Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

Gene Therapy volume 19, pages 494–503 (2012)Cite this article

Abstract

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Edelstein ML, Abedi MR, Wixon J . Gene therapy clinical trials worldwide to 2007- an update. J Gene Med 2007; 9: 833–842.

    Article  PubMed  Google Scholar 

  2. Walther W, Stein U . Therapeutic genes for cancer gene therapy. Mol Biotechnol 1999; 13: 21–28.

    Article  CAS  PubMed  Google Scholar 

  3. Lo HW, Day CP, Hung MC . Cancer-specific gene therapy. Adv Genet 2005; 54: 235–255.

    CAS  PubMed  Google Scholar 

  4. Michl P, Gress TM . Bacteria and bacterial toxins as therapeutic agents for solid tumors. Current Cancer Drug Targets 2004; 4: 689–702.

    Article  CAS  PubMed  Google Scholar 

  5. Martin V, Cortes ML, deFelipe P, Farsetti A, Calcaterra NB, Izquierdo M . Cancer gene therapy by thyroid hormone-mediated expression of toxin genes. Cancer Res 2000; 60: 3218–3224.

    CAS  PubMed  Google Scholar 

  6. Lee EJ, Jameson JL . Cell-specific cre-mediated activation of the diphteria toxin gene in pituitary tumor cells: potential for cytotoxic gene therapy. Hum Gene Ther 2002; 13: 533–542.

    Article  CAS  PubMed  Google Scholar 

  7. Li Y, McCadden J, Ferrer F, Kruszewski M, Carducci M, Simons J et al. Prostate-specific expression of the diphteria toxin A chain (DT-A): studies of inducibility and specificity of expression of prostate-specific antigen promoter-driven DT-A adenoviral-mediated gene transfer. Cancer Res 2002; 62: 2576–2582.

    CAS  PubMed  Google Scholar 

  8. Zheng JY, Chen D, Chan J, Yu D, Ko E, Pang S . Regression of prostate cancer xenografts by a lentiviral vecor specifically expresing diphteria toxin A. Cancer Gene Ther 2003; 10: 764–770.

    Article  CAS  PubMed  Google Scholar 

  9. Kreitman RJ . Chimeric fusion proteins—pseudomonas exotoxin based. Curr Opin Investig Drugs 2001; 2: 1282–1293.

    CAS  PubMed  Google Scholar 

  10. Candolfi M, Xiong W, Yagiz K, Liu C, Muhammed AK, Puntel M et al. Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics. Proc Natl Acad Sci USA 2010; 107: 20021–20026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Laske DW, Youle RJ, Oldfield EH . Tumor regression with regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors. Nat Med 1997; 3: 1362–1368.

    Article  CAS  PubMed  Google Scholar 

  12. Husain SR, Puri RK . Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. J Neurooncol 2003; 65: 37–48.

    Article  PubMed  Google Scholar 

  13. Zheng JY, Chen D, Chan J, Yu D, Ko E, Pang S . Regression of prostate cancer xenografts by a lentiviral vector specifically expressing diphtheria toxin A. Cancer Gene Ther 2003; 10: 764–770.

    Article  CAS  PubMed  Google Scholar 

  14. Ayesh B, Matouk I, Ohana P, Sughayer MA, Birman T, Ayesh S et al. Inhibition of tumor growth by DT-A expressed under the control of IGF2 P3 and P4 promoter sequences. Mol Ther 2003; 7: 535–541.

    Article  CAS  PubMed  Google Scholar 

  15. Bhakdi S, Bayley H, Valeva A, Walev I, Walker B, Kehoe M et al. Staphylococcal α-toxin, streptolysin O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. Arch Microbiol 1996; 165: 73–79.

    Article  CAS  PubMed  Google Scholar 

  16. Yang WS, Park SO, Yoon AR, Yoo JY, Kim MK, Yun CO et al. Suicide cancer gene therapy using pore-forming toxin, streptolysin O. Mol Cancer Ther 2006; 5: 1610–1619.

    Article  CAS  PubMed  Google Scholar 

  17. Michl P, Buchholz M, Rolke M, Kunsch S, Löhr M, McClane B et al. Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin. Gastroenterology 2001; 121: 678–684.

