Download PDF
CC BY-NC-ND 4.0 · Homeopathy 2023; 112(01): 040-049
DOI: 10.1055/s-0042-1747682
Original Research Article

In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album

Ana Catarina Viana Valle
1   Doctor Izao Soares Institute, Sao Paulo, Brazil
2   Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
,
Hilana dos Santos Sena Brunel
3   BioInnova, Brazil
,
Bruno Stéfano Lima Dallago
4   Brasilia University, Brazil
,
Lucas Santana Rodrigues
5   BioCell Cell Therapy, Brazil
,
Patrícia Furtado Malard
2   Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
5   BioCell Cell Therapy, Brazil
,
Rosiane Andrade da Costa
2   Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
,
Rafael Rossetto
2   Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
,
Rosângela Vieira de Andrade 
2   Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
› Author Affiliations Funding This research and the manuscript preparation did not receive any funding. RAC received a post-doctoral scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PNPD protocol 88882.315167/2019–01).
› Further Information
Also available at
   Permissions and Reprints

Abstract

Introduction The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties.

Objective This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10−1, 1×10−2 potency (VAD1D2).

Methods MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test.

Results At the concentration of 10 μL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 μL/mL presented absolute percentages of initial apoptosis twice as high as at 10 μL/mL, which was similar to the control (untreated group).

Conclusion The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

Keywords

apoptosis - cell viability - cell differentiation - complementary therapy

Authors' Contributions

A.C.V.V. conceptualized the study, and contributed to the methodology, investigation, formal analysis, and writing—original draft H.d.S.S.B. contributed to methodology, investigation, formal analysis, and writing—original draft. B.S.L.D. did the formal analysis and co-wrote the original draft. L.S.R. contributed to conceptualization, investigation, and acquisition of resources. P.F.M. contributed to conceptualization and investigation, formal analysis, and writing—original draft. R.R. contributed to conceptualization, writing—review and editing. R.A.C. contributed to co-writing—review and editing. R.V.A. contributed to conceptualization, methodology, supervision, and project administration. All authors critically reviewed the manuscript, contributed to its revision, and approved the final version submitted.


