Masafumi Imamura

Reconsideration of Postoperative Oral Intake Tolerance After Pancreaticoduodenectomy: Prospective Consecutive Analysis of Delayed Gastric Emptying According to the ISGPS Definition and the Amount of Dietary Intake

Objective:A prospective consecutive study was planned to evaluate the postpancreaticoduodenectomy (PD) oral intake tolerance. The occurrence of delayed gastric emptying (DGE), as defined by the International Study Group of Pancreatic Surgery (ISGPS), and the amount of dietary intake were analyzed. The risk factors for low oral intake tolerance were additionally determined. Summary Background Data:The causation of DGE after PD is still unclear. Several possible factors have been discussed, such as reconstruction methods and other complications. However, none of them has followed the definition of ISGPS. Methods:Between 2003 and 2007, 101 consecutive patients underwent PD-related surgery, and standard operative procedure was performed on 85 patients. Perioperative data were prospectively collected in all patients, and the patients postoperative dietary intake was recorded for all meals until discharge. As an indicator of early postoperative oral intake tolerance, we added up the dietary intake from postoperative day 1 to 21 and called this value the total amount of dietary intake (TDI). The postoperative outcomes were compared between non-DGE and DGE. The high-low of TDI values was also analyzed. Multivariate analysis for factors associated with the occurrence of DGE and TDI was performed. Results:The occurrence of DGE as defined by ISGPS was 42%. The postoperative outcomes of DGE patients were significantly poor compared with those of non-DGE patients. TDI values were significantly low in DGE patients, and non-DGE patients with low TDI values showed a significantly extended duration of parenteral nutrition and postoperative hospitalization. Operative bleeding (>1,000 mL) and pancreatic fistulas were likely to be associated with DGE occurrence. Gender (women), BMI (>25 kg/m2), postoperative intraabdominal infection, and DGE were significantly associated with low TDI values. Conclusions:The ISGPS definition of DGE seemed feasible for patient management. TDI values provided additional information for analyzing postoperative oral intake tolerance, especially when describing the differences among non-DGE patients. Substantial risk factors for low oral intake tolerance were high BMI, postoperative intraabdominal infection, and DGE.

Transforming growth factor-β induces epithelial to mesenchymal transition by down-regulation of claudin-1 expression and the fence function in adult rat hepatocytes

Background/Aims: Transforming growth factor‐β (TGF‐β) initiates and maintains epithelial–mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF‐β, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear.

Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-alpha inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-delta inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control.

Transcriptional regulation of claudin-18 via specific protein kinase C signaling pathways and modification of DNA methylation in human pancreatic cancer cells.

Since claudin‐18 (Cldn18) is overexpressed in precursor lesion PanIN and pancreatic duct carcinoma, it serves as a diagnostic marker and a target of immunotherapy. The stomach isoform of Cldn18, Cldn18a2 is regulated via a PKC/MAPK/AP‐1‐dependent pathway in PKC activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐stimulated gastric cancer cells. However, little is known about how Cldn18 is regulated, not only in pancreatic duct carcinoma but also in normal human pancreatic duct epithelial cells (HPDE cells). In the present study, four pancreatic cancer cell lines, HPAF‐II, HPAC, PANC‐1 and BXPC3, and hTERT‐HPDE cells in which the hTERT gene was introduced into HPDE cells in primary culture, were treated with TPA. In all human pancreatic cancer cell lines and hTERT‐HPDE cells, Cldn18 mRNA indicated as Cldn18a2 was markedly induced by TPA and in well‐ or moderately differentiated human pancreatic cancer cells HPAF‐II and HPAC and hTERT‐HPDE cells, the protein was also strongly increased. The upregulation of Cldn18 by TPA in human pancreatic cancer cell lines was prevented by inhibitors of PKCδ, PKCε, and PKCα, whereas the upregulation of Cldn18 by TPA in hTERT‐HPDE cells was prevented by inhibitors of PKCδ, PKCθ, and PKCα. Furthermore, a CpG island was identified within the coding sequence of the Cldn18 gene and treatment with the demethylating agent 5‐azadeoxycytidine enhanced upregulation of Cldn18 by TPA in HPAF‐II and HPAC, but not hTERT‐HPDE cells. Our findings suggest that in human pancreatic cancer cells, Cldn18 is primarily regulated at the transcriptional level via specific PKC signaling pathways and modified by DNA methylation. J. Cell. Biochem. 112: 1761–1772, 2011.

Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells

The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.

Acinar cell carcinoma of the pancreas with intraductal growth: Report of a case

Acinar cell carcinomas (ACCs) of the pancreas are rare neoplasms, accounting for approximately 1% of all exocrine pancreatic tumors. This type of tumor is known to be aggressive, although the survival rates are somewhat better than they are for ductal carcinoma. The tumor tends to present nonspecific symptoms. It occurs in older patients, and jaundice is infrequent. This report presents a case of ACC of the pancreas with intraductal papillary growth and lymph node metastasis.

Targeting tight junctions during epithelial to mesenchymal transition in human pancreatic cancer

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and -18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1, -7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition (EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

Pancreaticoduodenectomy for biliary tract carcinoma with situs inversus totalis: difficulties and technical notes based on two cases.

Situs inversus totalis (SIT) denotes complete right-left inversion of the thoracic and abdominal viscera. Diagnosis and surgical procedures for abdominal pathology in patients with SIT are technically more complicated because of mirror-image transposition of the visceral organs. Moreover, SIT is commonly associated with cardiovascular and hepatobiliary malformations, which make hepatobiliary-pancreatic surgery difficult. Two cases of pancreaticoduodenectomy for biliary tract carcinoma in patients with SIT are presented. Both patients had an anomaly of the hepatic artery. Advanced diagnostic imaging techniques were very important for careful preoperative planning and to prevent misunderstanding of the arrangement of the abdominal viscera. This facilitated the surgical team’s adaptation to the mirror image of the standard procedure and helped avoid intraoperative complications due to cardiovascular and hepatobiliary malformations associated with SIT. Pancreaticoduodenectomy in patients with SIT can be performed successfully with detailed preoperative assessment, use of effective techniques by the surgeon, and appropriate support by assistants.