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Anti-cancer Effect of Xao Tam Phan Paramignya trimera Methanol Root Extract on Human Breast Cancer Cell Line MCF-7 in 3D Model

  • Lam-Huyen Nguyen-Thi
  • Sinh Truong Nguyen
  • Thao Phuong Tran
  • Chinh-Nhan Phan-Lu
  • Trung The Van
  • Phuc Van PhamEmail author
Part of the Advances in Experimental Medicine and Biology book series


Background: Cancer is one of the leading causes of death in the world. A great deal of effort has been made to discover new agents for cancer treatment. Xao tam phan (Paramignya trimera) is a traditional medicine of Vietnam used in cancer treatment for a long time, yet there is not much scientific evidence proving its anticancer potency. The study aimed to evaluate the toxicity of Paramignya trimera extract (PTE) on multicellular tumor spheres (MCTS) of MCF-7 cells using hanging drop technique. Methods: Firstly, MCF-7 cells were seeded on hanging drop plates, spheroid size was tracked, and growth curve was measured by MTT assay and AlamarBlue® assay. The necrotic core of MCTS was evaluated by propidium iodide (PI) staining. Toxicity of doxorubicin (DOX) and tirapazamine (TPZ) was then tested on 3D model compared to 2D culture condition. Results: The results showed that the IC50 of DOX on 3D MCF-7 cells was nearly 50 times greater than monolayer MCF-7 cells. In contrast, TPZ (an agent which is specifically toxic under hypoxic conditions) had significantly lower IC50 in 3D condition than in 2D. The toxicity tests for PTE showed that PTE strongly inhibited MCF-7 cells in both 2D and 3D conditions. Interestingly, the IC50 of PTE in 3D model was remarkably lower than in 2D (IC50 value was 168.9 ± 11.65 μg/ml compared to 260.8 ± 16.54 μg/ml, respectively). The invasion assay showed that PTE completely inhibited invasion of MCF-7 cells at 250 μg/mL concentration. Also, flow cytometry results indicated that PTE effectively induced apoptosis in MCF-7 spheroids in 3D condition at 250 μg/mL concentration. Conclusion: The results from this study emphasize the promise of PTE in cancer therapy.


3D tumor sphere Anticancer Apoptosis Extract MCF-7 Multicellular tumor spheres Paramignya trimera Tirapazamine Toxicity TPZ Xao tam phan 





Extracellular matrix


Fetal bovine serum


Multicellular tumor spheres


Paramignya trimera extract


Propidium iodide




Funding and Grants

This research was funded by Vietnam National University, Ho Chi Minh city, Viet Nam under grant number A2015-18-01/HD-KHCN.


