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.
[1] Morin, P.J. Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res. 65 (2005) 9603–9606. http://dx.doi.org/10.1158/0008-5472.CAN-05-278210.1158/0008-5472.CAN-05-2782Search in Google Scholar PubMed
[2] Tsukita, S., Yamazaki, Y., Katsuno, T., Tamura, A. and Tsukita, S. Tight junction-based epithelial microenvironment and cell proliferation. Oncogene 27 (2008) 6930–6938. http://dx.doi.org/10.1038/onc.2008.34410.1038/onc.2008.344Search in Google Scholar PubMed
[3] Tsukita, S., Furuse, M. and Itoh, M. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2 (2001) 285–293. http://dx.doi.org/10.1038/3506708810.1038/35067088Search in Google Scholar PubMed
[4] Fujita, K., Katahira, J., Horiguchi, Y., Sonoda, N., Furuse, M. and Tsukita, S. Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein. FEBS Lett. 476 (2000) 258–261. http://dx.doi.org/10.1016/S0014-5793(00)01744-010.1016/S0014-5793(00)01744-0Search in Google Scholar PubMed
[5] McClane, B.A. and Chakrabarti, G. New insights into the cytotoxic mechanisms of Clostridium perfringens enterotoxin. Anaerobe 10 (2004) 107–114. http://dx.doi.org/10.1016/j.anaerobe.2003.11.00410.1016/j.anaerobe.2003.11.004Search in Google Scholar PubMed
[6] Michl, P., Buchholz, M., Rolke, M., Kunsch, S., Löhr, M., McClane, B., Tsukita, S., Leder, G., Adler, G. and Gress, T.M. Claudin-4: a new target for pancreatic cancer treatment using Clostridium Perfringens enterotoxin. Gastroenterology 121 (2001) 678–684. http://dx.doi.org/10.1053/gast.2001.2712410.1053/gast.2001.27124Search in Google Scholar PubMed
[7] Katahira, J., Sugiyama, H,, Inoue, N., Horiguchi, Y., Matsuda, M. and Sugimoto, N. Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo. J. Biol. Chem. 272 (1997) 26652–26658. http://dx.doi.org/10.1074/jbc.272.42.2665210.1074/jbc.272.42.26652Search in Google Scholar PubMed
[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. and Sawada, N. Transcriptional control of tight junction proteins via a PKC signal pathway in hTERT-transfected human pancreatic duct epithelial cells. Am. J. Pathol. 177 (2010) 698–712. http://dx.doi.org/10.2353/ajpath.2010.09122610.2353/ajpath.2010.091226Search in Google Scholar PubMed PubMed Central
[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. and 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. 225 (2010) 720–733. http://dx.doi.org/10.1002/jcp.2227310.1002/jcp.22273Search in Google Scholar PubMed
[10] Deer, E.L., González-Hernández, J., Coursen, J.D., Shea, J.E., Ngatia, J., Scaife, C.L., Firpo, M.A. and Mulvihill, S.J. Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39 (2010) 425–435. http://dx.doi.org/10.1097/MPA.0b013e3181c1596310.1097/MPA.0b013e3181c15963Search in Google Scholar PubMed PubMed Central
[11] 11 Winkler, L., Gehring, C., Wenzel, A., Müller, S.L., Piehl, C., Krause, G., Blasig, I.E. and Piontek, J. Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3. J. Biol. Chem. 284 (2009) 18863–18872. http://dx.doi.org/10.1074/jbc.M109.00862310.1074/jbc.M109.008623Search in Google Scholar PubMed PubMed Central
[12] Sonoda, N., Furuse, M., Sasaki, H., Yonemura, S., Katahira, J., Horiguchi, Y. and 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. 147 (1999) 195–204. http://dx.doi.org/10.1083/jcb.147.1.19510.1083/jcb.147.1.195Search in Google Scholar PubMed PubMed Central
© 2011 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
