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Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells

  • Blanca F. Iglesias-Figueroa
  • Tania S. Siqueiros-Cendón
  • Denisse A. Gutierrez
  • Renato J. Aguilera
  • Edward A. Espinoza-Sánchez
  • Sigifredo Arévalo-Gallegos
  • Armando Varela-RamirezEmail author
  • Quintín Rascón-CruzEmail author


Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC50 of 91.4 and 109.46 µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55–3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.


Lactoferrin Cytoskeleton Anti-cancer drug discovery Apoptosis Cancer Cell cycle 



BFIF thank the Consejo Nacional de Ciencia y Tecnología (CONACyT) for the Ph.D. studies grant. Funding for this work was supported by an internal grant (2016-2017) from the Facultad de Ciencias Químicas, the Universidad Autónoma de Chihuahua to QRC, and also, partially provided by the National Institute of General Medical Sciences-Support of Competitive Research (SCORE) grant 1SC3GM103713 to RJA. The authors thank the Cytometry, Screening and Imaging, the Biomolecule Analysis and the Genomic Analysis Core Facilities at the University of Texas at El Paso (UTEP). Those Core Facilities were supported by a Research Centers in Minority Institutions (RCMI) program grant 5G12MD007592 to the Border Biomedical Research Center (BBRC) in UTEP from the National Institute on Minority Health and Health Disparities, a component of the National Institutes of Health. The authors also thank Gladys Almodovar and Lisett Contreras (both with UTEP) for outstanding technical and cell culture expertise and to Professor Giulio Francia (with UTEP) for the generous gift of the MDA-MB-231/LM2-4 cell line.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    Actor JK, Hwang S-A, Kruzel ML (2009) Lactoferrin as a natural immune modulator, Curr Pharm Des 15:1956CrossRefGoogle Scholar
  2. 2.
    Van der Strate B, Harmsen M, Schäfer P, Swart P, Jahn G, Speer C, Meijer D, Hamprecht K (2001) Viral load in breast milk correlates with transmission of human cytomegalovirus to preterm neonates, but lactoferrin concentrations do not. Clin Diagn Lab Immunol 8:818–821Google Scholar
  3. 3.
    Iglesias-Figueroa BF, Espinoza-Sánchez EA, Siqueiros-Cendón TS, Rascón-Cruz Q (2018) Lactoferrin as a nutraceutical protein from milk, an overview, Int Dairy J. Google Scholar
  4. 4.
    García-Montoya IA, Cendón TS, Arévalo-Gallegos S, Rascón-Cruz Q (2012) Lactoferrin a multiple bioactive protein: an overview. Biochim Biophys Acta (BBA)-Gen Subj 1820:226–236CrossRefGoogle Scholar
  5. 5.
    Siqueiros-Cendón T, Arévalo-Gallegos S, Iglesias-Figueroa BF, García-Montoya IA, Salazar-Martínez J, Rascón-Cruz Q (2014) Immunomodulatory effects of lactoferrin. Acta Pharmacol Sin 35:557–566CrossRefGoogle Scholar
  6. 6.
    Giansanti F, Panella G, Leboffe L, Antonini G (2016) Lactoferrin from milk: nutraceutical and pharmacological properties. Pharmaceuticals 9:61CrossRefGoogle Scholar
  7. 7.
    W.O. (2019) “WHO | Cancer. Accessed 19 Feb 2019
  8. 8.
    American Cancer Society, Cancer Facts & Figures (2019). Accessed 19 Feb 2019
  9. 9.
    Abu-Serie MM, El-Fakharany EM (2017) Efficiency of novel nanocombinations of bovine milk proteins (lactoperoxidase and lactoferrin) for combating different human cancer cell lines. Sci Rep 7:16769CrossRefGoogle Scholar
  10. 10.
    Zhang Y, Lima CF, Rodrigues LR (2014) Anticancer effects of lactoferrin: underlying mechanisms and future trends in cancer therapy. Nutr Rev 72:763–773CrossRefGoogle Scholar
  11. 11.
    Yang N, Rekdal Ø, Stensen W, Svendsen J (2002) Enhanced antitumor activity and selectivity of lactoferrin-derived peptides. J Pept Res 60:187–197CrossRefGoogle Scholar
  12. 12.
    de Mejia EG, Dia VP (2010) The role of nutraceutical proteins and peptides in apoptosis, angiogenesis, and metastasis of cancer cells. Cancer Metastasis Rev 29:511–528CrossRefGoogle Scholar
  13. 13.
