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A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth

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Abstract

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

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Acknowledgements

The authors are grateful to Dr. Valerio Morelli from Farmanguinhos/FIOCRUZ for supplying C. cajan, Elettra Greene for English review, Gabriela Arcanjo for review the references, and the Proteomics and Mass Spectrometry Unit platform (UEMP) at the Federal University of Rio de Janeiro (UFRJ) run by Dr. Russolina Zingali for support in the use of the license Mascot Server for protein identification searches; and UFRJ Pharmaceutical Science postgraduate program. Financial support was provided by Fundação Oswaldo Cruz (FIOCRUZ, PROEP CNPq/FARMANGUINHOS-407849/2017-8 and 407839/2017-8) and FAPERJ (Programa e Apoio a Projetos Temáticos no Estado do RJ- E-26/010.001478/2019).

Funding

Funding was provided by FIOCRUZ, PROEP CNPq/FARMANGUINHOS (Grant Nos. 407839/2017-8, 407849/2017-8) and FAPERJ, Programa e Apoio a Projetos Temáticos no Estado do RJ (Grant No. E-26/010.001478/2019).

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Authors and Affiliations

  1. Departament of Natural Products, Institute of Pharmaceuticals Technology, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, Rio de Janeiro, 21045-900, Brazil

    Erika Maria Gomes Ferreira Teixeira, Patrícia Fernandes Ferreira, Thayane Aparecida Alves & Raquel Elisa Silva-López

  2. Interdisciplinary Laboratory of Medical Research, IOC—Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, Rio de Janeiro, CEP 21045-900, Brazil

    Dario Eluam Kalume

  3. Departament of Pharmacology, Institute of Pharmaceuticals Technology, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, Rio de Janeiro, CEP 21045-900, Brazil

    Ana Paula G. A. Fontão & André Luís Franco Sampaio

  4. Laboratory of Bioprospection and Applied Ethnopharmacology, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil

    Erika Maria Gomes Ferreira Teixeira & Danilo Ribeiro de Oliveira

  5. Cellular and Molecular Oncobiology Program, National Institute of Cancer (INCa), Rio de Janeiro, Brazil

    José Andrés Morgado-Díaz

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  1. Erika Maria Gomes Ferreira Teixeira
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  9. Raquel Elisa Silva-López
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Contributions

RESL designed the study, data analysis, wrote and revised the manuscript, and raised funds. EMGFT performed the experiments with protease inhibitor. DEK performed the EM assays, analyzed the data and revided the manuscript. PFF and TAA performed the experiments with cancer proteases. APGAF and ALFS supplied the melanoma cell and supernatants and did the cell cytotoxicity experiments, and raised funds. JAMD supplied the Caco-2 cell and supernatants, and raised funds. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Raquel Elisa Silva-López.

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Teixeira, E.M.G.F., Kalume, D.E., Ferreira, P.F. et al. A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth. Protein J (2024). https://doi.org/10.1007/s10930-023-10175-9

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