Toshiya Ochiai

Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer

Background:Several recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We tested miR-221 and miR-375, which are frequently reported to be highly and poorly expressed in pancreatic cancer (PCa), as candidates for plasma biomarkers in PCa.Methods:This study was divided into three parts: (1) Confirmation of higher miR-221 levels in primary PCa tissue and cell lines than normal pancreatic tissues. (2) Evaluation of plasma miR-221 and miR-375 concentrations by comparing results from 47 consecutive PCa patients and 30 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in PCa patients.Results:(1) Expression of miR-221 was significantly higher in PCa tissues and cell lines than normal pancreatic tissues. (2) Plasma miR-221 concentrations were significantly higher in PCa patients than that in benign pancreatic tumours (P=0.016) and controls (P<0.0005), while plasma miR-375 concentrations tended to be lower in PCa patients (P=0.064), and the miR-221/miR-375 ratio was significantly higher (P<0.0001) in PCa patients than in controls. (3) Plasma miR-221 concentrations were significantly reduced in postoperative samples (P=0.018). Furthermore, PCa patients with high plasma miR-221 concentrations had significant correlation with distant metastasis (P=0.041), and non-resectable status (P=0.021).Conclusion:Plasma miR-221 could be a useful biomarker for cancer detection, monitoring tumour dynamics and predicting malignant outcomes in PCa patients, and may contribute to clinical decision making in PCa treatments.

HGF regulates VEGF expression via the c-Met receptor downstream pathways, PI3K/Akt, MAPK and STAT3, in CT26 murine cells

In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K/Akt, MAPK and STAT3 pathways, downstream of HGF/c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c‑Met signalling may be a promising target for anti-angiogenic strategies.

Elevated serum CRP levels after induction chemoradiotherapy reflect poor treatment response in association with IL-6 in serum and local tumor site in patients with advanced esophageal cancer

Elevated serum CRP levels are associated with tumor progression and poor prognosis of esophageal cancer. The aim of this study was to clarify the clinical significance of CRP in relation to response to chemoradiotherapy in patients with esophageal cancer.

Application of Polyethylene Glycolic Acid Felt with Fibrin Sealant to Prevent Postoperative Pancreatic Fistula in Pancreatic Surgery

ObjectiveThe purpose of this nonrandomized retrospective study was to report our new procedures using polyethylene glycolic acid (PGA) felt with fibrin sealant to prevent severe pancreatic fistula in patients undergoing pancreatic surgery.MethodsFrom 2000 to 2008, 54 and 63 patients underwent pancreaticoduodenectomy (PD) and distal pancreatectomy (DP), respectively. Of those patients, we applied PGA felt with fibrin sealant to 18 PD patients and 26 DP patients. In PD patients, the PGA felt was wrapped around the pancreatic suture site, while in DP patients, the PGA felt was wrapped around the predictive division site. The pancreaticojejunostomy site in PD patients and the cut stump in DP patients were coated with fibrin sealant. We compared the occurrence rates for severe postoperative pancreatic fistula (POPF) that occurred after PD or DP both with and without our new procedures.ResultsBefore introduction of our procedures, severe POPF developed in 14 of 36 PD patients (39%) and 10 of 37 DP patients (27%). In contrast, after introduction of our procedures, the incidence of POPF was only one in both of 18 PD (6%; P = 0.016) and 26 DP (4%; P = 0.017) patients.ConclusionIn summary, our procedure using PGA felt with fibrin sealant may reduce the risk of severe POPF.

Attenuated response to liver injury in moesin-deficient mice: Impaired stellate cell migration and decreased fibrosis

Hepatic stellate cells (HSCs) respond to injury with a coordinated set of events (termed activation), which includes migration and upregulation of matrix protein production. Cell migration requires an intact actin cytoskeleton that is linked to the plasma membrane by ezrin-radixin-moesin (ERM) proteins. We have previously found that the linker protein in HSCs is exclusively moesin. Here, we describe HSC migration and fibrogenesis in moesin-deficient mice. We developed an acute liver injury model that involved focal thermal denaturation and common bile duct ligation. HSC migration and collagen deposition were assessed by immunohistology and quantitative real-time PCR. Activated HSCs were isolated from wild-type or moesin-deficient mice for direct examination of migration. Activated HSCs from wild-type mice were positive for moesin. Migration of moesin-deficient HSCs was significantly reduced. In a culture assay, 22.1% of normal HSCs migrated across a filter in 36 h. In contrast, only 1.3% of activated moesin-deficient HSCs migrated. Collagen deposition around the injury area similarly was reduced in moesin-deficient liver. The linker protein moesin is essential for HSC activation and migration in response to injury. Fibrogenesis is coupled to migration and reduced in moesin-deficient mice. Agents that target moesin may be beneficial for chronic progressive fibrosis.

