In vitro treatment of carcinoma cell lines with pancreatic (pro)enzymes suppresses the EMT programme and promotes cell differentiation

Abstract

Background

Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma).

Results

After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of β-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin.

Conclusions

Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

References

  1. 1.

    P.A. Andreasen, L. Kjoller, L. Christensen, M.J. Duffy, The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1–22 (1997)

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    J. Beard, The action of trypsin upon the living cells of jensen’s mouse-tumour: A preliminary note upon a research made (with a Grant from the Carnegie Trust). Br. Med. J. 1, 140–1 (1906)

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    R.W. Moss, An annotated bibliography of works by John Beard. Integr. Cancer Ther. 7, 317–21 (2008)

    PubMed  Article  Google Scholar 

  4. 4.

    J. Beard, The cancer problem. Lancet 168, 281–283 (1905)

    Article  Google Scholar 

  5. 5.

    J.F. Novak, F. Trnka, Proenzyme therapy of cancer. Anticancer. Res. 25, 1157–77 (2005)

    PubMed  CAS  Google Scholar 

  6. 6.

    M. Wald, T. Olejar, V. Sebkova, M. Zadinova, M. Boubelik, P. Pouckova, Mixture of trypsin, chymotrypsin and papain reduces formation of metastases and extends survival time of C57Bl6 mice with syngeneic melanoma B16. Cancer Chemother. Pharmacol. 47, 16–22 (2001)

    Article  Google Scholar 

  7. 7.

    J. Beuth, B. Ost, A. Pakdaman, E. Rethfeldt, P.R. Bock, J. Hanisch et al., Impact of complementary oral enzyme application on the postoperative treatment results of breast cancer patients–results of an epidemiological multicentre retrolective cohort study. Cancer Chemother. Pharmacol. 47, 45–54 (2001)

    Article  Google Scholar 

  8. 8.

    T. Popiela, J. Kulig, J. Hanisch, P.R. Bock, Influence of a complementary treatment with oral enzymes on patients with colorectal cancers–an epidemiological retrolective cohort study. Cancer Chemother. Pharmacol. 47, 55–63 (2001)

    Article  Google Scholar 

  9. 9.

    A. Sakalova, P.R. Bock, L. Dedik, J. Hanisch, W. Schiess, S. Gazova et al., Retrolective cohort study of an additive therapy with an oral enzyme preparation in patients with multiple myeloma. Cancer Chemother Pharmacol 47, 38–44 (2001)

    Article  Google Scholar 

  10. 10.

    W.J. Nelson, Regulation of cell-cell adhesion by the cadherin-catenin complex. Biochem. Soc. Trans. 36, 149–55 (2008)

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7, 415–28 (2007)

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    R. Kemler, From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends. Genet. 93, 17–21 (1993)

    Google Scholar 

  13. 13.

    M. Conacci-Sorrell, J. Zhurinsky, A. Ben-Ze’ev, The cadherin-catenin adhesion system in signaling and cancer. J. Clin. Invest. 109, 987–91 (2002)

    PubMed  CAS  Google Scholar 

  14. 14.

    B.M. Gumbiner, P.D. McCrea, Catenins as mediators of the cytoplasmic functions of cadherins. J. Cell Sci. Suppl. 17, 155–158 (1993)

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    B. Zhai, H.X. Yan, S.Q. Liu, L. Chen, M.C. Wu, H.Y. Wang, Reduced expression of P120 catenin in cholangiocarcinoma correlated with tumor clinicopathologic parameters. World J. Gastroenterol. 14, 3739–44 (2008)

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    E. Margineanu, C.E. Cotrutz, C. Cotrutz, Correlation between E-cadherin abnormal expressions in different types of cancer and the process of metastasis. Rev. Med. Chir. Soc. Med. Nat. Iasi. 112, 432–6 (2008)

    PubMed  Google Scholar 

  17. 17.

    I. Molina-Ortiz, R.A. Bartolome, P. Hernandez-Varas, G.P. Colo, J. Teixido, Overexpression of E-cadherin on melanoma cells inhibits chemokine-promoted invasion involving p190RhoGAP/p120ctn-dependent inactivation of RhoA. J. Biol. Chem. 284, 15147–57 (2009)

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    R.W. Pang, R.T. Poon, From molecular biology to targeted therapies for hepatocellular carcinoma: the future is now. Oncology 72, 30–44 (2007)

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    S. Tommasi, R. Pinto, B. Pilato, A. Paradiso, Molecular pathways and related target therapies in liver carcinoma. Curr. Pharm. Des. 13, 3279–87 (2007)

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    S. Boyault, D.S. Rickman, A. de Reynies, C. Balabaud, S. Rebouissou, E. Jeannot et al., Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology 45, 42–52 (2007)

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    T. Parasassi, R. Brunelli, L. Bracci-Laudiero, G. Greco, A.C. Gustafsson, E.K. Krasnowska et al., Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms. Cell Death Differ. 12, 1285–96 (2005)

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    J.C. Fleet, L. Wang, O. Vitek, B.A. Craig, H.J. Edenberg, Gene expression profiling of Caco-2 BBe cells suggests a role for specific signaling pathways during intestinal differentiation. Physiol. Genomics 13, 57–68 (2003)

    PubMed  CAS  Google Scholar 

  23. 23.

