In Silico and In Vitro Studies of the Antimetastatic and Antiangiogenic Activities of Cordyceps militaris Extracts on Breast Cancer Cells (MDA-MB-231)

Alyssa Pei Qi  Chang; Sabrina Xin Yi  Khor; Adeline Yoke Yin Chia; Sunita  Chamyuang; Yin-Quan Tang

doi:10.20883/medical.e1255

Authors

Alyssa Pei Qi Chang School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia https://orcid.org/0009-0005-0131-2633
Sabrina Xin Yi Khor School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia https://orcid.org/0000-0002-4053-5603
Adeline Yoke Yin Chia School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia; Digital Health and Medical Advancement Impact Lab, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia https://orcid.org/0000-0002-1856-7211
Sunita Chamyuang School of Science, Mae Fah Luang University, Chaing Rai, Thailand; Microbial Products and Innovation Research Group, Mae Fah Luang University, Chaing Rai, Thailand https://orcid.org/0000-0002-3063-742X
Yin-Quan Tang School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia; Digital Health and Medical Advancement Impact Lab, Taylor's University, Subang Jaya, Selangor Darul Ehsan, Malaysia https://orcid.org/0000-0001-7327-2830

DOI:

https://doi.org/10.20883/medical.e1255

Keywords:

metastasis, angiogenesis, Cordyceps militaris, breast cancer

Abstract

Introduction. Cordyceps militaris (CM), a traditional medicinal fungus in East Asia, has garnered increasing attention due to its potential anticancer properties. Despite extensive use in traditional medicine, the mechanisms underlying its antimetastatic and antiangiogenic effects in breast cancer remain unclear. This study aimed to explore the bioactive components of aqueous extracts derived from CM’s mycelium (Aq-CMM) and fruiting body (Aq-CMF), focusing on their potential inhibitory activities on metastasis and angiogenesis in triple-negative breast cancer cells (MDA-MB-231).
Materials and methods. In silico molecular docking was conducted to screen for key CM bioactive compounds and evaluate their binding affinities toward metastasis- and angiogenesis-related targets, NF-κB and VEGFR. In vitro cytotoxicity was assessed using 2D monolayer and 3D spheroid MDA-MB-231 cultures treated with Aq-CMM and Aq-CMF. Cell viability (IC₅₀) was determined at 48 hours, and microscopic evaluation of treated spheroids was performed to assess morphological disruption.
Results and conclusions. Docking analyses identified ergothioneine as a primary CM-derived compound with strong binding affinity to NF-κB (−7.83 kcal/mol) and VEGFR (−7.62 kcal/mol), suggesting potent inhibitory effects on metastatic and angiogenic pathways. In vitro assays showed that Aq-CMF exerted greater growth-inhibitory effects (IC₅₀ = 54 µg/mL in 2D; 46 µg/mL in 3D) than Aq-CMM at 48 hours. Microscopic observations confirmed notable disruption of spheroid architecture following treatment. These findings highlight the therapeutic potential of ergothioneine-rich CM extracts, particularly from the fruiting body, as promising anticancer agents that warrant further mechanistic and translational studies.

Downloads

Download data is not yet available.

References

Jin Y, Meng X, Qiu Z, Su Y, Yu P, Qu P. Anti-tumor and anti-metastatic roles of cordycepin, one bioactive compound of Cordyceps militaris. Saudi Journal of Biological Sciences 2018;25:991–5. https://doi.org/10.1016/j.sjbs.2018.05.016.

Tuli HS, Sandhu SS, Sharma AK. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin. 3 Biotech 2014;4:1–12. https://doi.org/10.1007/s13205-013-0121-9.

Thoe ES, Chia YY, Tang YQ, Chamyuang S. Implementation of Omics Approaches in Unravelling the Potency of Cordyceps militaris in Drug Discovery. NPJ 2025;15:e030424228575. https://doi.org/10.2174/0122103155294164240323134513.

Quan X, Kwak BS, Lee J-Y, Park JH, Lee A, Kim TH, Park S. Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer. Evidence-Based Complementary and Alternative Medicine 2020;2020:9053274. https://doi.org/10.1155/2020/9053274.

Lee HH, Lee S, Lee K, Shin YS, Kang H, Cho H. Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis. DARU J Pharm Sci 2015;23:35. https://doi.org/10.1186/s40199-015-0117-6.

Yang C-H, Kao Y-H, Huang K-S, Wang C-Y, Lin L-W. Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells. Cell Death Dis 2012;3:e431–e431. https://doi.org/10.1038/cddis.2012.172.

Park SE, Kim J, Lee Y-W, Yoo H-S, Cho C-K. Antitumor Activity of Water Extracts From Cordyceps Militaris in NCI-H460 Cell Xenografted Nude Mice. Journal of Acupuncture and Meridian Studies 2009;2:294–300. https://doi.org/10.1016/S2005-2901(09)60071-6.