Plasma level of metastasis‐associated lung adenocarcinoma transcript 1 is associated with liver damage and predicts development of hepatocellular carcinoma

Recent studies have shown that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) was overexpressed in many human solid cancers, however, its roles in plasma of hepatocellular carcinoma (HCC) patients were unclear. The aim of this study was to investigate the significance of plasma MALAT1 levels in HCC patients. Plasma samples were collected from pre‐operative HCC, hepatic disease patients, and healthy controls, and tissue samples from HCC patients and colorectal cancer patients with liver metastasis. Plasma and tissue MALAT1 levels were measured. Plasma MALAT1 levels were progressively and significantly higher in HCC patients than hepatic disease patients, and higher in hepatic disease patients than healthy controls. The expression of MALAT1 in HCC tissue was slightly higher than that in paired non‐cancerous liver tissue, but not significant. The expression of MALAT1 in the non‐cancerous liver tissue of 20 HCC patients was significantly higher than that in normal liver tissue of 13 colorectal cancer patients. In contrast, plasma MALAT1 levels were significantly low in HCC patients with hepatitis B infection, and significantly high in patients with liver damage B or liver cirrhosis. In a receiver–operator curve analysis of HCC and hepatic disease patients, the cut‐off value of plasma MALAT1 was 1.60 and the area under the curve was 0.66. Plasma MALAT1 levels were not correlated with α‐fetoprotein or protein induced by vitamin K absence II, whereas sensitivity and specificity for the detection of HCC with the combination of MALAT1, α‐fetoprotein, and protein induced by vitamin K absence II were 88.6% and 75%, respectively. In conclusion, the plasma MALAT1 level is associated with liver damage, and has clinical utility for predicting development of HCC.

Progression of remnant gastric cancer is associated with duration of follow-up following distal gastrectomy

AIM To re-evaluate the recent clinicopathological features of remnant gastric cancer (RGC) and to develop desirable surveillance programs. METHODS Between 1997 and 2008, 1149 patients underwent gastrectomy for gastric cancer at the Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Japan. Of these, 33 patients underwent gastrectomy with lymphadenectomy for RGC. Regarding the initial gastric disease, there were 19 patients with benign disease and 14 patients with gastric cancer. The hospital records of these patients were reviewed retrospectively. RESULTS Concerning the initial gastric disease, the RGC group following gastric cancer had a shorter interval [P < 0.05; gastric cancer vs benign disease: 12 (2-22) vs 30 (4-51) years] and were more frequently reconstructed by Billroth-I procedure than those following benign lesions (P < 0.001). Regarding reconstruction, RGC following Billroth-II reconstruction showed a longer interval between surgical procedures [P < 0.001; Billroth-II vs Billroth-I: 32 (5-51) vs 12 (2-36) years] and tumors were more frequently associated with benign disease (P < 0.001) than those following Billroth-I reconstruction. In tumor location of RGC, after Billroth-I reconstruction, RGC occurred more frequently near the suture line and remnant gastric wall. After Billroth-II reconstruction, RGC occurred more frequently at the anastomotic site. The duration of follow-up was significantly associated with the stage of RGC (P < 0.05). Patients diagnosed with early stage RGC such as stage I-II tended to have been followed up almost every second year. CONCLUSION Meticulous follow-up examination and early detection of RGC might lead to a better prognosis. Based on the initial gastric disease and the procedure of reconstruction, an appropriate follow-up interval and programs might enable early detection of RGC.

