was investigated by zymography. RESULTS Gene expression by RT-PCR showed that both genes increased in the process of liver fibrosis, then decreased gradually in the recovery phase. ISH revealed that distribution of positive cells changed quickly in the recovery phase. Positive cells were widely seen in the liver, mainly around fibrous septa, in the aggressive phase, but were exclusively observed at the interface between the resolving fibrous band and the parenchyma, then were diffusely located in the lobules in the recovery phase. Main cells expressing both mRNAs seemed to be stellate cells for their morphology, though they did not express characteristic cell markers. Some hepatocytes and Kupffer cells expressed both mRNAs in the recovery phase. Gelatinase activity of MMP-2 increased in the recovery phase of 8-week-treated rat liver by gelatin zymography. CONCLUSIONS The results of present study suggest that both enzymes participate in the destruction of extracellular matrix in coordination with MMP-13.

CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice

BACKGROUND & AIMS Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice. METHODS Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. RESULTS Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. CONCLUSIONS Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Prediction of metabolic syndrome using artificial neural network system based on clinical data including insulin resistance index and serum adiponectin

OBJECTIVE This study aimed to predict the 6-year incidence of metabolic syndrome (MetS) using an artificial neural network (ANN) system and multiple logistic regression (MLR) analysis based on clinical factors, including the insulin resistance index calculated by homeostasis model assessment (HOMA-IR). DESIGN Subjects were recruited from participants in annual health check-ups in both 2000 and 2006. A total of 410 Japanese male teachers and other workers at Keio University, 30-59 years of age at baseline, participated in this retrospective cohort study. MEASUREMENTS Clinical parameters were randomly divided into a training dataset and a validation dataset, and the ANN system and MLR analysis were applied to predict individual incidences. The leave some out cross validation method was used for validation. RESULTS The sensitivity of the prediction was 0.27 for the MLR model and 0.93 for the ANN system, while specificities were 0.95 and 0.91, respectively. Sensitivity analysis employing the ANN system identified BMI, age, diastolic blood pressure, HDL-cholesterol, LDL-cholesterol and HOMA-IR as important predictors, suggesting these factors to be non-linearly related to the outcome. CONCLUSION We successfully predicted the 6-year incidence of MetS using an ANN system based on clinical data, including HOMA-IR and serum adiponectin, in Japanese male subjects.

Pro-inflammatory cytokine-induced matrix metalloproteinase-1 (MMP-1) secretion in human pancreatic periacinar myofibroblasts

Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes in fibrolysis, a process closely related to tissue remodeling. In the present study, we investigated MMP-1 secretion from human pancreatic periacinar myofibroblasts in response to pro-inflammatory cytokines IL-1β and TNF-α. We also attempted to clarify the intracellular signaling pathways mediating the cytokine-induced MMP-1 secretion. MMP-1 secretion was measured by an enzyme-linked immunosorbent assay. MMP-1 molecules were analyzed by Western blotting. MMP-1 mRNA expression was evaluated by Northern blotting. IL-1β and TNF-α stimulated the MMP-1 secretion in a dose- and time- dependent manner. Ninety percent of MMP-1 was secreted as inactive form (pro-MMP-1). The effects of IL-1β and TNF-α were significantly inhibited by PD98059 MEK/ERK inhibitor). In contrast, SB203580 (p38 MAPK inhibitor), GF109203X (PKC inhibitor), and PDTC (NF-ĸB inhibitor) did not alter the MMP-1 secretion induced by IL-1β and TNF-α. These effects were also observed at the mRNA level. In conclusion, in human pancreatic periacinar myofibroblasts, MMP-1 secretion was regulated by the pro-inflammatory cytokines via the MEK/ERK cascade. Thus, human pancreatic periacinar myofibroblasts may play an important role in the remodeling of damaged pancreatic tissue in chronic pancreatitis via MMP-1 secretion.

Transforming growth factor β and Ras/MEK/ERK signaling regulate the expression level of a novel tumor suppressor lefty

Objectives The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-&bgr;) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-&bgr;–dependent Lefty up-regulation. Methods Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-&bgr;–mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal–regulated kinase (ERK) inhibitor LY294002. Results Transforming growth factor &bgr; upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-&bgr;–Lefty pathway. Conclusions Our data suggest that Lefty is a novel TGF-&bgr; target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-&bgr;–Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-&bgr;.

CCR2 knockout exacerbates cerulein-induced chronic pancreatitis with hyperglycemia via decreased GLP-1 receptor expression and insulin secretion.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b(+)Gr-1(low) macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b(+)/Gr-1(-) and CD11b(+)/Gr-1(high) cells, but not CD11b(+)/Gr-1(low) cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b(+)-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

Induction of matrix metalloproteinase-1 gene transcription by tumour necrosis factor α via the p50/p50 homodimer of nuclear factor-κ B in activated human hepatic stellate cells

Background/Aims: Liver injury results in the activation of hepatic stellate cells (HSCs), which in turn produce matrix metalloproteinase (MMP) in response to pro‐inflammatory cytokines for tissue remodelling. This study explored the transcriptional induction of the MMP‐1 gene by tumour necrosis factor‐α (TNF‐α) in HSCs.

Biliary Findings Assist in Predicting Enlargement of Intraductal Papillary Mucinous Neoplasms of the Pancreas

BACKGROUND & AIMS There is controversy over the optimal management strategy for patients with branch-duct type intraductal papillary mucinous neoplasms of the pancreas (BD-IPMNs), precursors to pancreatic cancer. We aimed to identify factors associated with the presence of BD-IPMNs and changes in their diameter. METHODS Two separate analyses were conducted in a cohort of patients who underwent magnetic resonance cholangiopancreatography (MRCP) in a single year (2006). MRCP findings and clinical outcomes of these patients were followed for a maximum of 6 years. We evaluated initial MRCP findings and demographics associated with the presence of BD-IPMNs at baseline and increase in BD-IPMN diameter over time. RESULTS During the follow-up period, 154 patients developed BD-IPMN and 322 patients did not. Older age, diabetes mellitus, gallbladder adenomyomatosis, and absence of gallstones were associated with the presence of BD-IPMNs at baseline. Increases in diameter of BD-IPMNs were associated with 3 baseline factors: BD-IPMN diameter greater than 17 mm, gallbladder adenomyomatosis, and a common bile duct diameter less than 5.5 mm. Patients with BD-IPMNs could be stratified into 4 groups with varying risk for the enlargement of BD-IPMNs over time: those with 3 risk factors (hazard ratio [HR], 11.4; 95% confidence interval [CI], 3.4-37.8), 2 risk factors (HR, 4.7; 95% CI, 1.7-12.8), or 1 risk factor (HR, 3.1; 95% CI, 1.2-8.2) compared with those without risk factors. CONCLUSIONS For patients with BD-IPMNs, careful follow-up evaluation is particularly important for those with BD-IPMN >17 mm in size, common bile duct diameter <5.5 mm, or gallbladder adenomyomatosis.