  1. Alvarez-Perez, J., Ballesteros, P., & Cerdan, S. (2005). Microscopic images of intraspheroidal pH by 1H magnetic resonance chemical shift imaging of pH sensitive indicators. Magma (New York, NY), 18, 293–301.Google Scholar
  2. Bonnier, F., Keating, M. E., Wrobel, T. P., Majzner, K., Baranska, M., Garcia-Munoz, A., Blanco, A., & Byrne, H. J. (2015). Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell culture models. Toxicology in vitro: an international journal published in association with BIBRA, 29, 124–131.Google Scholar
  3. Brown, J. M. (1999). The hypoxic cell: A target for selective cancer therapy--eighteenth Bruce F. Cain memorial award lecture. Cancer Research, 59, 5863–5870.Google Scholar
  4. Charoen, K. M., Fallica, B., Colson, Y. L., Zaman, M. H., & Grinstaff, M. W. (2014). Embedded multicellular spheroids as a biomimetic 3D cancer model for evaluating drug and drug-device combinations. Biomaterials, 35, 2264–2271.Google Scholar
  5. Edmondson, R., Broglie, J. J., Adcock, A. F., & Yang, L. (2014). Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and Drug Development Technologies, 12, 207–218.Google Scholar
  6. Ferro, F., Shields Baheney, C., & Spelat, R. (2014). Three-dimensional (3D) cell culture conditions, present and future improvements. Razavi Int Journal of Medicine, 2, e17803.Google Scholar
  7. Gong, X., Lin, C., Cheng, J., Su, J., Zhao, H., Liu, T., Wen, X., & Zhao, P. (2015). Generation of multicellular tumor spheroids with microwell-based agarose scaffolds for drug testing. PLoS One, 10, e0130348.Google Scholar
  8. Hamilton, G. (1998). Multicellular spheroids as an in vitro tumor model. Cancer Letters, 131, 29–34.Google Scholar
  9. Ho, W. Y., Yeap, S. K., Ho, C. L., Rahim, R. A., & Alitheen, N. B. (2012). Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay. PLoS One, 7, e44640.Google Scholar
  10. Hsiao, A. Y., Tung, Y.-C., Qu, X., Patel, L. R., Pienta, K. J., & Takayama, S. (2012). 384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids. Biotechnology and Bioengineering, 109, 1293–1304.Google Scholar
  11. Hutmacher, D. W. (2010). Biomaterials offer cancer research the third dimension. Nature Materials, 9, 90–93.Google Scholar
  12. Ivascu, A., & Kubbies, M. (2006). Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. Journal of Biomolecular Screening, 11, 922–932.Google Scholar
  13. Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., & Nielsen, L. K. (2003). Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnology and Bioengineering, 83, 173–180.Google Scholar
  14. Kijanska M. K. J. (2016). In vitro 3D spheroids and microtissues: ATP-based cell viability and toxicity assays. Assay Guidance Manual [Internet].Google Scholar
  15. Kim, J. W., Ho, W. J., & Wu, B. M. (2011). The role of the 3D environment in hypoxia-induced drug and apoptosis resistance. Anticancer Research, 31, 3237–3245.Google Scholar
  16. Lin, R. Z., & Chang, H. Y. (2008). Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnology Journal, 3, 1172–1184.Google Scholar
  17. Mahassni, S. H., & Al-Reemi, R. M. (2013). Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium Sativum) seeds. Saudi Journal of Biological Sciences, 20, 131–139.Google Scholar
  18. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.Google Scholar
  19. Newman, D. J., & Cragg, G. M. (2012). Natural products as sources of new drugs over the 30 years from 1981 to 2010. Journal of Natural Products, 75, 311–335.Google Scholar
  20. Nguyen, H. T.-L., Nguyen, S. T., & Pham, P. V. (2016). Concise review: 3D cell culture systems for anticancer drug screening. Biomedical Research and Therapy, 3, 625–632.Google Scholar
  21. Nguyen, V. T., Sakoff, J. A., & Scarlett, C. J. (2017). Physicochemical properties, antioxidant and anti-proliferative capacities of dried leaf and its extract from Xao tam phan (Paramignya trimera). Chemistry & Biodiversity, 14.Google Scholar
  22. O’Brien, J., Wilson, I., Orton, T., & Pognan, F. (2000). Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry, 267, 5421–5426.Google Scholar
  23. Pampaloni, F., Reynaud, E. G., & Stelzer, E. H. (2007). The third dimension bridges the gap between cell culture and live tissue. Nature Reviews Molecular Cell Biology, 8, 839–845.Google Scholar
  24. Pham, P. V. (2015). Breast cancer stem cell culture and proliferation. In Breast cancer stem cells & therapy resistance. Cham: Springer.Google Scholar
  25. Reddy, S. B., & Williamson, S. K. (2009). Tirapazamine: A novel agent targeting hypoxic tumor cells. Expert Opinion on Investigational Drugs, 18, 77–87.Google Scholar
  26. Sant, S., & Johnston, P. A. (2017). The production of 3D tumor spheroids for cancer drug discovery. Drug Discovery Today: Technologies, 23, 27–36.Google Scholar
  27. Schmeichel, K. L., & Bissell, M. J. (2003). Modeling tissue-specific signaling and organ function in three dimensions. Journal of Cell Science, 116, 2377–2388.Google Scholar
  28. Smith, S. J., Wilson, M., Ward, J. H., Rahman, C. V., Peet, A. C., Macarthur, D. C., Rose, F. R., Grundy, R. G., & Rahman, R. (2012). Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition. PLoS One, 7, e52335.Google Scholar
  29. Strese, S., Fryknas, M., Larsson, R., & Gullbo, J. (2013). Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer, 13, 331.Google Scholar
  30. Sutherland, R. M. (1988). Cell and environment interactions in tumor microregions: The multicell spheroid model. Science (New York, N.Y.), 240, 177–184.Google Scholar
  31. Timmins, N. E., & Nielsen, L. K. (2007). Generation of multicellular tumor spheroids by the hanging-drop method. Methods in Molecular Medicine, 140, 141–151.Google Scholar
  32. Tung, Y. C., Hsiao, A. Y., Allen, S. G., Torisawa, Y. S., Ho, M., & Takayama, S. (2011). High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. The Analyst, 136, 473–478.Google Scholar
  33. Vaupel, P., & Mayer, A. (2007). Hypoxia in cancer: Significance and impact on clinical outcome. Cancer Metastasis Reviews, 26, 225–239.Google Scholar
  34. Vinci, M., Box, C., & Eccles, S. A. (2015). Three-dimensional (3D) tumor spheroid invasion assay. Journal of Visualized Experiments: JoVE, 1, 52686.Google Scholar
  35. Xu, Z., Chen, X., Zhong, Z., Chen, L., & Wang, Y. (2011). Ganoderma lucidum polysaccharides: Immunomodulation and potential anti-tumor activities. The American Journal of Chinese Medicine, 39, 15–27.Google Scholar
  36. Yin, S.-Y., Wei, W.-C., Jian, F.-Y., & Yang, N.-S. (2013). Therapeutic applications of herbal medicines for cancer patients. Evidence-based Complementary and Alternative Medicine, 2013, 15.Google Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Lam-Huyen Nguyen-Thi
    • 1
    • 2
  • Sinh Truong Nguyen
    • 1
    • 2
  • Thao Phuong Tran
    • 1
  • Chinh-Nhan Phan-Lu
    • 1
    • 2
  • Trung The Van
    • 4
  • Phuc Van Pham
    • 1
    • 2
    • 3
    Email author
  1. 1.Stem Cell InstituteUniversity of Science, VNUHCMHo Chi Minh CityVietnam
  2. 2.Laboratory of Cancer ResearchUniversity of Science, VNUHCMHo Chi Minh CityVietnam
  3. 3.Laboratory of Stem Cell Research and ApplicationUniversity of Science, VNUHCMHo Chi Minh CityVietnam
  4. 4.Ho Chi Minh City Medicine and Pharmacy University, Hospital of Dermatology – Ho Chi Minh CityHo Chi Minh CityVietnam

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