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Homeopathy 2022; 111(04): 278-287
DOI: 10.1055/s-0041-1740967
Original Research Article

Phenotypic Changes in Mammary Adenocarcinoma (4T1) cells In Vitro after Treatment with Carcinosinum

Thaís Cristina da Silva
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
,
William Alves dos Santos
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
,
Sandra A.G. Pinto
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
,
Paulo Ricardo Dell'Armelina Rocha
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
,
Elizabeth Cristina Perez Hurtado
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
,
Leoni Villano Bonamin 
1   Research Center, Graduation Program in Environmental and Experimental Pathology, University Paulista—UNIP, São Paulo, Brazil
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Abstract

Objective The present study aimed to identify possible phenotypic changes in 4T1 (murine mammary adenocarcinoma) cells in vitro, including viability, HER-2 (human epidermal growth factor receptor-type 2) expression, and metastatic potential, after treatment with Carcinosinum in different homeopathic dilutions (12cH, 30cH, 200cH) shaken mechanically in pure, sterile, water from a commercial stock dilution.

Methods Treated cells were cultured in R10 medium, using 24-well plates, 105 cells per well, and treated with vehicle, Carcinosinum 12cH, 30cH or 200cH; untreated cells were used as the baseline control. After 24 hours of treatment, the percentage of apoptotic cells was analyzed by annexin V. Cell morphology was evaluated by microscopy after hematoxylin-eosin and Giemsa staining, whilst HER-2 expression was assessed using immunocytochemistry. The metastatic potential was determined by the expression and activity of the enzyme matrix metalloproteinase 9 (MMP-9) using zymography. The cytokine profile was established using the cytometric bead array method.

Result Treatment of 4T1 cells in vitro with Carcinosinum 30cH produced an increase in the number of annexin V-positive cells (apoptosis) and decreased expression of proactivated MMP-9. Cells treated with Carcinosinum 200cH presented hyper-expression of HER-2 on the plasma membrane, identified by immunocytochemistry. There were no differences in cytokine production among treatments.

Conclusion The data show promising results for Carcinosinum 30cH in vitro, but in vivo studies are also required to evaluate the role of tumor microenvironment in its effects.