Supplementary Material



Publication History

Received: 15 October 2021

Accepted: 14 January 2022

Article published online:
21 August 2022

© 2022. The Faculty of Homeopathy. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 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
  • 2 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
  • 3 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
  • 4 Ostermann T, Raak C, Büssing A. Survival of cancer patients treated with mistletoe extract (Iscador): a systematic literature review. BMC Cancer 2009; 9: 451
    CrossrefPubMedSearch in Google Scholar
  • 5 Menke K, Schwermer M, Schramm A, Zuzak TJ. Preclinical evaluation of antitumoral and cytotoxic properties of Viscum album fraxini extract on pediatric tumor cells. Planta Med 2019; 85: 1150-1159
    Search in Google Scholar
  • 6 Kissel D, Jurin M, Zarcovic N. Über die zytostatischen und immunologischen Effekte von Viscum album. Erfahrunsheilkunde 1990; 39: 973-988
    PubMedSearch in Google Scholar
  • 7 Gabius HJ, Gabius S, Joshi SS. et al. From ill-defined extracts to the immunomodulatory lectin: will there be a reason for oncological application of mistletoe?. Planta Med 1994; 60: 2-7
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Lyu SY, Park WB. Mistletoe lectin transport by M-cells in follicle-associated epithelium (FAE) and IL-12 secretion in dendritic cells situated below FAE in vitro. Arch Pharm Res 2010; 33: 1433-1441
    CrossrefPubMedSearch in Google Scholar
  • 9 Zarković N, Kališnik T, Loncarić I. et al. Comparison of the effects of Viscum album lectin ML-1 and fresh plant extract (Isorel) on the cell growth in vitro and tumorigenicity of melanoma B16F10. Cancer Biother Radiopharm 1998; 13: 121-131
    PubMedSearch in Google Scholar
  • 10 Schötterl S, Huber SM, Lentzen H, Mittelbronn M, Naumann U. Adjuvant therapy using mistletoe containing drugs boosts the T-cell-mediated killing of glioma cells and prolongs the survival of glioma bearing mice. Evid Based Complement Alternat Med 2018; 2018: 3928572
    CrossrefPubMedSearch in Google Scholar
  • 11 Schaller G, Urech K, Grazi G, Giannattasio M. Viscotoxin composition of the three European subspecies of Viscum album. Planta Med 1998; 64: 677-678
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Khil LY, Kim W, Lyu S, Park WB, Yoon JW, Jun HS. Mechanisms involved in Korean mistletoe lectin-induced apoptosis of cancer cells. World J Gastroenterol 2007; 13: 2811-2818
    CrossrefPubMedSearch in Google Scholar
  • 13 Lee C-H, Kim J-K, Kim H-Y, Park S-M, Lee S-M. Immunomodulating effects of Korean mistletoe lectin in vitro and in vivo. Int Immunopharmacol 2009; 9: 1555-1561
    CrossrefPubMedSearch in Google Scholar
  • 14 Nazaruk J, Orlikowski P. Phytochemical profile and therapeutic potential of Viscum album L. Nat Prod Res 2016; 30: 373-385
    Search in Google Scholar
  • 15 Delebinski CI, Twardziok M, Kleinsimon S. et al. A natural combination extract of Viscum album L. Containing both triterpene acids and lectins is highly effective against AML in vivo. PLoS One 2015; 10: e0133892
    Search in Google Scholar
  • 16 Hegde P, Maddur MS, Friboulet A, Bayry J, Kaveri SV. Viscum album exerts anti-inflammatory effect by selectively inhibiting cytokine-induced expression of cyclooxygenase-2. PLoS One 2011; 6: e26312
    CrossrefPubMedSearch in Google Scholar
  • 17 Komath SS, Kavitha M, Swamy MJ. Beyond carbohydrate binding: new directions in plant lectin research. Org Biomol Chem 2006; 4: 973-988
    CrossrefPubMedSearch in Google Scholar
  • 18 Chikramane PS, Suresh AK, Bellare JR, Kane SG. Extreme homeopathic dilutions retain starting materials: a nanoparticulate perspective. Homeopathy 2010; 99: 231-242
    Thieme ConnectPubMedSearch in Google Scholar
  • 19 Maha N, Shaw A. Academic doctors' views of complementary and alternative medicine (CAM) and its role within the NHS: an exploratory qualitative study. BMC Complement Altern Med 2007; 7: 17
    CrossrefPubMedSearch in Google Scholar
  • 20 de Almeida Andrade F, Schlechta Portella CF. Research methods in complementary and alternative medicine: an integrative review. J Integr Med 2018; 16: 6-13