Risk Factors and Management of Postoperative Bile Leakage after Hepatectomy without Bilioenteric Anastomosis

Background/Aims: Bile leakage frequently causes major complications after hepatic resection. We investigated perioperative risk factors and management of postoperative bile leakage after hepatic resection without extrahepatic biliary resection and reconstruction. Methods: We included 247 consecutive patients who underwent elective hepatic resection without bilioenteric anastomosis at our institution between 2002 and 2009. Perioperative risk factors, including patient and surgical variables, were evaluated using univariate and logistic regression analyses. Results: Postoperative bile leakage occurred in 26 patients (10.5%). The surgical drain was retained in 6 patients (23%); 9 (35%) underwent drain salvage and 11 (42%) underwent percutaneous puncture under computed tomography or ultrasound guidance. Eight patients underwent endoscopic nasobiliary drainage (ENBD) for postoperative bile leakage, and bile leakage healed at a median interval of 19.5 days after ENBD. By univariate analysis, postoperative bile leakage was associated with central bisectionectomy, surgical time and intraoperative blood loss. Logistic regression analysis identified central bisectionectomy as an independent risk factor for postoperative bile leakage (p = 0.0003, odds ratio 16.724). Conclusion: Meticulous procedures are necessary during parenchymal hepatic resection, especially during central bisectionectomy. Drain management should be precise in the case of postoperative bile leakage. We believe ENBD may rapidly cure postoperative major bile leakage.

Plasma microRNA profiles: identification of miR-744 as a novel diagnostic and prognostic biomarker in pancreatic cancer

Background:This study aims to explore novel microRNAs in plasma for screening cancer and predicting clinical outcomes in pancreatic cancer (PCa) patients using a microRNA array-based approach.Methods:We used the Toray 3D-Gene microRNA array-based approach to compare plasma levels between PCa patients and healthy volunteers.Results:(1) Six oncogenic microRNAs (miR-615-5p, -744, -575, -557, -675, and -550a) with high expression in plasma were selected. (2) By quantitative RT–PCR using plasma samples from 94 PCa patients and 68 healthy volunteers, a significantly higher level of plasma miR-744 in PCa patients than in healthy volunteers was validated in small-scale analysis (P=0.0038), two independent cohort analyses, and large-scale analysis (P<0.0001, AUC 0.8307). (3) miR-744 expression was significantly higher in PCa tissues (P=0.0069) and PCa cell lines (P=0.0074) than in normal tissues and fibroblasts, respectively. Preoperative plasma level of miR-744 was significantly reduced in postoperative samples (P=0.0063). (4) A high level of plasma miR-744, which was correlated with lymph node metastasis (P=0.0407) and recurrences (P=0.0376), was an independent poor prognostic factor of PCa patients after pancreatectomy (P=0.0007, HR 21.2 (3.17–436)). Furthermore, a high level of plasma miR-744 contributed to poorer progression-free survival of non-operable PCa patients who underwent gemcitabine-based chemotherapy (P=0.0533). Overexpression of miR-744 in PCa cells induced significant chemoresistance to gemcitabine in vitro.Conclusions:Plasma miR-744 might be useful biomarker for screening PCa, monitoring, and predicting poor prognosis and chemoresistance in PCa patients.

Efficacy of 5-aminolevulinic acid-mediated photodynamic therapy using light-emitting diodes in human colon cancer cells.

5-Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) (ALA-PDT) is a highly selective treatment for malignant cells. ALA-PDT has the potential to develop into a novel therapeutic strategy for various types of cancer. Recently, light-emitting diodes (LEDs), which are inexpensive, stable and easier to handle compared to lasers, have been used in PDT as a light source. However, in colorectal cancer (CRC), the efficacy of ALA-PDT in combination with LEDs has not been fully assessed. Therefore, in this study, we evaluated the antitumor effect of ALA-PDT using various LEDs in colon cancer cells. The HT-29 human colon cancer cell line was used both in vitro and in vivo. HT-29 cells were seeded in 96-well plates. Following 5-ALA administration, cells were irradiated using LEDs at different wavelengths. Three types of LEDs, blue (peak wavelength, 456 nm), white (broad-band) and red (635 nm) were used. Twenty-four hours after irradiation, the cytotoxic effects of ALA-PDT were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In order to evaluate the antitumor effect of ALA-PDT in vivo, nude mice were inoculated with HT-29 cells. Xenograft mice were injected intraperitoneally with 5-ALA and irradiated with 3 types of LEDs at a measured fluence rate of 96 mW/cm2 and fluence of 32 J/cm2. Each group comprised 6 mice. ALA-PDT was repeated 3 times at weekly intervals. Tumor weights were measured. Compared to the controls, ALA-PDT using LEDs showed significant antitumor effects in vitro and in vivo. The blue and white LEDs demonstrated greater antitumor effects compared to the red LEDs in vitro and in vivo. In particular, tumor inhibition rates in the blue and white LED groups were approximately 88% to those of the control group in the mouse models. In conclusion, ALA-PDT using LEDs is effective and useful in the treatment of CRC cells. This method could be a novel treatment modality for CRC.