    Rosa L, Cutone A, Lepanto MS, Paesano R, Valenti P (2017) Lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis. Int J Mol Sci 18:1985CrossRefGoogle Scholar
  14. 14.
    Kazan HH, Urfali-Mamatoglu C, Gunduz U (2017) Iron metabolism and drug resistance in cancer. Biometals 30:629–641CrossRefGoogle Scholar
  15. 15.
    Legendre C, Garcion E (2015) Iron metabolism: a double-edged sword in the resistance of glioblastoma to therapies. Trends Endocrinol Metab 26:322–331CrossRefGoogle Scholar
  16. 16.
    Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149:1060–1072CrossRefGoogle Scholar
  17. 17.
    Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156:317–331CrossRefGoogle Scholar
  18. 18.
    Iglesias-Figueroa B, Valdiviezo-Godina N, Siqueiros-Cendón T, Sinagawa-García S, Arévalo-Gallegos S, Rascón-Cruz Q (2016) High-level expression of recombinant bovine lactoferrin in Pichia pastoris with antimicrobial activity. Int J Mol Sci 17:902CrossRefGoogle Scholar
  19. 19.
    Cailleau R, Young R, Olive M, Reeves W Jr (1974) Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 53:661–674CrossRefGoogle Scholar
  20. 20.
    Munoz R, Man S, Shaked Y, Lee CR, Wong J, Francia G, Kerbel RS (2006) Highly efficacious nontoxic preclinical treatment for advanced metastatic breast cancer using combination oral UFT-cyclophosphamide metronomic chemotherapy. Cancer Res. 66:3386–3391CrossRefGoogle Scholar
  21. 21.
    Cailleau R, Olive M, Cruciger QV (1978) Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In vitro 14:911–915CrossRefGoogle Scholar
  22. 22.
    Lee AV, Oesterreich S, Davidson NE (2015) MCF-7 cells—changing the course of breast cancer research and care for 45 years. JNCI: J Natl Cancer Inst. Google Scholar
  23. 23.
    Gazdar AF, Kurvari V, Virmani A, Gollahon L, Sakaguchi M, Westerfield M, Kodagoda D, Stasny V, Cunningham HT, Wistuba II (1998) Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer. Int J Cancer 78:766–774CrossRefGoogle Scholar
  24. 24.
    Keydar I, Chen L, Karby S, Weiss F, Delarea J, Radu M, Chaitcik S, Brenner H (1979) Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer (1965) 15:659–670CrossRefGoogle Scholar
  25. 25.
    Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, McGrath CM, Russo J, Pauley RJ, Jones RF, Brooks S (1990) Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50:6075–6086Google Scholar
  26. 26.
    Robles-Escajeda E, Das U, Ortega NM, Parra K, Francia G, Dimmock JR, Varela-Ramirez A, Aguilera RJ (2016) A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells. Cellular Oncol 39:265–277CrossRefGoogle Scholar
  27. 27.
    Nunes LM, Robles-Escajeda E, Santiago-Vazquez Y, Ortega NM, Lema C, Muro A, Almodovar G, Das U, Das S, Dimmock JR (2014) The gender of cell lines matters when screening for novel anti-cancer drugs. AAPS J 16:872–874CrossRefGoogle Scholar
  28. 28.
    Lema C, Varela-Ramirez A, Aguilera RJ (2011) Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds. Curr Cell Biochem 1:1Google Scholar
  29. 29.
    Varela-Ramirez A, Costanzo M, Carrasco YP, Pannell KH, Aguilera RJ (2011) Cytotoxic effects of two organotin compounds and their mode of inflicting cell death on four mammalian cancer cells. Cell Biol Toxicol 27:159–168CrossRefGoogle Scholar
  30. 30.
    Santiago-Vázquez Y, Das U, Varela-Ramirez A, Baca ST, Ayala-Marin Y, Lema C, Das S, Baryyan A, Dimmock JR, Aguilera RJ (2016) Tumor-selective cytotoxicity of a novel pentadiene analogue on human leukemia/lymphoma cells. Clin Cancer Drugs 3:138–146CrossRefGoogle Scholar
  31. 31.
    Gutierrez DA, DeJesus RE, Contreras L, Rodriguez-Palomares IA, Villanueva PJ, Balderrama KS, Monterroza L, Larragoity M, Varela-Ramirez A, Aguilera RJ (2019) A new pyridazinone exhibits potent cytotoxicity on human cancer cells via apoptosis and poly-ubiquitinated protein accumulation, Cell Biol Toxicol. Google Scholar
  32. 32.
    Sierra-Fonseca JA, Najera O, Martinez-Jurado J, Walker EM, Varela-Ramirez A, Khan AM, Miranda M, Lamango NS, Roychowdhury S (2014) Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction. BMC Neurosci 15:132CrossRefGoogle Scholar
