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Chlorophyllin abrogates canonical Wnt/β-catenin signaling and angiogenesis to inhibit the development of DMBA-induced hamster cheek pouch carcinomas

Cellular Oncology volume 35, pages 385–395 (2012)Cite this article

An Erratum to this article was published on 16 February 2013

Abstract

Background

Chlorophyllin, a water soluble semi-synthetic food-grade derivative is reported to exhibit a wide range of beneficial health effects. We investigated the effect of chlorophyllin supplementation on Wnt/β-catenin and vascular endothelial growth factor (VEGF) signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model.

Methods and results

Hamsters were divided into 4 groups. The right buccal pouches of group 1 and 2 hamsters were painted with 0.5 % DMBA for 14 weeks. Group 2 animals received in addition chlorophyllin (4 mg/kg bw) in the diet. Group 3 animals received chlorophyllin alone and group 4 animals served as control. mRNA and protein expression of components of Wnt, VEGF, and PI3K/Akt signaling pathways were analyzed by RT-PCR and Western blot analysis. Dietary chlorophyllin administration suppressed the development of HBP carcinomas by altering the expression of several components of the Wnt/β-catenin signaling pathway. This was associated with inhibition of angiogenesis as evidenced by decreased expression of the proangiogenic factors HIF-1α, VEGF, and VEGFR2. Chlorophyllin administration also downregulated the expression of histone deacetylases involved in epigenetic regulation of tumor angiogenesis.

Conclusion

Dietary chlorophyllin that abrogates Wnt/β-catenin and VEGF signaling by targeting a multitude of key signaling molecules is an attractive candidate for preventing tumor progression.

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References

  1. R. Dashwood, Chlorophylls as anticarcinogens. Int. J. Oncol. 10, 721–727 (1997)

    PubMed  CAS  Google Scholar 

  2. R. Dashwood, T. Negishi, H. Hayatsu, V. Breinholt, J. Hendricks, G. Bailey, Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mutat. Res. 399, 245–253 (1998)

    PubMed  Article  CAS  Google Scholar 

  3. L.C. Chiu, C.K. Kong, V.E. Ooi, The chlorophyllin-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells is associated with ERK deactivation and cyclin D1 depletion. Int. J. Mol. Med. 16, 735–740 (2005)

    PubMed  CAS  Google Scholar 

  4. K. Chimploy, G.D. Diaz, Q. Li, O. Carter, W.M. Dashwood, C.K. Mathews, D.E. Williams, G.S. Bailey, R.H. Dashwood, E2F4 and ribonucleotide reductase mediate S phase arrest in colon cancer cells treated with chlorophyllin. Int. J. Cancer 125, 2086–2094 (2009)

    PubMed  Article  CAS  Google Scholar 

  5. X.W. Ding, X.L. Ding, S. Zheng, H.J. Yang, CHL prevent colon neoplasms in mice and its selective inhibition on COX-2. Ai Zheng 23, 1409–1413 (2004)

    PubMed  Google Scholar 

  6. M.T. Simonich, T. McQuistan, C. Jubert, C. Pereira, J.D. Hendricks, M. Schimerlik, B. Zhu, R.H. Dashwood, D.E. Williams, G.S. Bailey, Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout. Food Chem. Toxicol. 46, 1014–1024 (2008)

    PubMed  Article  CAS  Google Scholar 

  7. A.P. Reddy, U. Harttig, M.C. Barth, W.M. Baird, M. Schimerlik, J.D. Hendricks, G.S. Bailey, Inhibition of dibenzo[a, l]pyrene-induced multi-organ carcinogenesis by dietary chlorophyllin in rainbow trout. Carcinogenesis 20, 1919–1926 (1999)

    PubMed  Article  CAS  Google Scholar 

  8. P. Thiyagarajan, R. Senthil Murugan, K. Kavitha, P. Anitha, D. Prathiba, S. Nagini, Dietary chlorophyllin inhibits the canonical NF-κB signaling pathway and induces intrinsic apoptosis in a hamster model of oral oncogenesis. Food Chem. Toxicol. 50, 867–876 (2012)