    Article  CAS  PubMed  Google Scholar 

  18. Kominsky SL, Vali M, Korz D, Gabig TG, Weitzman SA, Agani P et al. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4. Am J Pathol 2004; 164: 1627–1633.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Johnson EA . Clostridial toxins as therapeutic agents: benefits of nature's most toxic proteins. Annu Rev Microbiol 1999; 53: 551–575.

    Article  CAS  PubMed  Google Scholar 

  20. Czeczulin JR, Hanna PC, McClane BA . Cloning, nucleotide sequencing and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli. Infect Immun 1993; 61: 3429–3439.

    CAS  PubMed Central  PubMed  Google Scholar 

  21. McClane BA . The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions. Toxicon 2001; 39: 1781–1791.

    Article  CAS  PubMed  Google Scholar 

  22. Smedley III JG, Uzal FA, McClane BA . Indentification of a prepore large-complex in the mechanism of action of Clostridium perfringens enterotoxin. Infect Immun 2007; 75: 2381–2390.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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.

    Article  CAS  PubMed  Google Scholar 

  24. Kokai-Kun JF, McClane BA . Deletion analysis of the Clostridium perfringens enterotoxin. Clin Infect Dis 1997; 65: 1014–1022.

    CAS  Google Scholar 

  25. Kokai-Kun JF, Benton K, Wieckowski EU, McClane BA . Identification of a Clostridium perfringens enterotoxin region required for large complex formation and cytotoxicity by random mutagenesis. Infect Immun 1999; 67: 5634–5641.

    CAS  PubMed Central  PubMed  Google Scholar 

  26. 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: 258–2561.

    Article  CAS  PubMed  Google Scholar 

  27. Morita K, Furuse M, Fujimoto K, Tsukita S . Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 1999; 96: 511–516.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Long H, Crean CD, Lee WH, Cummings OW, Gabig TG . Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium. Cancer Res 2001; 61: 7878–7881.

    CAS  PubMed  Google Scholar 

  29. Rangel LB, Agarwal R, D′Souza T, Pizer ES, Alo PL, Lancaster WD et al. Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas. Clin Cancer Res 2003; 9: 2567–2575.

    CAS  PubMed  Google Scholar 

  30. Morin PJ . Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res 2005; 65: 9603–9606.

    Article  CAS  PubMed  Google Scholar 

  31. Soini Y . Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumors. Histopathol 2005; 46: 551–560.

    Article  CAS  Google Scholar 

  32. Hewitt KJ, Agarwal R, Morin PJ . The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 2006; 6: 186.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kominsky SL, Tyler B, Sosnowski J, Brady K, Doucet M, Nell D et al. Clostridium perfringens enterotoxin as a novel-targeted therapeutic for brain metastasis. Cancer Res 2007; 67: 7977–7982.

    Article  CAS  PubMed  Google Scholar 

  34. Smedley III JG, McLane BA . Fine mapping of the N-terminal cytotoxicity region of Clostridium perfringens enterotoxin by site-directed mutagenesis. Infect Immun 2004; 72: 6914–6923.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Santin AD, Cane S, Bellone S, Palmieri M, Siegel ER, Thomas M et al. Treatment of chemotherapy-resistant human ovarian cancer xenografts in C.B-17/SCID mice by intraperitoneal administration of Clostridium perfringens enterotoxin. Cancer Res 2005; 65: 4334–4342.

    Article  CAS  PubMed  Google Scholar 

  36. Santin AD, Bellone S, Siegel ER, McKenney JK, Thomas M, Roman JJ et al. Overexpression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in uterine carcinomas. Clin Cancer Res 2007; 13: 3339–3346.

    Article  CAS  PubMed  Google Scholar 

  37. Santin AD, Bellone S, Marizzoni M, Palmieri M, Siegel ER, McKenney JK et al. Overexpression of claudin-3 and claudin-4 receptors in uterine serous papillary carcinoma. Cancer 2007; 109: 1312–1322.

    Article  CAS  PubMed  Google Scholar 

  38. Jain RK . Delivey of novel therapeutic agents in tumors: physiological barriers and strategies. J Natl Cancer Inst 1989; 81: 570–576.

    Article  CAS  PubMed  Google Scholar 

  39. Saeki R, Kondoh M, Kakutani H, Tsunoda S-I, Mochizuki Y, Hamakubo T et al. A novel tumor-targeted therapy using a claudin-4-targeting molecule. Mol Pharmacol 2009; 76: 918–926.

