Daisuke Kyuno

Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.

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.

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to Clostridium perfringens enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and −4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.

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.

Tight junctions in human pancreatic duct epithelial cells

Tight junctions of the pancreatic duct are essential regulators of physiologic secretion of the pancreas and disruption of the pancreatic ductal barrier is known to contribute to the pathogenesis of pancreatitis and progression of pancreatic cancer. Various inflammatory mediators and carcinogens can trigger tight junction disassembly and disruption of the pancreatic barrier, however signaling events that mediates such barrier dysfunctions remain poorly understood. This review focuses on structure and regulation of tight junctions in normal pancreatic epithelial cells and mechanisms of junctional disruption during pancreatic inflammation and cancer. We will pay special attention to a novel model of human telomerase reverse transcriptase-transfected human pancreatic ductal epithelial cells and will describe the roles of major signaling molecules such as protein kinase C and c-Jun N-terminal kinase in formation and disassembly of the pancreatic ductal barrier.

Targeting claudin-4 in human pancreatic cancer

Introduction: Pancreatic cancer is one of the most malignant human diseases and there is an urgent need to develop novel diagnostic and therapeutic strategies. Claudin-4, overexpressed in pancreatic cancer and its precursor lesions, is a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Areas covered: This review describes and discusses the studies targeting claudin-4 in normal human pancreatic duct epithelial (HPDE) cells and cancer cells. Expert opinion: Claudin-4 is in part regulated via a PKCα signal transduction pathway in pancreatic cancer cell lines. PKCα inhibitors may represent potential therapeutic agents against human pancreatic cancer cells by the use of CPE cytotoxicity via claudin-4. The COOH-terminal half fragment of CPE (C-CPE) enhances the effectiveness of clinically relevant chemotherapies and can be used as a carrier for drugs and other bacterial toxins to claudin-4-positive cancer cells. hTERT-HPDE cells, in which the human telomerase reverse transcriptase (hTERT) gene is introduced into normal HPDE cells, 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.

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.

Claudin‐4 binder C‐CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway

The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) modulates the tight junction protein claudin and disrupts the tight junctional barrier. It also can enhance the effectiveness of anticancer agents. However, the detailed mechanisms of the effects of C‐CPE remain unclear in both normal and cancerous cells. The C‐CPE mutant called C‐CPE 194 binds only to claudin‐4, but the C‐CPE 194 mutant called C‐CPE m19 binds not only to claudin‐4 but also to claudin‐1. In the present study, to investigate the mechanisms of the effects of C‐CPE on claudin expression, the tight junctional functions and the cytotoxicity of anticancer agents, human pancreatic cancer cells, and normal human pancreatic duct epithelial cells (HPDEs) were treated with C‐CPE 194 and C‐CPE m19. In well‐differentiated cells of the pancreatic cancer cell line HPAC, C‐CPE 194 and C‐CPE m19 disrupted both the barrier and fence functions without changes in expression of claudin‐1 and ‐4, together with an increase of MAPK phosphorylation. C‐CPE 194, but not C‐CPE m19, enhanced the cytotoxicity of the anticancer agents gemcitabine and S‐1. In poorly differentiated pancreatic cancer cell line PANC‐1, C‐CPE 194, but not C‐CPE m19, decreased claudin‐4 expression and enhanced MAPK activity and the cytotoxicity of the anticancer agents. In normal HPDEs, C‐CPE 194 and C‐CPE m19 decreased claudin‐4 expression and enhanced the MAPK activity, whereas they did not affect the cytotoxicity of the anticancer agents. Our findings suggest that the claudin‐4 binder C‐CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.