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Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 390, Issue 10, pp 1005–1013 | Cite as

Cytotoxicity against tumor cell lines and anti-inflammatory properties of chitinases from Calotropis procera latex

  • Carolina Araújo Viana
  • Márcio V. Ramos
  • José Delano Barreto Marinho Filho
  • Letícia Veras Costa Lotufo
  • Ingrid Samantha Tavares Figueiredo
  • Jefferson Soares de Oliveira
  • Pietro Mastroeni
  • José Vitor Lima-FilhoEmail author
  • Nylane Maria Nunes AlencarEmail author
Original Article

Abstract

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 μg/mL but induced high messenger RNA expression of IL-6, IL1-β, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 μg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-β were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

Keywords

Anticancer activity Folk medicine Laticifer proteins Nitric oxide 

Notes

Acknowledgements

This study is part of the consortium Molecular Biotechnology of Plant Latex supported by the Northeast Biotechnology Network (RENORBIO-Brazil) and funded by the Brazilian National Counsel of Technological and Scientific Development (CNPq). The authors are also grateful to CAPES for scholarship support to Carolina Viana, and Ceará State Foundation (FUNCAP—Program PPSUS). We thank Panagiotis Tourlomousis, Kate Fitzgerald, and Clare Bryant for providing the macrophage cell lineage used in the present study.