    Article  CAS  PubMed  Google Scholar 

  40. Saeki R, Kondoh M, Kakutani H, Matsuhisa K, Takahashi A, Suzuki H et al. A claudin-targeting molecule as an inhibitor of tumor metastasis. J Pharmacol Exp Ther 2010; 334: 576–582.

    Article  CAS  PubMed  Google Scholar 

  41. Kakutani H, Kondoh M, Saeki R, Fuji M, Watanabe Y, Mizoguchi H et al. Claudin-4-targeting of diphteria toxin fragment A using a C-terminal fragment of Clostridium perfringens entrotoxin. Eur J Pharm Biopharm 2010; 75: 213–217.

    Article  CAS  PubMed  Google Scholar 

  42. Yao Q, Cao S, Li C, Mengesha A, Low P, Kong B et al. Turn a diarrhoea toxin into a receptor-mediated therapy for plethora of CLDN-4-overexpressing cancers. Biochem Biophys Re Comm 2010; 398: 413–419.

    Article  CAS  Google Scholar 

  43. Yuan X, Lin X, Manorek G, Kanatani I, Cheung LH, Rosenblum MG et al. Recombinant CPE fused to tumor necrosis factor targets human ovarian cancer cell expressing the claudin-3 and claudin-4 receptors. Mol Cancer Ther 2009; 8: 1906–1915.

    Article  CAS  PubMed  Google Scholar 

  44. Rello S, Stockert JC, Moreno V, Gamez A, Pacheco M, Juarranz A et al. Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments. Apoptosis 2005; 10: 201–208.

    Article  CAS  PubMed  Google Scholar 

  45. McDonel JL . The molecular mode of action of Clostridium perfringens enterotoxin. Am J Clin Nutr 1997; 32: 210–218.

    Article  Google Scholar 

  46. Bergsbaken T, Fink SL, Cookson BT . Pyroptosis: host cell death and inflammation. Nature Rev Microbiol 2009; 7: 99–109.

    Article  CAS  Google Scholar 

  47. Chakrabarti G, Zhou X, McLane BA . Death pathways activated in Caco-2 cells by Clostridium perfringens enterotoxin. Infect Immun 2003; 71: 4260–4270.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Walther W, Stein U, Schlag PM . Local gene therapy for cancer. In: Stein U, Schlag PM (eds). Regional Therapy For Cancer. Humana Press: Totowa, 2007, pp 181–196.

    Chapter  Google Scholar 

  49. Walther W, Siegel R, Kobelt D, Knösel T, Dietel M, Bembenek A et al. Nonviral intratumoral jet-injection gene transfer in metastatic melanoma and breast cancer: results of phase I clinical trial. Clin Cancer Res 2008; 14: 7545–7553.

    Article  CAS  PubMed  Google Scholar 

  50. Winkler L, Gehring C, Wenzel A, Müller SL, Piehl C, Krause G et al. Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3. J Biol Chem 2009; 284: 18863–18872.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank MR Popoff (Institut Pasteur, Paris) for kindly providing the Clostridium perfringens DNA; R Fischer and P Sander (R-Biopharm) for providing recombinant CPE and technical support. This work was supported by the Berliner Krebsgesellschaft, Grant WAFF200822 (to WW).

Author information

Authors and Affiliations

  1. Experimental and Clinical Research Center, Charité University Medicine Berlin, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

    W Walther, S Petkov, O N Kuvardina, J Aumann, U Stein & P M Schlag

  2. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

    D Kobelt, I Fichtner, M Lemm & P M Schlag

  3. Leibnitz-Institut für Molekulare Pharmakologie, Berlin, Germany

    J Piontek & I E Blasig

  4. Charité Comprehensive Cancer Center,, Berlin, Germany

    P M Schlag

Authors
  1. W Walther
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Petkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. O N Kuvardina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J Aumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D Kobelt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. I Fichtner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Lemm
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J Piontek
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. I E Blasig
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. U Stein
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. P M Schlag
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to W Walther.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Walther, W., Petkov, S., Kuvardina, O. et al. Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors. Gene Ther 19, 494–503 (2012). https://doi.org/10.1038/gt.2011.136

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.136

Keywords

This article is cited by

Search

Advanced search

Quick links