Park SE, Yoo HS, Jin C-Y, Hong SH, Lee Y-W, Kim BW, Lee SH, Kim W-J, Cho CK, Choi YH. Induction of apoptosis and inhibition of telomerase activity in human lung carcinoma cells by the water extract of Cordyceps militaris. Food and Chemical Toxicology 2009;47:1667–75. https://doi.org/10.1016/j.fct.2009.04.014.

Chamyuang S, Owatworakit A, Honda Y. New insights into cordycepin production in Cordyceps militaris and applications. Ann Transl Med 2019;7:S78–S78. https://doi.org/10.21037/atm.2019.04.12.

Kapałczyńska M, Kolenda T, Przybyła W, Zajączkowska M, Teresiak A, Filas V, Ibbs M, Bliźniak R, Łuczewski Ł, Lamperska K. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Aoms 2016. https://doi.org/10.5114/aoms.2016.63743.

Liu Y, Guo Z-J, Zhou X-W. Chinese Cordyceps: Bioactive Components, Antitumor Effects and Underlying Mechanism—A Review. Molecules 2022;27:6576. https://doi.org/10.3390/molecules27196576.

Tuli HS, Sharma AK, Sandhu SS, Kashyap D. Cordycepin: A bioactive metabolite with therapeutic potential. Life Sciences 2013;93:863–9. https://doi.org/10.1016/j.lfs.2013.09.030.

Chen L, Zhang L, Ye X, Deng Z, Zhao C. Ergothioneine and its congeners: anti-ageing mechanisms and pharmacophore biosynthesis. Protein & Cell 2024;15:191–206. https://doi.org/10.1093/procel/pwad048.

Shi C, Zhang N, Feng Y, Cao J, Chen X, Liu B. Aspirin Inhibits IKK-β-mediated Prostate Cancer Cell Invasion by Targeting Matrix Metalloproteinase-9 and Urokinase-Type Plasminogen Activator. Cell Physiol Biochem 2017;41:1313–24. https://doi.org/10.1159/000464434.

Al-Sanea MM, Chilingaryan G, Abelyan N, Sargsyan A, Hovhannisyan S, Gasparyan H, Gevorgyan S, Albogami S, Ghoneim MM, Farag AK, Mohamed AAB, El-Damasy AK. Identification of Novel Potential VEGFR-2 Inhibitors Using a Combination of Computational Methods for Drug Discovery. Life 2021;11:1070. https://doi.org/10.3390/life11101070.

Hu W-H, Duan R, Xia Y-T, Xiong Q-P, Wang H-Y, Chan GK-L, Liu S-Y, Dong TT-X, Qin Q-W, Tsim KW-K. Binding of Resveratrol to Vascular Endothelial Growth Factor Suppresses Angiogenesis by Inhibiting the Receptor Signaling. J Agric Food Chem 2019;67:1127–37. https://doi.org/10.1021/acs.jafc.8b05977.

Yang T, Yao H, He G, Song L, Liu N, Wang Y, Yang Y, Keller ET, Deng X. Effects of Lovastatin on MDA-MB-231 Breast Cancer Cells: An Antibody Microarray Analysis. J Cancer 2016;7:192–9. https://doi.org/10.7150/jca.13414.

Yang Y, Ren L, Li W, Zhang Y, Zhang S, Ge B, Yang H, Du G, Tang B, Wang H, Wang J. GABAergic signaling as a potential therapeutic target in cancers. Biomedicine & Pharmacotherapy 2023;161:114410. https://doi.org/10.1016/j.biopha.2023.114410.

Lim J, Gew L, Tang Y-Q. Biocomputational-mediated screening and molecular docking platforms for discovery of coumarin-derived antimelanogenesis agents. Dermatol Sin 2023;41:8. https://doi.org/10.4103/ds.DS-D-22-00087.

Grosdidier A, Zoete V, Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Research 2011;39:W270–7. https://doi.org/10.1093/nar/gkr366.

Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. IJMS 2021;22:12827. https://doi.org/10.3390/ijms222312827.

Li L, Hu M, Wang T, Chen H, Xu L. Repositioning Aspirin to Treat Lung and Breast Cancers and Overcome Acquired Resistance to Targeted Therapy. Front Oncol 2020;9:1503. https://doi.org/10.3389/fonc.2019.01503.

Putra WE, Salma, WO, Widiastuti, D. The Potency of Boesenbergin A and Boesenbergin B Compounds from Kaempferia pandurata as Anti-metastasis Agent: in silico study. Malaysian Journal of Biochemistry & Molecular Biology (MJBMB). 2020;23(3):128-32.

