Masaya Oikawa

Intraductal papillary mucinous neoplasms of the pancreas showing fistula formation into other organs

BackgroundThis study assessed the mechanism of fistula formation in intraductal papillary mucinous neoplasm (IPMN) of the pancreas.MethodsA total of 274 patients with IPMN who had been diagnosed by endoscopic retrograde cholangiopancreatography and endoscopic ultrasonography (EUS) at our center were enrolled. The patients with IPMN which had fistula formation into other organs were investigated retrospectively as to (1) clinical prevalence and the organs penetrated by IPMN, (2) analysis of the mechanism of fistula formation by immunohistopathological study, (3) efficacy of EUS in progression assessment, and (4) prognosis.ResultsAmong the subjects, fistula formation into other organs was observed in 18 patients (6.6%) and into 28 organs. There were 7 patients (39%) in whom multiple organs were penetrated. Of 16 patients who had undergone investigation of the expression of mucin markers, 94% had an intestinal-type tumor. Of 9 patients who had undergone surgery or autopsy, 67% showed mechanical penetration without invasion around the fistula. Only papillary protrusions were seen by EUS in 4 of these patients with noninvasive papillary adenocarcinoma showing mechanical penetration. All 5 patients who had pancreatic parenchymal invasion showed a mass with a mixed-echo pattern in addition to papillary protrusions shown by EUS, corresponding to colloid carcinoma.ConclusionsThere were 2 processes in the development of fistulas in IPMN. Of those patients showing fistula formation, 94% had intestinal-type IPMN, and 67% showed mechanical penetration. Delineation of a mass with the mixed-echo pattern suggested an invasive penetration due to colloid carcinoma.

GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent RhoA activation

The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.

Farnesoid X receptor, hepatocyte nuclear factors 1α and 3β are essential for transcriptional activation of the liver-specific organic anion transporter-2 gene

BackgroundWe isolated the human liver-specific organic anion transporter gene, LST-2 (OATP8/SLCO1B3), which is exclusively expressed in the basolateral membrane of the hepatocytes. In this study, we analyzed the transcriptional regulation of the LST-2 gene in hepatocyte-derived cells and the effect of bile acid.MethodsTranscriptional activity of the LST-2 gene was measured using a human LST-2 promoter–luciferase reporter plasmid under various concentrations of bile acids. Electrophoresis mobility shift assays of farnesoid X receptor (FXR), hepatocyte nuclear factor (HNF) 1α, and HNF3β were performed.ResultsLuciferase analysis showed that the 5′-flanking region from −180 to −20 bp is responsible for LST-2 transcriptional activity. By site-directed mutation analysis, it was revealed that the consensus binding sites for FXR, HNF1α, and HNF3β play important roles in the transcriptional activity of the LST-2 gene. By electrophoresis mobility shift assay, we observed specific protein–DNA complexes of FXR, HNF1α, and HNF-3β. Luciferase activity was increased fivefold when chenodeoxycholate or deoxycholate were added. Northern blot analyses revealed that the expression of LST-2 was increased by addition of chenodeoxycholate or deoxycholate in a dose-dependent manner.ConclusionsThis study demonstrated that the transcription of the LST-2 gene is regulated by three transcription factors, FXR, HNF1α, and HNF3β. HNF1α and HNF3β might contribute to its liver-specific expression, and FXR might play a role in its transcriptional activation by bile acids.

Bile acids repress E‐cadherin through the induction of Snail and increase cancer invasiveness in human hepatobiliary carcinoma