  33. 33.
    Contreras L, Calderon RI, Varela-Ramirez A, Zhang H-Y, Quan Y, Das U, Dimmock JR, Skouta R, Aguilera RJ (2018) Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones, Cell Oncol. Google Scholar
  34. 34.
    Robles-Escajeda E, Lerma D, Nyakeriga AM, Ross JA, Kirken RA, Aguilera RJ, Varela-Ramirez A (2013) Searching in mother nature for anti-cancer activity: anti-proliferative and pro-apoptotic effect elicited by green barley on leukemia/lymphoma cells. PLoS ONE 8:e73508CrossRefGoogle Scholar
  35. 35.
    Valdez B, Carr K, Norman J (2016) Violet-excited nim-DAPI allows efficient and reproducible cell cycle analysis on the Gallios flow cytometer. Beckman Coulter Life Sciences. Houston, TX. Accessed Jan 2016
  36. 36.
    Varela-Ramirez A (2014) Female versus male cells in anti-cancer drug discovery: the winner is … AAPS Blog. Accessed Sept 2018
  37. 37.
    Clayton JA, Collins FS (2014) Policy: NIH to balance sex in cell and animal studies. Nat News 509:282CrossRefGoogle Scholar
  38. 38.
    García-Mata R, Bebök Z, Sorscher EJ, Sztul ES (1999) Characterization and dynamics of aggresome formation by a cytosolic Gfp-chimera. J Cell Biol 146:1239–1254CrossRefGoogle Scholar
  39. 39.
    Lázaro-Diéguez F, Aguado C, Mato E, Sánchez-Ruíz Y, Esteban I, Alberch J, Knecht E, Egea G (2008) Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide. J Cell Sci 121:1415–1425CrossRefGoogle Scholar
  40. 40.
    Leventis PA, Grinstein S (2010) The distribution and function of phosphatidylserine in cellular membranes. Ann Rev Biophys 39:407–427CrossRefGoogle Scholar
  41. 41.
    Villanueva PJ, Martinez A, Baca ST, DeJesus RE, Larragoity M, Contreras L, Gutierrez DA, Varela-Ramirez A, Aguilera RJ (2018) Pyronaridine exerts potent cytotoxicity on human breast and hematological cancer cells through induction of apoptosis, PLoS ONE 13:e0206467CrossRefGoogle Scholar
  42. 42.
    Pereira CS, Guedes JP, Gonçalves M, Loureiro L, Castro L, Gerós H, Rodrigues LR, Côrte-Real M (2016) Lactoferrin selectively triggers apoptosis in highly metastatic breast cancer cells through inhibition of plasmalemmal V-H+-ATPase. Oncotarget 7:62144Google Scholar
  43. 43.
    Zhang Y, Lima CF, Rodrigues LR (2014) Bovine lactoferrin induces cell cycle arrest and inhibits mTOR signaling in breast cancer cells. Nutr Cancer 66:1371–1385CrossRefGoogle Scholar
  44. 44.
    Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3:339CrossRefGoogle Scholar
  45. 45.
    Fadeel B, Xue D (2009) The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Crit Rev Biochem Mol Biol 44:264–277CrossRefGoogle Scholar
  46. 46.
    Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2:965CrossRefGoogle Scholar
  47. 47.
    Guedes JP, Pereira CS, Rodrigues LR, Côrte-Real M (2018) Bovine milk lactoferrin selectively kills highly metastatic prostate cancer PC-3 and osteosarcoma MG-63 cells in vitro. Front Oncol. Google Scholar
  48. 48.
    Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96CrossRefGoogle Scholar
  49. 49.
    Dolbeare F, Gratzner H, Pallavicini M, Gray J (1983) Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci 80:5573–5577CrossRefGoogle Scholar
  50. 50.
    Xiao Y, Monitto CL, Minhas KM, Sidransky D (2004) Lactoferrin down-regulates G1 cyclin-dependent kinases during growth arrest of head and neck cancer cells. Clin Cancer Res 10:8683–8686CrossRefGoogle Scholar
  51. 51.
    Damiens E, El Yazidi I, Mazurier J, Duthille I, Spik G, Boilly-Marer Y (1999) Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells. J Cell Biochem 74:486–498CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratorio de Biotecnología I, Facultad de Ciencias QuímicasUniversidad Autónoma de ChihuahuaChihuahuaMexico
  2. 2.The Cytometry, Screening and Imaging Core Facility, Border Biomedical Research Center, Department of Biological SciencesThe University of Texas at El PasoEl PasoUSA

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