Ferreira L, Dos Santos R, Oliva G, Andricopulo A. Molecular Docking and Structure-Based Drug Design Strategies. Molecules 2015;20:13384–421. https://doi.org/10.3390/molecules200713384.

Chen S-Y, Ho K-J, Hsieh Y-J, Wang L-T, Mau J-L. Contents of lovastatin, γ-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia. LWT 2012;47:274–8. https://doi.org/10.1016/j.lwt.2012.01.019.

Lin S-Y, Chen Y-K, Yu H-T, Barseghyan GS, Asatiani MD, Wasser SP, Mau J-L. Comparative Study of Contents of Several Bioactive Components in Fruiting Bodies and Mycelia of Culinary-Medicinal Mushrooms. Int J Med Mushr 2013;15:315–23. https://doi.org/10.1615/IntJMedMushr.v15.i3.80.

Kajal K, Panda AK, Bhat J, Chakraborty D, Bose S, Bhattacharjee P, Sarkar T, Chatterjee S, Kar SK, Sa G. Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis. Sci Rep 2019;9:4073. https://doi.org/10.1038/s41598-019-40626-2.

Bhuiyan MR, Ahmed KS, Reza MS, Hossain H, Siam SMM, Nayan S, Jafrin S, Shuvo SR, Ud Daula AFMS. Prediction of angiogenesis suppression by myricetin from Aeginetia indica via inhibiting VEGFR2 signaling pathway using computer-aided analysis. Heliyon 2025;11:e41749. https://doi.org/10.1016/j.heliyon.2025.e41749.

Ghith A, Youssef KM, Ismail NSM, Abouzid KAM. Design, synthesis and molecular modeling study of certain VEGFR-2 inhibitors based on thienopyrimidne scaffold as cancer targeting agents. Bioorganic Chemistry 2019;83:111–28. https://doi.org/10.1016/j.bioorg.2018.10.008.

Jenkham P, Wibunkul K, Jantakee K, Katasai A, Thephinlap C, Khoothiam K, Suwannasom N, Panya A, Thepmalee C. Effect of aqueous extract from cordyceps militaris on the immune cell activation to kill breast cancer and hepatocellular carcinoma cell lines. Health Science, Science and Technology Reviews 2023;16:30–45.

Berrouet C, Dorilas N, Rejniak KA, Tuncer N. Comparison of Drug Inhibitory Effects ($$hbox {IC}_{50}$$) in Monolayer and Spheroid Cultures. Bull Math Biol 2020;82:68. https://doi.org/10.1007/s11538-020-00746-7.

Jędrejko KJ, Lazur J, Muszyńska B. Cordyceps militaris: An Overview of Its Chemical Constituents in Relation to Biological Activity. Foods 2021;10:2634. https://doi.org/10.3390/foods10112634.

Yow HY, Tang YQ, Lee JS. Antiproliferative effect of aspirin and diclofenac on MDA-MD-231 triple-negative breast cancer cells. Malaysian Journal of Biochemistry & Molecular Biology (MJBMB). 2022;25: 88-92.

Chen B-X, Xue L-N, Wei T, Ye Z-W, Li X-H, Guo L-Q, Lin J-F. Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris. Microb Cell Fact 2022;21:169. https://doi.org/10.1186/s12934-022-01891-5.

Lan Y-H, Lu Y-S, Wu J-Y, Lee H-T, Srinophakun P, Canko GN, Chiu C-C, Wang H-MD. Cordyceps militaris Reduces Oxidative Stress and Regulates Immune T Cells to Inhibit Metastatic Melanoma Invasion. Antioxidants 2022;11:1502. https://doi.org/10.3390/antiox11081502.

Yi Leong HJ. Identification of Potentially Therapeutic Target Genes in Metastatic Breast Cancer via Integrative Network Analysis. EJMO 2023:371–87. https://doi.org/10.14744/ejmo.2023.80452.

Chan JSL, Barseghyan GS, Asatiani MD, Wasser SP. Chemical Composition and Medicinal Value of Fruiting Bodies and Submerged Cultured Mycelia of Caterpillar Medicinal Fungus Cordyceps militaris CBS-132098 (Ascomycetes). Int J Med Mushrooms 2015;17:649–59. https://doi.org/10.1615/IntJMedMushrooms.v17.i7.50.

Wiley JL, Razdan RK, Martin BR. Evaluation of the role of the arachidonic acid cascade in anandamide’s in vivo effects in mice. Life Sciences 2006;80:24–35. https://doi.org/10.1016/j.lfs.2006.08.017.

Saydé T, El Hamoui O, Alies B, Gaudin K, Lespes G, Battu S. Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology. Nanomaterials 2021;11:481. https://doi.org/10.3390/nano11020481.