    S. Sell, Stem cell origin of cancer and differentiation therapy. Crit. Rev. Oncol. Hematol. 51, 1–28 (2004)

    PubMed  Article  Google Scholar 

  24. 24.

    R.L. Eckert, J.F. Crish, T. Efimova, S. Balasubramanian, Antioxidants regulate normal human keratinocyte differentiation. Biochem. Pharmacol. 68, 1125–31 (2004)

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    J. Leipner, R. Saller, Systemic enzyme therapy in oncology: Effect and mode of action. Drugs 59, 769–80 (2000)

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    E.R. Camp, V.J. Findlay, S.G. Vaena, J. Walsh, D.N. Lewin, D.P. Turner et al., Slug expression enhances tumor formation in a noninvasive rectal cancer model. J. Surg. Res. 170(1), 56–63 (2011)

    PubMed  Article  Google Scholar 

  27. 27.

    P.B. Gupta, T.T. Onder, G. Jiang, K. Tao, C. Kuperwasser, R.A. Weinberg et al., Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138, 645–59 (2009)

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    A. Voulgari, A. Pintzas, Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim. Biophys. Acta 1796, 75–90 (2009)

    PubMed  CAS  Google Scholar 

  29. 29.

    J. Fogh, J.M. Fogh, T. Orfeo, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59, 221–6 (1977)

    PubMed  CAS  Google Scholar 

  30. 30.

    M. Pinto, S. Robineleon, M.D. Appay, M. Kedinger, N. Triadou, E. Dussaulx et al., Enterocyte-like differentiation and polarization of the human-colon carcinoma cell-line caco-2 in culture. Biol. Cell 47, 323–330 (1983)

    Google Scholar 

  31. 31.

    M.D. Basson, G.A. Turowski, Z. Rashid, F. Hong, J.A. Madri, Regulation of human colonic cell line proliferation and phenotype by sodium butyrate. Dig. Dis. Sci. 41, 1989–93 (1996)

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    S.R. Wood, Q. Zhao, L.H. Smith, C.K. Daniels, Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression. Tissue Cell 35, 47–58 (2003)

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    S.K. Kurdistani, P. Arizti, C.L. Reimer, M.M. Sugrue, S.A. Aaronson, S.W. Lee, Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage. Cancer Res. 58, 4439–44 (1998)

    PubMed  CAS  Google Scholar 

  34. 34.

    J.P. Brunet, N. Jourdan, J. Cotte-Laffitte, C. Linxe, M. Geniteau-Legendre, A. Servin et al., Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration. J. Virol. 74, 10801–6 (2000)

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    R.J. Guang, J.L. Ford, Y.N. Fu, Y.Z. Li, L.M. Shaw, A.B. Pardee, Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer. Cancer Res. 60, 749–755 (2000)

    Google Scholar 

  36. 36.

    J.M. Anderson, Molecular structure of tight junctions and their role in epithelial transport. News Physiol. Sci. 16, 126–30 (2001)

    PubMed  CAS  Google Scholar 

  37. 37.

    Y. Kang, J. Massague, Epithelial-mesenchymal transitions: Twist in development and metastasis. Cell 118, 277–9 (2004)

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    A.A. Hardikar, B. Marcus-Samuels, E. Geras-Raaka, B.M. Raaka, M.C. Gershengorn, Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates. Proc. Natl. Acad. Sci. U. S. A. 100, 7117–22 (2003)

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Y. Wu, J. Li, S. Saleem, S.P. Yee, A.A. Hardikar, R. Wang, c-Kit and stem cell factor regulate PANC-1 cell differentiation into insulin- and glucagon-producing cells. Lab. Invest. 90(1373–84) (2010)

  40. 40.

    J.A. Marchal, J. Prados, J. Campos, F. González, C. Melguizo, C. Velez et al., Therapeutic potential of differentiation in cancer and normal stem cells, in New Cell Differentiation Research Topics, ed. by H. Saitama (Nova Science Publisher, Inc, New York, 2008), pp. 7–77

    Google Scholar 

  41. 41.

    J. Czyzewska, K. Guzinska-Ustymowicz, M. Ustymowicz, A. Pryczynicz, A. Kemona, The expression of E-cadherin-catenin complex in patients with advanced gastric cancer: role in formation of metastasis. Folia Histochem. Cytobiol. 48, 37–45 (2010)

    PubMed  Article  Google Scholar 

  42. 42.