Keywords

homeopathy - mammary cancer - experimental models


Publication History

Received: 23 July 2021

Accepted: 25 October 2021

Article published online:
27 April 2022

© 2022. Faculty of Homeopathy. This article is published by Thieme.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646-674
    CrossrefPubMedSearch in Google Scholar
  • 2 Cavalcante SAM, Barbosa da Silva F, Vilanova Marques CA, Níglio de Figueiredo E, Rivero de Gutiérrez MG. Ações do Enfermeiro no rastreamento e Diagnóstico do Câncer de Mama no Brasil. Rev Bras Cancerol 2013; 59: 459-466
    CrossrefPubMedSearch in Google Scholar
  • 3 Tapia C, Glatz K, Novotny H. et al. Close association between HER-2 amplification and overexpression in human tumors of non-breast origin. Mod Pathol 2007; 20: 192-198
    CrossrefPubMedSearch in Google Scholar
  • 4 Wu J, Ma R, Cao H. et al. Intraoperative imaging of metastatic lymph nodes using a fluorophore-conjugated antibody in a HER2/neu-expressing orthotopic breast cancer mouse model. Anticancer Res 2013; 33: 419-424
    PubMedSearch in Google Scholar
  • 5 Souza CM. Carcinoma mamário murino 4t1: características morfológicas, imunofenotípicas, bioquímicas e ensaios pré-clínicos com talidomida/ carboplatina. Tese (Patologia). Belo Horizonte, Minas Gerais: Faculdade de Medicina da Universidade Federal de Minas Gerais; 2013: 12
    Search in Google Scholar
  • 6 NIH (National Institutes of Health). What is Cancer? National Cancer Institute, 2010. Accessed on July 20th, 2021 at: http://www.cancer.gov/cancertopics/what-is-cancer
    PubMed
  • 7 Sorrentino L, Piraneo S, Riggio E. et al. Is there a role for homeopathy in breast cancer surgery? A first randomized clinical trial on treatment with Arnica montana to reduce post-operative seroma and bleeding in patients undergoing total mastectomy. J Intercult Ethnopharmacol 2017; 6: 1-8
    CrossrefPubMedSearch in Google Scholar
  • 8 Monteiro DA, Iriart JAB. Homeopatia no Sistema Único de Saúde: representações dos usuários sobre o tratamento homeopático. Cad Saude Publica 2007; 23: 1903-1912
    CrossrefPubMedSearch in Google Scholar
  • 9 Denez KB. Percepção dos usuários da clínica pública ou privada sobre a homeopatia e o acesso aos medicamentos homeopáticos. [Dissertação de mestrado (Assistência Farmacêutica)]. Florianópolis, SC: Universidade Federal de Santa Catarina; 2015: 220
    Search in Google Scholar
  • 10 Bonamin LV. ed. Discovering How Homeopathy Works. Amazon-Kindle; 2018: 70
    Search in Google Scholar
  • 11 Nogueira L. Estudo dos efeitos de preparações homeopáticas de Phytolacca decandra na evolução de adenocarcinoma mamário em modelo murino. [Dissertação de mestrado (Patologia Ambiental e Experimental)]. São Paulo, São Paulo: Universidade Paulista—UNIP; 2015: 63
    Search in Google Scholar
  • 12 Dos Santos AP, Cardoso TN, Waisse S, Bonamin LV. Homeopathy in experimental cancer models: a systematic review. Homeopathy 2021; 110: 76-85
    Thieme ConnectPubMedSearch in Google Scholar
  • 13 Nwabudike LC, Miulescu M, Tatu AL. Case series of an alternative therapy for generalised lichen planus: four case studies. Exp Ther Med 2019; 18: 943-948
    PubMedSearch in Google Scholar
  • 14 Preethi K, Ellanghiyil S, Kuttan G, Kuttan R. Induction of apoptosis of tumor cells by some potentiated homeopathic drugs: implications on mechanism of action. Integr Cancer Ther 2012; 11: 172-182
    CrossrefPubMedSearch in Google Scholar
  • 15 Remya V, Kuttan G. Homeopathic remedies with antineoplastic properties have immunomodulatory effects in experimental animals. Homeopathy 2015; 104: 211-219
    Thieme ConnectPubMedSearch in Google Scholar
  • 16 Amaral JG. Efeitos de medicamentos homeopáticos no tumor de Ehrlich em camundongos: uma abordagem experimental. Tese, Patologia Ambiental e Experimental. São Paulo, SP: Universidade Paulista; 2015: 71
    Search in Google Scholar
  • 17 Bonamin LV, Carvalho AC, Amaral J, Cardoso TN, Perez EC, Peres GB. Combination of homeopathic potencies, immune response, and tumour microenvironment. In: Bonamin LV, Waisse S. eds. Transdisciplinarity and Translational in High Dilution Research. Signals and Images GIRI Series. Cambridge: Cambridge Scholars Publishing; 2019: 211-243
    Search in Google Scholar
  • 18 Kupai K, Szucs G, Cseh S. et al. Matrix metalloproteinase activity assays: Importance of zymography. J Pharmacol Toxicol Methods 2010; 61: 205-209
    CrossrefPubMedSearch in Google Scholar
  • 19 Wu Q-W, Yang Q-M, Huang Y-F. et al. Expression and clinical significance of matrix metalloproteinase-9 in lymphatic invasiveness and metastasis of breast cancer. PLoS One 2014; 9: e97804
    CrossrefPubMedSearch in Google Scholar