    CrossrefPubMedSearch in Google Scholar
  • 21 Evans M, Shaw A, Thompson EA. et al. Decisions to use complementary and alternative medicine (CAM) by male cancer patients: information-seeking roles and types of evidence used. BMC Complement Altern Med 2007; 7: 25
    CrossrefPubMedSearch in Google Scholar
  • 22 Augustin M, Bock PR, Hanisch J, Karasmann M, Schneider B. Safety and efficacy of the long-term adjuvant treatment of primary intermediate- to high-risk malignant melanoma (UICC/AJCC stage II and III) with a standardized fermented European mistletoe (Viscum album L.) extract: Results from a multicenter, comparative, epidemiological cohort study in Germany and Switzerland. Arzneimittelforschung/Drug Res 2005; 55: 38-49
    PubMedSearch in Google Scholar
  • 23 Lima LF, Rocha RMP, Alves AMCV. et al. Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue. Complement Ther Med 2016; 25: 39-44
    CrossrefPubMedSearch in Google Scholar
  • 24 Bonamin LV, de Carvalho AC, Waisse S. Viscum album (L.) in experimental animal tumors: a meta-analysis. Exp Ther Med 2017; 13: 2723-2740
    Search in Google Scholar
  • 25 Christen-Clottu O, Klocke P, Burger D, Straub R, Gerber V. Treatment of clinically diagnosed equine sarcoid with a mistletoe extract (Viscum album austriacus). J Vet Intern Med 2010; 24: 1483-1489
    CrossrefPubMedSearch in Google Scholar
  • 26 Cesar B, Abud APR, de Oliveira CC. et al. Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication. Micron 2008; 39: 461-470
    CrossrefPubMedSearch in Google Scholar
  • 27 Pedalino CMV. Homeopathic medicines in “potency chords”. Cultura Homeopática 2006; 16: 18-21
    PubMedSearch in Google Scholar
  • 28 Schnädelbach D. Die dritte Ausgabe des Europäischen Arzneibuchs und das Deutsche Arzneibuch. Pharm Unserer Zeit 1997; 26: 76-80
    CrossrefPubMedSearch in Google Scholar
  • 29 De FrancescoF, Ricci G, D'Andrea F, Nicoletti GF, Ferraro GA. Human adipose stem cells: from bench to bedside. Tissue Eng Part B Rev 2015; 21: 572-584
    CrossrefPubMedSearch in Google Scholar
  • 30 Abud APR, Zych J, Reus TL. et al. The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test. Regul Toxicol Pharmacol 2015; 73: 992-998
    CrossrefPubMedSearch in Google Scholar
  • 31 Rostock M, Huber R, Greiner T. et al. Anticancer activity of a lectin-rich mistletoe extract injected intratumorally into human pancreatic cancer xenografts. Anticancer Res 2005; 25 (3B): 1969-1975
    PubMedSearch in Google Scholar
  • 32 Morrison B, Lilford RJ, Ernst E. Methodological rigour and results of clinical trials of homoeopathic remedies. Perfusion (Nürnberg) 2000; 13: 132-138
    PubMedSearch in Google Scholar
  • 33 Horneber MA, Bueschel G, Huber R, Linde K, Rostock M. Mistletoe therapy in oncology. Cochrane Database Syst Rev 2008; CD003297
    PubMedSearch in Google Scholar
  • 34 Marvibaigi M, Supriyanto E, Amini N, Abdul Majid FA, Jaganathan SK. Preclinical and clinical effects of mistletoe against breast cancer. BioMed Res Int 2014; 2014: 785479
    CrossrefPubMedSearch in Google Scholar
  • 35 Estko M, Baumgartner S, Urech K. et al. Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro. BMC Complement Altern Med 2015; 15: 130
    CrossrefPubMedSearch in Google Scholar
  • 36 Laszczyk MN. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy. Planta Med 2009; 75: 1549-1560
    Thieme ConnectPubMedSearch in Google Scholar
  • 37 Cassetta L, Cassol E, Poli G. Macrophage polarization in health and disease. ScientificWorldJournal 2011; 11: 2391-2402
    CrossrefPubMedSearch in Google Scholar
  • 38 Strüh CM, Jäger S, Kersten A, Schempp CM, Scheffler A, Martin SF. Triterpenoids amplify anti-tumoral effects of mistletoe extracts on murine B16.f10 melanoma in vivo. PLoS One 2013; 8: e62168
    CrossrefPubMedSearch in Google Scholar
  • 39 Ahmad S, Rehman T, Abbasi WM. Homoeopathic approach for the treatment of cancer. Indian J Res Homoeopath 2018; 12: 157-163
    CrossrefPubMedSearch in Google Scholar
  • 40 Sunila ES, Kuttan R, Preethi KC, Kuttan G. Dynamized preparations in cell culture. Evid Based Complement Alternat Med 2009; 6: 257-263