    PubMed  Article  CAS  Google Scholar 

  9. D.T. Shaughnessy, L.M. Gangarosa, B. Schliebe, D.M. Umbach, Z. Xu, B. MacIntosh, M.G. Knize, P.P. Matthews, A.E. Swank, R.S. Sandler, D.M. DeMarini, J.A. Taylor, Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt. PLoS One 6, e18707 (2011)

    PubMed  Article  CAS  Google Scholar 

  10. P.A. Egner, J.B. Wang, Y.R. Zhu, B.C. Zhang, Y. Wu, Chlorophyllin intervention reduces aflatoxin–DNA adducts in individuals at high risk for liver cancer. Proc. Natl. Acad. Sci. USA 98, 14601–14606 (2001)

    PubMed  Article  CAS  Google Scholar 

  11. J.P. Kamat, K.K. Boloor, T.P. Devasagayam, Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo. Biochim. Biophys. Acta 1487, 113–127 (2000)

    PubMed  Article  CAS  Google Scholar 

  12. S. Arimoto, S. Fukuoka, C. Itome, H. Nakano, H. Rai, H. Hayatsu, Binding of polycyclic planar mutagens to chlorophyllin resulting in inhibition of the mutagenic activity. Mutat. Res. 287, 293–305 (1993)

    PubMed  Article  CAS  Google Scholar 

  13. M. Hirose, A. Nishikawa, M. Shibutani, T. Imai, T. Shirai, Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ. Mol. Mutagen. 39, 271–278 (2002)

    PubMed  Article  CAS  Google Scholar 

  14. V. Breinholt, M. Schimerlik, R. Dashwood, G. Bailey, Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen. Chem. Res. Toxicol. 8, 506–514 (1995)

    PubMed  Article  CAS  Google Scholar 

  15. K. John, R.L. Divi, C. Keshava, C.C. Orozco, M.E. Schockley, D.L. Richardson, M.C. Poirier, J. Nath, A. Weston, CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin. Cancer Lett. 292, 254–260 (2010)

    PubMed  Article  CAS  Google Scholar 

  16. Y. Zhang, L. Guan, X. Wang, T. Wen, J. Xing, J. Zhao, Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free. Radic. Res. 42, 362–371 (2008)

    PubMed  Article  Google Scholar 

  17. N. Tachino, D. Guo, W.M. Dashwood, S. Yamane, R. Larsen, R. Dashwood, Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen. Mutat. Res. 308, 191–203 (1994)

    PubMed  Article  CAS  Google Scholar 

  18. G.D. Diaz, Q. Li, R.H. Dashwood, Caspase-8 and apoptosis-inducing factor mediate a cytochrome c-independent pathway of apoptosis in human colon cancer cells induced by the dietary phytochemical chlorophyllin. Cancer Res. 63, 1254–1261 (2003)

    PubMed  CAS  Google Scholar 

  19. M.S. Khan, D. Halagowder, S.N. Devaraj, Methylated chrysin induces co-ordinated attenuation of the canonical Wnt and NF-κB signaling pathway and upregulates apoptotic gene expression in the early hepatocarcinogenesis rat model. Chem. Biol. Interact. 193, 12–21 (2011)

    PubMed  Article  CAS  Google Scholar 

  20. S. Huang, C.A. Pettaway, H. Uehara, C.D. Bucana, I.J. Fidler, Blockade of NfkappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 20, 4188–4197 (2001)

    PubMed  Article  CAS  Google Scholar 

  21. H.L. Pahl, Activators and target genes of Rel/NF-κB transcription factors. Oncogene 18, 6853–6866 (1999)

    PubMed  Article  CAS  Google Scholar 

  22. R.S. Tarapore, I.A. Siddiqui, H. Mukhtar, Modulation of Wnt/β-catenin signalling pathway by bioactive food components. Carcinogenesis 33, 483–491 (2012)