Rolver MG, Elingaard-Larsen LO, Pedersen SF. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. JoVE 2019:59714. https://doi.org/10.3791/59714.

Aggarwal V, Tuli H, Varol A, Thakral F, Yerer M, Sak K, Varol M, Jain A, Khan Md, Sethi G. Role of Reactive Oxygen Species in Cancer Progression: Molecular Mechanisms and Recent Advancements. Biomolecules 2019;9:735. https://doi.org/10.3390/biom9110735.

Virtanen SS, Kukkonen-Macchi A, Vainio M, Elima K, Härkönen PL, Jalkanen S, Yegutkin GG. Adenosine Inhibits Tumor Cell Invasion via Receptor-Independent Mechanisms. Molecular Cancer Research 2014;12:1863–74. https://doi.org/10.1158/1541-7786.MCR-14-0302-T.

Kanbara K, Otsuki Y, Watanabe M, Yokoe S, Mori Y, Asahi M, Neo M. GABAB receptor regulates proliferation in the high-grade chondrosarcoma cell line OUMS-27 via apoptotic pathways. BMC Cancer 2018;18:263. https://doi.org/10.1186/s12885-018-4149-4.

Zhang D, Li X, Yao Z, Wei C, Ning N, Li J. GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation. Cancer Letters 2014;348:100–8. https://doi.org/10.1016/j.canlet.2014.03.006.

Wang W, Nag SA, Zhang R. Targeting the NFκB signaling pathways for breast cancer prevention and therapy. Curr Med Chem. 2015;22(2):264-89. doi: 10.2174/0929867321666141106124315. n.d.

Choi H-W, Shin P-G, Lee J-H, Choi W-S, Kang M-J, Kong W-S, Oh M-J, Seo Y-B, Kim G-D. Anti-inflammatory effect of lovastatin is mediated via the modulation of NF-κB and inhibition of HDAC1 and the PI3K/Akt/mTOR pathway in RAW264.7 macrophages. Int J Mol Med 2017. https://doi.org/10.3892/ijmm.2017.3309.

Xie L, Zhu G, Shang J, Chen X, Zhang C, Ji X, Zhang Q, Wei Y. An overview on the biological activity and anti-cancer mechanism of lovastatin. Cellular Signalling 2021;87:110122. https://doi.org/10.1016/j.cellsig.2021.110122.

Khandelwal Gilman KA, Han S, Won Y-W, Putnam CW. Complex interactions of lovastatin with 10 chemotherapeutic drugs: a rigorous evaluation of synergism and antagonism. BMC Cancer 2021;21:356. https://doi.org/10.1186/s12885-021-07963-w.

Wong YY, Moon A, Duffin R, Barthet-Barateig A, Meijer HA, Clemens MJ, De Moor CH. Cordycepin Inhibits Protein Synthesis and Cell Adhesion through Effects on Signal Transduction. Journal of Biological Chemistry 2010;285:2610–21. https://doi.org/10.1074/jbc.M109.071159.

Li J, Cai H, Sun H, Qu J, Zhao B, Hu X, Li W, Qian Z, Yu X, Kang F, Wang W, Zou Z, Gu B, Xu K. Extracts of Cordyceps sinensis inhibit breast cancer growth through promoting M1 macrophage polarization via NF-κB pathway activation. Journal of Ethnopharmacology 2020;260:112969. https://doi.org/10.1016/j.jep.2020.112969.

Bjornsti M-A, Houghton PJ. The tor pathway: a target for cancer therapy. Nat Rev Cancer 2004;4:335–48. https://doi.org/10.1038/nrc1362.

Feng C, Chen R, Fang W, Gao X, Ying H, Zheng X, Chen L, Jiang J. Synergistic effect of CD47 blockade in combination with cordycepin treatment against cancer. Front Pharmacol 2023;14:1144330. https://doi.org/10.3389/fphar.2023.1144330.

Wang D, Zhang Y, Lu J, Wang Y, Wang J, Meng Q, Lee RJ, Wang D, Teng L. Cordycepin, a Natural Antineoplastic Agent, Induces Apoptosis of Breast Cancer Cells via Caspase-dependent Pathways. Nat Prod Commun. 2016 Jan;11(1):63-8. PMID: 26996021.

Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. ASSAY and Drug Development Technologies 2014;12:207–18. https://doi.org/10.1089/adt.2014.573.

Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FDP. 3D Cell Culture Systems: Advantages and Applications. Journal Cellular Physiology 2015;230:16–26. https://doi.org/10.1002/jcp.24683.

Lv D, Hu Z, Lu L, Lu H, Xu X. Three‑dimensional cell culture: A powerful tool in tumor research and drug discovery (Review). Oncol Lett 2017. https://doi.org/10.3892/ol.2017.7134.