  • 20 Hu X, Beeton C. Detection of functional matrix metalloproteinases by zymography. J Vis Exp 2010; 45: e2445
    PubMedSearch in Google Scholar
  • 21 Farmacopéia homeopática brasileira. 3ª ed.—ANVISA—Agência Nacional de Vigilância Sanitária. 2011. Accessed July 20th, 2021 in English at: https://www.gov.br/anvisa/pt-br/assuntos/farmacopeia/farmacopeia-homeopatica/arquivos/8056json-file-1
    PubMed
  • 22 de Santana FR, Dalboni LC, Nascimento KF. et al. High dilutions of antimony modulate cytokines production and macrophage—Leishmania (L.) amazonensis interaction in vitro. Cytokine 2017; 92: 33-47
    CrossrefPubMedSearch in Google Scholar
  • 23 Frenkel M, Mishra BM, Sen S. et al. Cytotoxic effects of ultra-diluted remedies on breast cancer cells. Int J Oncol 2010; 36: 395-403
    PubMedSearch in Google Scholar
  • 24 Tang H, Qiao J, Fu YX. Immunotherapy and tumor microenvironment. Cancer Lett 2016; 370: 85-90
    CrossrefPubMedSearch in Google Scholar
  • 25 Xu N, Lei Z, Li XL. et al. Clinical study of tumor angiogenesis and perfusion imaging using multi-slice spiral computed tomography for breast cancer. Asian Pac J Cancer Prev 2013; 14: 429-433
    CrossrefPubMedSearch in Google Scholar
  • 26 Onuchic AC, Chammas R. Câncer e o microambiente tumoral. Rev Med (São Paulo) 2010; 89: 21-31
    CrossrefPubMedSearch in Google Scholar
  • 27 Shi J, Ren Y, Zhen L, Qiu X. Exosomes from breast cancer cells stimulate proliferation and inhibit apoptosis of CD133+ cancer cells in vitro. Mol Med Rep 2015; 11: 405-409
    CrossrefPubMedSearch in Google Scholar
  • 28 Lee HS, Ha AW, Kim WK. Effect of resveratrol on the metastasis of 4T1 mouse breast cancer cells in vitro and in vivo. Nutr Res Pract 2012; 6: 294-300
    CrossrefPubMedSearch in Google Scholar
  • 29 Hermann PC, Huber SL, Herrler T. et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007; 1: 313-323
    CrossrefPubMedSearch in Google Scholar
  • 30 Oskarsson T, Batlle E, Massagué J.. Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell 2014; 14: 306-321
    CrossrefPubMedSearch in Google Scholar
  • 31 Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med 2013; 19: 1423-1437
    CrossrefPubMedSearch in Google Scholar
  • 32 Bernhard EJ, Gruber SB, Muschel RJ. Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc Natl Acad Sci U S A 1994; 91: 4293-4297
    CrossrefPubMedSearch in Google Scholar
  • 33 Zhang Y, Sun X, Nan N. et al. Elemene inhibits the migration and invasion of 4T1 murine breast cancer cells via heparanase. Mol Med Rep 2017; 16: 794-800
    CrossrefPubMedSearch in Google Scholar
  • 34 Barillari G. The impact of matrix metalloproteinase-9 on the sequential steps of the metastatic process. Int J Mol Sci 2020; 21: 4526
    CrossrefPubMedSearch in Google Scholar
  • 35 Zhao S, Ma W, Zhang M. et al. High expression of CD147 and MMP-9 is correlated with poor prognosis of triple-negative breast cancer (TNBC) patients. Med Oncol 2013; 30: 335
    CrossrefPubMedSearch in Google Scholar
  • 36 Liu Y, Xin T, Jiang QY. et al. CD147, MMP9 expression and clinical significance of basal-like breast cancer. Med Oncol 2013; 30: 366
    CrossrefPubMedSearch in Google Scholar
  • 37 Cupić DF, Tesar EC, Ilijas KM, Nemrava J, Kovacević M. Expression of matrix metalloproteinase 9 in primary and recurrent breast carcinomas. Coll Antropol 2011; 35: 7-10
    PubMedSearch in Google Scholar
  • 38 Zeng Y, Liu C, Dong B. et al. Inverse correlation between Naa10p and MMP-9 expression and the combined prognostic value in breast cancer patients. Med Oncol 2013; 30: 562
    CrossrefPubMedSearch in Google Scholar
  • 39 Nascimento ESM. Cinética de migração de células do sistema imune após desafio com células de adenocarcinoma mamário 4T1. [Dissertação de mestrado (Patologia Ambiental e Experimental)]— São Paulo, São Paulo: Universidade Paulista—UNIP; 2014: 38
    Search in Google Scholar
  • 40 Zamarron BF, Chen W. Dual roles of immune cells and their factors in cancer development and progression. Int J Biol Sci 2011; 7: 651-658
    CrossrefPubMedSearch in Google Scholar
  • 41 Viana CTR. Caracterização do perfil inflamatório e angiogênico associados ao desenvolvimento de tumores murinos. [Thesis (Ciências Biológicas: Fisiologia e Farmacologia)]— Belo Horizonte, Minas Gerais: Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais; 2014: 149
    Search in Google Scholar
  • 42 Conze D, Weiss L, Regen PS. et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Res 2001; 61: 8851-8858
    PubMedSearch in Google Scholar
  • 43 Wang Y, Qu Y, Zhang XL. et al. Autocrine production of interleukin-6 confers ovarian cancer cells resistance to tamoxifen via ER isoforms and SRC-1. Mol Cell Endocrinol 2014; 382: 791-803
    CrossrefPubMedSearch in Google Scholar