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.
Key words: Tight junction, Claudin-4, CPE, Human pancreatic duct epithelial cells, Human pancreatic cancer cells
Full Text
The Full Text of this article is available as a PDF (2.9 MB).
Abbreviations used
- 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
References
- 1.Morin P.J. Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res. 2005;65:9603–9606. doi: 10.1158/0008-5472.CAN-05-2782. [DOI] [PubMed] [Google Scholar]
- 2.Tsukita S., Yamazaki Y., Katsuno T., Tamura A., Tsukita S. Tight junction-based epithelial microenvironment and cell proliferation. Oncogene. 2008;27:6930–6938. doi: 10.1038/onc.2008.344. [DOI] [PubMed] [Google Scholar]
- 3.Tsukita S., Furuse M., Itoh M. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2001;2:285–293. doi: 10.1038/35067088. [DOI] [PubMed] [Google Scholar]
- 4.Fujita K., Katahira J., Horiguchi Y., Sonoda N., Furuse M., Tsukita S. Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein. FEBS Lett. 2000;476:258–261. doi: 10.1016/S0014-5793(00)01744-0. [DOI] [PubMed] [Google Scholar]
- 5.McClane B.A., Chakrabarti G. New insights into the cytotoxic mechanisms of Clostridium perfringens enterotoxin. Anaerobe. 2004;10:107–114. doi: 10.1016/j.anaerobe.2003.11.004. [DOI] [PubMed] [Google Scholar]
- 6.Michl P., Buchholz M., Rolke M., Kunsch S., Löhr M., McClane B., Tsukita S., Leder G., Adler G., Gress T.M. Claudin-4: a new target for pancreatic cancer treatment using Clostridium Perfringens enterotoxin. Gastroenterology. 2001;121:678–684. doi: 10.1053/gast.2001.27124. [DOI] [PubMed] [Google Scholar]
- 7.Katahira J., Sugiyama H., Inoue N., Horiguchi Y., Matsuda M., Sugimoto N. Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo. J. Biol. Chem. 1997;272:26652–26658. doi: 10.1074/jbc.272.42.26652. [DOI] [PubMed] [Google Scholar]
- 8.Yamaguchi H., Kojima T., Ito T., Kimura Y., Imamura M., Son S., Koizumi J., Murata M., Nagayama M., Nobuoka T., Tanaka S., Hirata K., Sawada N. Transcriptional control of tight junction proteins via a PKC signal pathway in hTERT-transfected human pancreatic duct epithelial cells. Am. J. Pathol. 2010;177:698–712. doi: 10.2353/ajpath.2010.091226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kojima T., Fuchimoto J., Takasawa A., Yamaguchi H., Ito T., Ninomiya T., Kikuchi S., Ogasawara N., Ohkuni T., Masaki T., Hirata K., Himi T., Sawada N. c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells. J. Cell. Physiol. 2010;225:720–733. doi: 10.1002/jcp.22273. [DOI] [PubMed] [Google Scholar]
- 10.Deer E.L., González-Hernández J., Coursen J.D., Shea J.E., Ngatia J., Scaife C.L., Firpo M.A., Mulvihill S.J. Phenotype and genotype of pancreatic cancer cell lines. Pancreas. 2010;39:425–435. doi: 10.1097/MPA.0b013e3181c15963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Winkler L., Gehring C., Wenzel A., Müller S.L., Piehl C., Krause G., Blasig I.E., Piontek J. Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3. J. Biol. Chem. 2009;284:18863–18872. doi: 10.1074/jbc.M109.008623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sonoda N., Furuse M., Sasaki H., Yonemura S., Katahira J., Horiguchi Y., Tsukita S. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier. J. Cell Biol. 1999;147:195–204. doi: 10.1083/jcb.147.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]