    PubMed  Article  CAS  Google Scholar 

  23. R. Jr, Roskoski, Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit. Rev. Oncol. Hematol. 62, 179–213 (2007)

    Article  Google Scholar 

  24. A. Surazynski, P. Steven, S.P. Donald, K. Sandra, S.K. Cooper, M.A. Whiteside, K. Salnikow, Y. Liu, J.M. Phang, Extracellular matrix and HIF-1 signaling: the role of prolidase. Int. J. Cancer 22, 1435–1440 (2008)

    Google Scholar 

  25. H.P. Gerber, A. McMurtrey, J. Kowalski, M. Yan, B.A. Keyt, V. Dixit, N. Ferrara, Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem. 273, 30336–30343 (1998)

    PubMed  Article  CAS  Google Scholar 

  26. I.H. Koumakpayi, C. Le Page, A.M. Mes-Masson, F. Saad, Hierarchical clustering of immunohistochemical analysis of the activated erbB/PI3K/Akt/NF-kappaB signalling pathway and prognostic significance in prostate cancer. Br. J. Cancer 102, 1163–1173 (2010)

    PubMed  Article  CAS  Google Scholar 

  27. O. Witt, H.E. Deubzer, T. Milde, I. Oehme, HDAC family: what are the cancer relevant targets? Cancer Lett. 77, 8–21 (2009)

    Article  Google Scholar 

  28. G. Shklar, Development of experimental oral carcinogenesis and its impact on current oral cancer research. J. Dent. Res. 78, 1768–1772 (1999)

    PubMed  Article  CAS  Google Scholar 

  29. E. Madrigal-Bujaidar, N. Velazquez-Guadarrama, S. Diaz-Barriga, Inhibitory effect of chlorophyllin on the frequency of sister chromatid exchanges produced by benzo[a]pyrene in vivo. Mutat. Res. 388, 79–83 (1997)

    PubMed  Article  CAS  Google Scholar 

  30. S. Nagini, P. Vidjaya Letchoumy, A. Thangavelu, R. Cr, Of humans and hamsters: a comparative evaluation of carcinogen activation, DNA damage, cell proliferation, apoptosis, invasion, and angiogenesis in oral cancer patients and hamster buccal pouch carcinomas. Oral. Oncol. 45, e31–e37 (2009)

    PubMed  Article  CAS  Google Scholar 

  31. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)

    PubMed  Article  CAS  Google Scholar 

  32. S. Legrand-Poels, S. Schoonbrodt, J. Piette, Regulation of interleukin-6 gene expression by pro-inflammatory cytokines in a colon cancer cell line. Biochem. J. 349, 765–773 (2000)

    PubMed  CAS  Google Scholar 

  33. Y. Fu, S. Zheng, N. An, T. Athanasopoulos, L. Popplewell, A. Liang, K. Li, C. Hu, Y. Zhu, β-catenin as a potential key target for tumor suppression. Int. J. Cancer 129, 1541–1551 (2011)

    PubMed  Article  CAS  Google Scholar 

  34. A. Bafico, G. Liu, L. Goldin, V. Harris, S.A. Aaronson, An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell. 6, 497–506 (2004)

    PubMed  Article  CAS  Google Scholar 

  35. R. Najdi, R.F. Holcombe, M.L. Waterman, Wnt signaling and colon carcinogenesis: beyond APC. J. Carcinog. 10, 5 (2011)

    PubMed  Article  CAS  Google Scholar 

  36. C. Gaston-Massuet, C.L. Andoniadou, M. Signore, S.A. Jayakody, N. Charolidi, R. Kyeyune, B. Vernay, T.S. Jacques, M.M. Taketo, P. Le Tissier, M.T. Dattani, J.P. Martinez-Barbera, Increased wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans. Nutr. Cancer 63, 558–564 (2011)

