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

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Abstract

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.

Abbreviations

CA-II:

carbonic anhydrase isozyme 2

CK:

cytokeratin

CPE:

Clostridium perfringens enterotoxin

DAPI:

4′,6-diamidino-2-phenylindole dihydrochloride

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

fetal bovine serum

HPDE:

human pancreatic duct epithelial

hTERT:

human telomerase reverse transcriptase

PBS:

phosphate-buffered saline

RT:

room temperature

siRNAs:

small interference RNAs

TBS:

Tris-buffered saline

TER:

transepithelial electrical resistance

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Correspondence to Takashi Kojima.

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Yamaguchi, H., Kojima, T., Ito, T. et al. Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells. Cell Mol Biol Lett 16, 385–397 (2011) doi:10.2478/s11658-011-0014-z

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Key words

  • Tight junction
  • Claudin-4
  • CPE
  • Human pancreatic duct epithelial cells
  • Human pancreatic cancer cells