References

  1. Alencar NMN, Figueiredo IST, Vale MR, Bitencurt FS, Oliveira JS, Ribeiro RA, Ramos MV (2004) Anti-inflammatory effect of the latex from Calotropis procera in three different experimental models: peritonitis, paw edema and hemorrhagic cystitis. Planta Med 70:1144–1149CrossRefPubMedGoogle Scholar
  2. Alencar NMN, Oliveira JS, Mesquita RO, Lima MW, Vale MR, Etchells JP, Freitas CDT, Ramos MV (2006) Pro- and anti-inflammatory activities of the latex from Calotropis procera (Ait.) R.Br. are triggered by compounds fractionated by dialysis. Inflamm Res 55:559–564CrossRefPubMedGoogle Scholar
  3. Ali AMM, Kawasakil T, Yamada T (2007) Characterization of a chitinase gene encoded by virus-sensitive Chlorella strains and expressed during vírus infection. Arab J Biotech 10:81–96Google Scholar
  4. Arya S, Kumar VL (2005) Anti-inflammatory efficacy of extracts of latex of Calotropis procera against different mediators of inflammation. Mediat Inflamm 2005:228–232CrossRefGoogle Scholar
  5. Benjamim CF, Silva JS, Fortes ZB, Oliveira MA, Ferreira SH, Cunha FQ (2002) Inhibition of leukocyte rolling by nitric oxide during sepsis leads to reduced migration of active microbicidal neutrophils. Infect Immun 70:3602–3610CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal Biochemist 72:248–254CrossRefGoogle Scholar
  7. Chaudhary P, de Araújo VC, Ramos MV, Kumar VL (2015) Antiedematogenic and antioxidant properties of high molecular weight protein sub-fraction of Calotropis procera latex in rat. J Basic Clin Pharm 6:69–73CrossRefPubMedPubMedCentralGoogle Scholar
  8. de Souza GEP, Ferreira SH (1985) Blockade by antimacrophage serum of the migration of PMN neutrophils into the inflamed peritoneal cavity. Agents Actions 17:97–103CrossRefPubMedGoogle Scholar
  9. Dewan S, Kumar S, Kumar VL (2000a) Antipyretic effect of latex of Calotropis procera. Indian J Pharmacol 32:247–252Google Scholar
  10. Dewan S, Sangraula H, Kumar VL (2000b) Preliminary studies on the analgesic activity of latex of Calotropris procera. J Ethnopharmacol 73:307–311CrossRefPubMedGoogle Scholar
  11. Di Rosa M, Distefano G, Zorena K, Malaguarnera L (2015) Chitinases and immunity: ancestral molecules with new functions. Immunobiology 221:399–411CrossRefPubMedGoogle Scholar
  12. Elmonem MA, Van Den Heuvel LP, Levtchenko EN (2016) Immunomodulatory effects of chitotriosidase enzyme. Enzyme Res. doi: 10.1155/2016/2682680
  13. Frederiksen RF, Paspaliari DK, Larsen T, Storgaard BG, Larsen MH, Ingmer H, Palcic MM, Leisner JJ (2013) Bacterial chitinases and chitin-binding proteins as virulence factors. Microbiol 159:833–847CrossRefGoogle Scholar
  14. Freitas CDT, Viana CA, Vasconcelos IM, Moreno FBB, Lima-Filho JV, Oliveira HD, Moreira RA, Monteiro-Moreira ACO, Ramos MV (2016) First insights into the diversity and functional properties of chitinases of the latex of Calotropis procera. Plant Physiol Bioch 2016:361–371CrossRefGoogle Scholar
  15. Geginat J, Larghi P, Paroni M, Nizzoli G, Penatti A, Pagani M, Gagliani N, Meroni P, Abrignani S, Flavell RA (2016) The light and the dark sides of interleukin-10 in immune-mediated diseases and cancer. Cytokine Growth Factor RevGoogle Scholar
  16. Hoedon T, Mathan G, Arya S, Kumar VL, Kumar V (2006) Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma. World J Gastroenterol 12:2517–2522CrossRefGoogle Scholar
  17. Horobin RW (2011) How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotech Histochem 86:36–51. doi: 10.3109/10520295.2010.515491
  18. Kawada M, Hachiya Y, Arihiro A, Mizoguchi E (2007) Role of mammalian chitinases in inflammatory conditions. Keio J Med 56:21–27CrossRefPubMedGoogle Scholar
  19. Kesari P, Patil DN, Kumar P, Tomar S, Sharma AK, Kumar P (2015) Structural and functional evolution of chitinase-like proteins from plants. Proteomics 15:1693–1705CrossRefPubMedGoogle Scholar
  20. Ku BM, Lee YK, Ryu J, Jeong JY, Choi J, Eun KM, Shin HY, Kim DJ, Hwang EM, Yoo JC, Park JY, Roh GS, Kim HJ, Cho GJ, Choi WS, Paek SH, Kang SS (2011) CHI3L1 (YKL-40) is expressed in human gliomas and regulates the invasion, growth and survival of glioma cells. Int J Cancer 128:1316–1326CrossRefPubMedGoogle Scholar
  21. Kzhyshkowska J, Yin S, Liu T, Riabov V, Mitrofanova I (2016) Role of chitinase-like proteins in cancer. Biol Chem 397:231–247CrossRefPubMedGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  23. Lam SK, Ng TB (2010) Acaconin, a chitinase-like antifungal protein with cytotoxic and anti-HIV-1 reverse transcriptase activities from Acacia confusa seeds. Acta Biochem Pol 57:299–304Google Scholar
  24. Lee CG, Da Silva CA, Dela Cruz CS, Ahangari F, Ma B, Kang JM, He C, Takyar S, Elias JÁ (2011) Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodelling, and injury. Annu Rev Physiol 73:479–501CrossRefPubMedGoogle Scholar