    Article  Google Scholar 

  37. L. Queimado, D. Obeso, M.D. Hatfield, Y. Yang, D.M. Thompson, A.M. Reis, Dysregulation of Wnt pathway components in human salivary gland tumors. Arch. Otolaryngol. Head Neck Surg. 134, 94–101 (2008)

    PubMed  Article  Google Scholar 

  38. I. Gehrke, R.K. Gandhirajan, K.A. Kreuzer, Targeting the WNT/b-catenin/TCF/LEF1 axis in solid and haematological cancers: multiplicity of therapeutic options. Eur. J. Cancer 45, 2759–2767 (2009)

    PubMed  Article  Google Scholar 

  39. R. Vidya Priyadarsini, R. Senthil Murugan, S. Nagini, Aberrant activation of Wnt/β-catenin signaling pathway contributes to the sequential progression of DMBA induced HBP carcinomas. Oral Oncol. 48, 33–39 (2012)

    PubMed  Article  Google Scholar 

  40. J.K. Sethi, A. Vidal-Puig, Wnt signalling and the control of cellular metabolism. Biochem. J. 427, 1–17 (2010)

    PubMed  Article  CAS  Google Scholar 

  41. B.W. Doble, J.R. Woodgett, GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell. Sci. 116, 1175–1186 (2003)

    PubMed  Article  CAS  Google Scholar 

  42. R. Mishra, Gliycogen synthase kinase 3 beta: can it be a target for oral cancer. Mol. Cancer 9, 144 (2010)

    PubMed  Article  Google Scholar 

  43. J. Luo, Glycogen synthase kinase 3beta (GSK3beta) in tumorigenesis and cancer chemotherapy. Cancer Lett. 273, 194–200 (2009)

    PubMed  Article  CAS  Google Scholar 

  44. O. Carter, G.S. Bailey, R.H. Dashwood, The dietary phytochemical chlorophyllin alters E-cadherin and beta-catenin expression in human colon cancer cells. J. Nutr. 134, 3441–344 (2004)

    Google Scholar 

  45. S. Song, N. Mazurek, C. Liu, Y. Sun, Q.Q. Ding, K. Liu, M.C. Hung, R.S. Bresalier, Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity. Cancer Res. 69, 1343–1349 (2009)

    PubMed  Article  CAS  Google Scholar 

  46. A. Iamaroon, S. Krisanaprakornkit, Overexpression and activation of Akt2 protein in oral squamous cell carcinoma. Oral Oncol. 45, e175–e179 (2009)

    PubMed  Article  CAS  Google Scholar 

  47. J.A. Morrison, M.L. Gulley, R. Pathmanathan, N. Raab-Traub, Differential signalling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma. Cancer Res. 64, 5251–5260 (2004)

    PubMed  Article  CAS  Google Scholar 

  48. K.H. Chun, H.Y. Lee, K. Hassan, F. Khuri, W.K. Hong, R. Lotan, Implication of protein kinase B/Akt and Bcl-2/Bcl-XL suppression by the farnesyl transferase inhibitor SCH66336 in apoptosis induction in squamous carcinoma cells. Cancer Res. 63, 4796–4800 (2003)

    PubMed  CAS  Google Scholar 

  49. P. Amornphimoltham, V. Sriuranpong, V. Patel, F. Benavides, C.J. Conti, J. Sauk, E.A. Sausville, A.A. Molinolo, J.S. Gutkind, Persistent activation of the Akt pathway in head and neck squamous cell carcinoma: a potential target for UCN-01. Clin. Cancer Res. 10, 4029–4037 (2004)

    PubMed  Article  CAS  Google Scholar 

  50. Y. Li, Z. Wang, D. Kong, R. Li, S.H. Sarkar, F.H. Sarkar, Regulation of Akt/FOXO3a/GSK-3beta/AR signaling network by isoflavone in prostate cancer cells. J. Biol. Chem. 283, 27707–27716 (2008)