Although some kinds of bile acids have been implicated in colorectal cancer development, the mechanism of cancer progression remains unexplored in hepatobiliary cancer. From our personal results using complementary DNA microarray, we found that chenodeoxycholic acid (CDCA) induced Snail expression in human carcinoma cell lines derived from hepatocellular carcinoma and cholangiocarcinoma. Snail expression plays an important role in the regulation of E‐cadherin and in the acquisition of invasive potential in many types of human cancers including hepatocellular carcinoma. We found that CDCA and lithocholic acid (LCA) induced Snail expression in a concentration‐dependent manner and down‐regulated E‐cadherin expression in hepatocellular carcinoma and cholangiocarcinoma cell lines. Moreover, Snail short interference RNA (siRNA) treatment reduced the down‐regulation of E‐cadherin by CDCA or LCA. Luciferase analysis demonstrated that the promoter region from –111 to –24 relative to the transcriptional start site was necessary for this induction and, at least in part, nuclear factor Y (NF‐Y) and stimulating protein 1 (Sp1) might be an inducer of Snail expression in response to bile acids. In addition, using an in vitro wound healing assay and invasion assay, we observed that CDCA and LCA induced cell migration and invasion. These results suggest that bile acids repress E‐cadherin through the induction of transcription factor Snail and increase cancer invasiveness in human hepatocellular carcinoma and cholangiocarcinoma. Inhibition of this bile acid‐stimulated pathway may prove useful as an adjuvant in the therapy of hepatocellular carcinoma. (Cancer Sci 2008; 99: 1785–1792)

Inhibition of heme oxygenase ameliorates sepsis-induced liver dysfunction in rats.

Abstract.Purpose: The disintegration of heme produces carbon monoxide (CO), a known vasodilator, which is catalyzed by heme oxygenase (HO). This study aimed to clarify the effect of HO inhibition on septicrat livers using two types of HO inhibitors; Sn-protoporphyrin (Sn-PP) and Zn-protoporphyrin (Zn-PP). Methods: Sepsis was induced in male Sprague-Dawley rats by cecal ligation and puncture (CLP). Either NaOH or HO inhibitors were injected intraperitoneally; first 18 h prior to CLP, then immediately after CLP. The animals were killed 12 and 24 h after CLP and the liver tissue and plasma were harvested. Results: Using Northern blotting, we found that mRNA of the stress-inducible isozyme, HO-1, was dramatically induced 12 h after CLP. Administering the HO inhibitors, Sn-PP and Zn-PP (5 μmol/kg), induced a significant inhibition of the elevation of aspartate aminotransferase plasma levels, the elevation of cyclic guanosine monophosphete (cGMP) in the liver tissue, and the increase in the sinusoidal space ratio, 24 h after CLP. Both Sn-PP and Zn-PP decreased the mortality rate 24 h after CLP compared with normal saline. Conclusions: CO produced by excessively induced HO-1 after CLP promotes an immoderate dilation of the sinusoidal space through the up-regulation of cGMP, resulting in liver dysfunction. Therefore, administering HO inhibitors at appropriate doses could be beneficial for the amelioration of sepsis-induced liver dysfunction.

Silencing of LRRFIP1 reverses the epithelial–mesenchymal transition via inhibition of the Wnt/β-catenin signaling pathway

The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial-mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.

GC-binding factor 2 interacts with dishevelled and regulates Wnt signaling pathways in human carcinoma cell lines.

GC‐binding factor 2 (GCF2), a transcriptional repressor that decreases the activity of several genes is capable of binding directly to the GC‐rich sequence of the EGFR promoter and repressing the transcriptional activity of EGFR. In addition to its function as a transcriptional repressor, GCF2 can directly interact with other proteins such as flightless‐1 (Fli‐1). Many previous findings pertaining to the function of Fli‐1 have suggested a role for fli‐1 in providing a direct link between molecules involved in signal transduction pathways and the actin cytoskeleton. We hypothesized that GCF2, together with Fli‐1, plays a role in regulating cytoskeleton function, cell migration, and/or morphology. In our study, we observed that GCF2 is crucial for the activation of RhoA, a small GTPase that plays a key role in the regulation of the actin cytoskeleton. RhoA was markedly inactivated as a result of the decreased expression of GCF2. Co‐immunoprecipitations were subsequently performed to further investigate the mechanism for the repressive function. We identified dishevelled (Dvl), which is the key mediator for the Wnt pathway, as a binding partner with GCF2. These results strongly suggest that GCF2 plays a role in the Wnt‐noncanonical planar cell polarity (PCP) signaling pathway. Consequently, GCF2 may regulate the cytoskeleton or migration via Dvls and RhoA.