    CrossrefPubMedSearch in Google Scholar
  • 41 Müthing J, Meisen I, Bulau P. et al. Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues. Biochemistry 2004; 43: 2996-3007
    CrossrefPubMedSearch in Google Scholar
  • 42 Twardziok M, Kleinsimon S, Rolff J. et al. Multiple active compounds from Viscum album L. synergistically converge to promote apoptosis in Ewing sarcoma. PLoS One 2016; 11: e0159749
    CrossrefPubMedSearch in Google Scholar
  • 43 Choi SH, Lyu SY, Park WB. Mistletoe lectin induces apoptosis and telomerase inhibition in human A253 cancer cells through dephosphorylation of Akt. Arch Pharm Res 2004; 27: 68-76
    CrossrefPubMedSearch in Google Scholar
  • 44 Duong Van Huyen, Sooryanarayana J-P, Delignat S, Bloch M-F, Kazatchkine MD, Kaveri SV. Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin. Chemotherapy 2001; 47: 366-376
    CrossrefPubMedSearch in Google Scholar
  • 45 Möckel B, Schwarz T, Zinke H, Eck J, Langer M, Lentzen H. Effects of mistletoe lectin I on human blood cell lines and peripheral blood cells. Cytotoxicity, apoptosis and induction of cytokines. Arzneimittelforschung 1997; 47: 1145-1151
    PubMedSearch in Google Scholar
  • 46 Ribéreau-Gayon G, Jung ML, Frantz M, Anton R. Modulation of cytotoxicity and enhancement of cytokine release induced by Viscum album L. extracts or mistletoe lectins. Anticancer Drugs 1997; 8: S3-S8
    CrossrefPubMedSearch in Google Scholar
  • 47 Hoessli D, Ahmad I. Mistletoe lectins: carbohydrate-specific apoptosis inducers and immunomodulators. Curr Org Chem 2008; 12: 918-925
    CrossrefPubMedSearch in Google Scholar
  • 48 Bantel H, Engels IH, Voelter W, Schulze-Osthoff K, Wesselborg S. Mistletoe lectin activates caspase-8/FLICE independently of death receptor signaling and enhances anticancer drug-induced apoptosis. Cancer Res 1999; 59: 2083-2090
    PubMedSearch in Google Scholar
  • 49 Doehmer J, Eisenbraun J. Assessment of extracts from mistletoe (Viscum album) for herb-drug interaction by inhibition and induction of cytochrome P450 activities. Phytother Res 2012; 26: 11-17
    CrossrefPubMedSearch in Google Scholar
  • 50 Engdal S, Nilsen OG. In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer patients. Phytother Res 2009; 23: 906-912
    CrossrefPubMedSearch in Google Scholar
  • 51 Steinborn C, Klemd AM, Sanchez-Campillo AS. et al. Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model. PLoS One 2017; 12: e0181553
    Search in Google Scholar
  • 52 Kim MJ, Shin KS, Jeon JH. et al. Human chorionic-plate-derived mesenchymal stem cells and Wharton's jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells. Cell Tissue Res 2011; 346: 53-64
    CrossrefPubMedSearch in Google Scholar
  • 53 Choi JH, Lyu SY, Lee HJ, Jung J, Park WB, Kim GJ. Korean mistletoe lectin regulates self-renewal of placenta-derived mesenchymal stem cells via autophagic mechanisms. Cell Prolif 2012; 45: 420-429
    CrossrefPubMedSearch in Google Scholar
  • 54 Bellin M, Marchetto MC, Gage FH, Mummery CL. Induced pluripotent stem cells: the new patient?. Nat Rev Mol Cell Biol 2012; 13: 713-726
    CrossrefPubMedSearch in Google Scholar
  • 55 Behar RZ, Bahl V, Wang Y. et al. A method for rapid dose-response screening of environmental chemicals using human embryonic stem cells. J Pharmacol Toxicol Methods 2012; 66: 238-245
    CrossrefPubMedSearch in Google Scholar
  • 56 Rajamohan D, Matsa E, Kalra S. et al. Current status of drug screening and disease modelling in human pluripotent stem cells. BioEssays 2013; 35: 281-298
    CrossrefPubMedSearch in Google Scholar
  • 57 Coleman RA. Human-based systems in drug and chemical safety testing—toward replacement, the 'single R'. Altern Lab Anim 2014; 42: 357-366
    CrossrefPubMedSearch in Google Scholar
  • 58 Melzer J, Iten F, Hostanska K, Saller R. Efficacy and safety of mistletoe preparations (Viscum album) for patients with cancer diseases. A systematic review. Forsch Komplementmed 2009; 16: 217-226
    PubMedSearch in Google Scholar
  • 59 Halle W. The Registry of Cytotoxicity: toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals. Altern Lab Anim 2003; 31: 89-198
    CrossrefPubMedSearch in Google Scholar