    I.J. Chalmers, M. Aubele, E. Hartmann, E. Braungart, M. Werner, H. Hofler, M.J. Atkinson, Mapping the chromosome 16 cadherin gene cluster to a minimal deleted region in ductal breast cancer. Cancer Genet. Cytogenet. 126, 39–44 (2001)

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    W. Feng, R. Orlandi, N. Zhao, M.L. Carcangiu, E. Tagliabue, J. Xu et al., Tumor suppressor genes are frequently methylated in lymph node metastases of breast cancers. B.M.C. Cancer 10(378–88) (2010)

    Google Scholar 

  44. 44.

    M.T. Debies, D.R. Welch, Genetic basis of human breast cancer metastasis. J. Mammary Gland. Biol. Neoplasia 6, 441–51 (2001)

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    R.B. Hazan, R. Qiao, R. Keren, I. Badano, K. Suyama, Cadherin switch in tumor progression. Ann. N. Y. Acad. Sci. 1014, 155–63 (2004)

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Y. Uchikado, H. Okumura, S. Ishigami, T. Setoyama, M. Matsumoto, T. Owaki et al., Increased Slug and decreased E-cadherin expression is related to poor prognosis in patients with gastric cancer. Gastric Cancer 14, 41–9 (2011)

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Y. Li, C.Q. Chen, Y.L. He, S.R. Cai, D.J. Yang, W.L. He et al., Abnormal expression of E-cadherin in tumor cells is associated with poor prognosis of gastric carcinoma. J. Surg. Oncol. 106(3), 304–10 (2012)

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    X. Tian, Z. Liu, B. Niu, J. Zhang, T.K. Tan, S.R. Lee et al., E-cadherin/beta-catenin complex and the epithelial barrier. J. Biomed. Biotechnol. 2011, 1–6 (2011)

    Google Scholar 

  49. 49.

    J.A. Marchal, M.C. Nunez, A. Aranega, M.A. Gallo, A. Espinosa, J.M. Campos, Acyclonucleosides, modified seco-nucleosides, and salicyl- or catechol-derived acyclic 5-fluorouracil O, N-acetals: antiproliferative activities, cellular differentiation and apoptosis. Curr. Med. Chem. 16, 1166–83 (2009)

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    J.P. Thiery, J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131–42 (2006)

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    V. Bolos, H. Peinado, M.A. Perez-Moreno, M.F. Fraga, M. Esteller, A. Cano, The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J. Cell. Sci. 116, 499–511 (2003)

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    C. Castro Alves, E. Rosivatz, C. Schott, R. Hollweck, I. Becker et al., Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin. J. Pathol. 211, 507–15 (2007)

    PubMed  Article  CAS  Google Scholar 

  53. 53.

    P. Jethwa, M. Naqvi, R.G. Hardy, N.A. Hotchin, S. Roberts, R. Spychal et al., Overexpression of Slug is associated with malignant progression of esophageal adenocarcinoma. World J. Gastroenterol. 14, 1044–52 (2008)

    PubMed  Article  CAS  Google Scholar 

  54. 54.

    K. Zhang, D. Chen, X. Jiao, S. Zhang, X. Liu, J. Cao et al., Slug enhances invasion ability of pancreatic cancer cells through upregulation of matrix metalloproteinase-9 and actin cytoskeleton remodeling. Lab. Invest. 91, 426–38 (2011)

    PubMed  Article  CAS  Google Scholar 

  55. 55.

    M.G. Mendez, S. Kojima, R.D. Goldman, Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 24, 1838–51 (2010)

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

Macarena Peran was supported by a grant from Jaen University, Spain (“Plan de Apoyo a la Investigación, Desarrollo Tecnológico e Innovación. IV. Programa de Ayudas a los Investigadores”). Work in the laboratory of Bath University was funded by Propanc Pty Ltd. Work in the laboratory of J.A. Marchal at Granada University was funded by the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria, FEDER, grant number PI10/02295).

Conflict of interest

Dr Julian Kenyon is the Founder and Scientific Director of Propanc Pty Ltd and owns stock in the company. Propanc Pty Ltd and the University of Bath have filed a joint patent from the scientific research undertaken in this report

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Macarena Perán or Julian Kenyon.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Perán, M., Marchal, J.A., García, M.A. et al. In vitro treatment of carcinoma cell lines with pancreatic (pro)enzymes suppresses the EMT programme and promotes cell differentiation. Cell Oncol. 36, 289–301 (2013). https://doi.org/10.1007/s13402-013-0134-8

Download citation

Keywords

  • Cell adhesion
  • Cell differentiation
  • Pancreatic (pro)enzymes
  • Epithelial-mesenchymal transition
  • Cancer treatment