Peroxisome proliferator-activated receptor α activates cyclooxygenase-2 gene transcription through bile acid transport in human colorectal cancer cell lines

BackgroundEvidence is accumulating that bile acids are involved in colon cancer development, but their molecular mechanisms remain unexplored. Bile acid has been reported to be associated with induction of the cyclooxygenase-2 (COX-2) gene. Because the human liver-specific organic anion transporter-2 (LST-2/OATP8/OATP1B3) is expressed in gastrointestinal cancers and might transport bile acids to the intracellular space, we studied the molecular mechanisms by which bile acids induce the transcription of COX-2, and the role of LST-2 in colonic cell lines.MethodsTranscriptional activity of COX-2 was measured using a human COX-2 promoter-luciferase assay under various concentrations of bile acids. Electrophoresis mobility shift assays (EMSAs) for peroxisome proliferators-activated receptor (PPAR) α and cyclic AMP responsive element (CRE) were performed.ResultsThe COX-2 promoter was induced by lithocholic acid (LCA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA). Deletion and site-directed mutation analyses showed that CRE is the responsive element for LCA. An adenovirus expression system revealed that LST-2 is responsible for induction of COX-2. By EMSA using oligonucleotides of CRE, we observed formation of a specific protein-DNA complex, which was inhibited by a specific antibody against PPARα and CRE. A PPARα-specific agonist induced transcription of COX-2.ConclusionThese results indicate that COX-2 is transcriptionally activated by the addition of LCA, CDCA, and DCA and that LST-2 plays an important role by transporting bile acid to the intracellular space. Moreover, LCA-dependent COX-2 gene activation consists of a transcriptional complex including PPARα and CRE-binding protein. Thus, this induction of COX-2 may participate in carcinogenesis and progression of colorectal cancer cells.

Clinical outcome of the laparoscopic surgery for stage II and III colorectal cancer

BackgroundLaparoscopic colorectal cancer surgery has become widely accepted recently. However, the oncological validity of this surgery has not yet been well analyzed, especially for advanced cancer. The aim of this study is to assess the clinical outcome of laparoscopic surgery for stage II/III colorectal cancer in our hospital.Patients and methodsBetween June 1999 and August 2006, 321 patients underwent laparoscopic colorectal cancer surgery in our hospital; of those 121 cases whose pathological findings revealed stage II/III were included in this study. Among these cases, we assessed a short-term outcome and a medium-term outcome in terms of survival evaluation.ResultsThe male:female ratio was 73:48, and mean age of patients was 62.4 years. Thirteen tumors were located in the cecum, 29 in the ascending colon, five in the transverse colon, one in the descending colon, 43 in the sigmoid colon, and 30 in the rectum. Average duration of operation was 184 minutes, and mean estimated blood loss was 53.5 ml. Five patients (4.1%) were converted to open procedures. No intraoperative complication was observed but eight complications (6.6%) occurred postoperatively. Forty-two cases were classified as stage II, 62 as stage IIIA /B, and 17 as stage IIIC. Five patients died of cancer relapse (4.1%), and 18 cases had recurrence of disease (14.9%), to date. No port-site recurrence was detected. Overall five-year survival was 95.7% in stage II, 84.1% in stage IIIA/B, 70.0% in stage IIIC. Meanwhile disease-free five-year survival was 75.6% in stage II, 80.1% in stage IIIA/B, and 66.8% in stage IIIC. No significant difference was observed between stages, in terms of either overall or disease-free survival.ConclusionAlthough further evaluation is required, laparoscopic surgery for stage II/III colorectal cancer is safe and would be an oncologically adequate procedure.

Prospective randomized controlled study comparing cell block method and conventional smear method for bile cytology.

There is a paucity of data on the cell block (CB) method for bile cytology. We compared the diagnostic efficacy of the CB method with that of conventional smear cytology for bile obtained by endoscopic retrograde cholangiopancreatography (ERCP) in a randomized controlled trial manner.