  25. Lima-Filho JV, Patriota JM, Silva AFB, Pontes-Filho NT, Oliveira RSB, Alencar NMN, Ramos MV (2010) Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium. J Ethnopharmacol 129:327–334CrossRefPubMedGoogle Scholar
  26. Lin J, Lin E, Nemunaitis J (2004) Bacteria in the treatment of cancer. Curr Opin Mol Ther 6:629–639PubMedGoogle Scholar
  27. Malysheva EV, Kruglov SV, Khomenko IP, Bakhtina LY, Pshennikova MG, Manukhina EB, Malyshev IY (2006) Role of extracellular and intracellular nitric oxide in the regulation of macrophage responses. Bull Exp Biol Med 141:404–406CrossRefPubMedGoogle Scholar
  28. Nagpal K, Plantinga TS, Wong J, Monks BG, Gay NJ, Netea MG, Fitzgerald KA, Golenbock DT (2009) A TIR domain variant of MyD88 adapter-like (mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling. J Biol Chem 284:25742–25748CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nascimento DCO, Ralph MT, Batista JEC, Silva DMF, Gomes-Filho MA, Alencar NM, Leal NC, Ramos MV, Lima-Filho JV (2016) Latex protein extracts from Calotropis procera with immunomodulatory properties protect against experimental infections with Listeria monocytogenes. Phytomedicine 23:745–753CrossRefPubMedGoogle Scholar
  30. Oliveira JS, Bezerra DP, Freitas CDT, Filho DBM, Moraes MO, Pessoa C, Costa-Lotufo LV, Ramos MV (2007) In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br. Toxicol Vitr 21:1563–1573CrossRefGoogle Scholar
  31. Oliveira RSB, Figueiredo IST, Freitas LBN, Pinheiro RSP, Brito GAC, Alencar NMN, Ramos MV, Ralph MT, Lima-Filho JV (2012) Inflammation induced by phytomodulatory proteins from the latex of Calotropis procera (Asclepiadaceae) protects against Salmonella infection in a murine model of typhoid fever. Inflamm Res 61:689–698CrossRefPubMedGoogle Scholar
  32. Pan XQ (2012) The mechanism of the anticancer function of M1 macrophages and their use in the clinic. Chin J Cancer 31:557–563PubMedPubMedCentralGoogle Scholar
  33. Pan XQ, Shih CC, Harday J (2005) Chitinase induces lysis of MCF-7 cells in culture and of human breast cancer xenograft B11-2 in SCID mice. Anticancer Res 25:3167–3172PubMedGoogle Scholar
  34. Ramos MV, Oliveira JS, FigueiredoJG FIST, Kumar VL, Bitencourt FS, Cunha FQ, Oliveira RSB, Bomfim LR, Lima-Filho JV, Alencar NMN (2009) Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model. J Ethnopharmacol 125:387–392CrossRefPubMedGoogle Scholar
  35. Riabov V, Gudima A, Wang N, Mickley A, Orekhov A, Kzhyshkowska J (2014) Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis. Front Physiol. doi: 10.3389/fphys.2014.00075
  36. Rivera A, Siracusa MC, Yap GS, Gause WC (2016) Innate cell communication kick-starts pathogen-specific immunity. Nat Immunol 17:356–363CrossRefPubMedPubMedCentralGoogle Scholar
  37. Schähs M, Strasser R, Stadlmann J, Kunert R, Rademacher T, Steinkellner H (2007) Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern. Plant Biotechnol J 5:657–663CrossRefPubMedGoogle Scholar
  38. Secco DD, Paron JA, de Oliveira SHP, Ferreira SH, Silva JS, Cunha FDQ (2003) Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis. Nitric Oxide - Biol Chem 9:153–164CrossRefGoogle Scholar
  39. van Eijk M, van Roomen CP, Renkema GH, Bussink AP, Andrews L, Blommaart EF, Sugar A, Verhoeven AJ, Boot RG, Aerts JM (2005) Characterization of human phagocyte-derived chitotriosidase, a component of innate immunity. Int Immunol 17:1505–1512CrossRefPubMedGoogle Scholar
  40. Xu L, Wang Y, Wang L, Gao Y, An C (2008) TYchi, a novel chitinase with RNA N-glycosidase and anti-tumor activities. Front Biosci 13:3127CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Carolina Araújo Viana
    • 1
  • Márcio V. Ramos
    • 1
  • José Delano Barreto Marinho Filho
    • 2
  • Letícia Veras Costa Lotufo
    • 3
  • Ingrid Samantha Tavares Figueiredo
    • 4
  • Jefferson Soares de Oliveira
    • 5
  • Pietro Mastroeni
    • 6
  • José Vitor Lima-Filho
    • 7
    • 8
    Email author
  • Nylane Maria Nunes Alencar
    • 4
    • 9
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyFederal University of CearáFortalezaBrazil
  2. 2.Medical SchoolFederal University of PiauíParnaíbaBrazil
  3. 3.Department of PharmacologyUniversity of São PauloSão PauloBrazil
  4. 4.Department of Physiology and PharmacologyFederal University of CearáFortalezaBrazil
  5. 5.Biomedicine SchoolFederal University of PiauíParnaíbaBrazil
  6. 6.Department of Veterinary MedicineUniversity of CambridgeCambridgeUK
  7. 7.Department of BiologyFederal Rural University of PernambucoRecifeBrazil
  8. 8.Departamento de Biologia, Laboratório de Microbiologia e ImunologiaUniversidade Federal Rural de PernambucoRecifeBrazil
  9. 9.Departamento de Fisiologia e FarmacologiaUniversidade Federal do CearáFortalezaBrazil

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