    PubMed  Article  CAS  Google Scholar 

  51. P. Roy, N. Kalra, S. Prasad, J. George, Y. Shukla, Chemopreventive potential of resveratrol in mouse skin tumors through regulation of mitochondrial and PI3K/AKT signaling pathways. Pharm. Res. 26, 211–217 (2009)

    PubMed  Article  CAS  Google Scholar 

  52. C. Hope, K. Planutis, M. Planutiene, M.P. Moyer, K.S. Johal, J. Woo, C. Santoso, J.A. Hanson, R.F. Holcombe, Low concentrations of resveratrol inhibit Wnt signal throughput in colon-derived cells: implications for colon cancer prevention. Mol. Nutr. Food Res. 52, S52–S61 (2008)

    PubMed  Google Scholar 

  53. M. Potente, H. Gerhardt, P. Carmeliet, Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887 (2011)

    PubMed  Article  CAS  Google Scholar 

  54. X. Lu, Y. Kang, Hypoxia and hypoxia-inducible factors: master regulators of metastasis. Clin. Cancer Res. 16, 5928–5935 (2011)

    Article  Google Scholar 

  55. L. Ellis, H. Hammers, R. Pili, Targeting tumor angiogenesis with histone deacetylase inhibitors. Cancer Lett. 280, 145–153 (2009)

    PubMed  Article  CAS  Google Scholar 

  56. D.Z. Qian, S.K. Kachhap, S.J. Collis, H.M. Verheul, M.A. Carducci, P. Atadja, R. Pili, Class II histone deacetylases are associated with VHL independent regulation of hypoxia-inducible factor 1α. Cancer Res. 66, 668814–668821 (2006)

    Google Scholar 

  57. P. Anitha, R. Vidya Priyadarsini, K. Kavitha, P. Thiyagarajan, S. Nagini, Ellagic acid coordinately attenuates Wnt/β-catenin and NF-κB signaling pathways to induce intrinsic apoptosis in an animal model of oral oncogenesis. Eur. J. Nutr. (2012).

  58. T.A. Bhat, R.P. Singh, Tumor angiogenesis—a potential target in cancer chemoprevention. Food Chem. Toxicol. 6, 1334–1345 (2008)

    Google Scholar 

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Acknowledgements

We gratefully acknowledge the technical assistance of Mr. P. Thiyagarajan. This work was supported by a grant from the Department of Biotechnology, New Delhi, India under the 7th FP of the Indo-EU Joint Collaborative Project on ‘FUNCFOOD’ (BT/IN/EU/17/SN/2009).

Ethical standards

This research complies with the ethical guidelines of the Indian Council of Medical Research, and has been approved by the institutional ethical committee.

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Authors and Affiliations

  1. Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, 608 002, Tamil Nadu, India

    Nagini Siddavaram & Vidya Priyadarsini Ramamurthi

  2. Department of Oral & Maxillofacial Pathology, Rajah Muthiah Dental College & Hospital, Annamalainagar, 608 002, Tamil Nadu, India

    Veeravarmal Veeran

  3. Centre for Life Sciences, Central University of Jharkhand, Ranchi, 835205, Jharkhand, India

    Rajakishore Mishra

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  1. Nagini Siddavaram
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  2. Vidya Priyadarsini Ramamurthi
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Correspondence to Nagini Siddavaram.

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Siddavaram, N., Ramamurthi, V.P., Veeran, V. et al. Chlorophyllin abrogates canonical Wnt/β-catenin signaling and angiogenesis to inhibit the development of DMBA-induced hamster cheek pouch carcinomas. Cell Oncol. 35, 385–395 (2012). https://doi.org/10.1007/s13402-012-0099-z

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Keywords

  • Angiogenesis
  • Chlorophyllin
  • Hamster buccal pouch
  • PI3K
  • Wnt