Silibinin inhibits migration and invasion of breast cancer MDA-MB-231 cells through induction of mitochondrial fusion

  • Lingling Si
  • Jianing Fu
  • Weiwei Liu
  • Toshihiko Hayashi
  • Yuheng Nie
  • Kazunori Mizuno
  • Shunji Hattori
  • Hitomi Fujisaki
  • Satoshi Onodera
  • Takashi IkejimaEmail author


Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30–90 μM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin’s negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-β proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells.


Silibinin Mitochondrial fusion ROS Migration and invasion Inflammation MDA-MB-231 cells 



This research was supported by National Natural Science Foundation of China (No. 81703528).


  1. 1.
    Masso-Welch P, Girald Berlingeri S, King-Lyons ND, Mandell L, Hu J, Greene CJ, Federowicz M, Cao P, Connell TD, Heakal Y (2018) LT-IIc, a bacterial type II heat-labile enterotoxin, induces specific lethality in triple negative breast cancer cells by modulation of autophagy and induction of apoptosis and necroptosis. Int J Mol Sci. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lee H, Yoon Y (2016) Mitochondrial fission and fusion. Biochem Soc Trans 44:1725–1735. CrossRefPubMedGoogle Scholar
  3. 3.
    Campello S, Lacalle RA, Bettella M, Manes S, Scorrano L, Viola A (2006) Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J Exp Med 203:2879–2886. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Han XJ, Yang ZJ, Jiang LP, Wei YF, Liao MF, Qian Y, Li Y, Huang X, Wang JB, Xin HB, Wan YY (2015) Mitochondrial dynamics regulates hypoxia-induced migration and antineoplastic activity of cisplatin in breast cancer cells. Int J Oncol 46:691–700. CrossRefPubMedGoogle Scholar
  5. 5.
    Zhao J, Zhang J, Yu M, Xie Y, Huang Y, Wolff DW, Abel PW, Tu Y (2013) Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Oncogene 32:4814–4824. CrossRefPubMedGoogle Scholar
  6. 6.
    Qiu Y, Liu Y, Li WH, Zhang HQ, Tian XX, Fang WG (2018) P2Y2 receptor promotes the migration and invasion of breast cancer cells via EMT-related genes Snail and E-cadherin. Oncol Rep 39:138–150. CrossRefPubMedGoogle Scholar
  7. 7.
    Chio IIC, Tuveson DA (2017) ROS in cancer: the burning question. Trends Mol Med 23:411–429. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Arumugam A, Subramani R, Nandy S, Powell S, Velazquez M, Orozco A, Galvez A, Lakshmanaswamy R (2016) Desacetyl nimbinene inhibits breast cancer growth and metastasis through reactive oxygen species mediated mechanisms. Tumour Biol 37:6527–6537. CrossRefPubMedGoogle Scholar
  9. 9.
    Seol MA, Park JH, Jeong JH, Lyu J, Han SY, Oh SM (2017) Role of TOPK in lipopolysaccharide-induced breast cancer cell migration and invasion. Oncotarget 8:40190–40203. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ye K, Chen QW, Sun YF, Lin JA, Xu JH (2018) Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-kappaB signaling. J Cell Biochem 119:1922–1930. CrossRefPubMedGoogle Scholar
  11. 11.
    Zhong Z, Liang S, Sanchez-Lopez E, He F, Shalapour S, Lin XJ, Wong J, Ding S, Seki E, Schnabl B, Hevener AL, Greenberg HB, Kisseleva T, Karin M (2018) New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature 560:198–203. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jiang K, Wang W, Jin X, Wang Z, Ji Z, Meng G (2015) Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol Rep 33:2711–2718. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Byun HJ, Darvin P, Kang DY, Sp N, Joung YH, Park JH, Kim SJ, Yang YM (2017) Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells. Oncol Rep 37:3270–3278. CrossRefPubMedGoogle Scholar
  14. 14.
    Li X, Lin H, Jiang F, Lou Y, Ji L, Li S (2019) Knock-down of HOXB8 prohibits proliferation and migration of colorectal cancer cells via Wnt/beta-catenin signaling pathway. Med Sci Monit 25:711–720. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mao W, Yan S, Zhang H, Cao L, Wang J, He P (2016) A combined modality of carboplatin and photodynamic therapy suppresses epithelial–mesenchymal transition and matrix metalloproteinase-2 (MMP-2)/MMP-9 expression in HEp-2 human laryngeal cancer cells via ROS-mediated inhibition of MEK/ERK signalling pathway. Lasers Med Sci 31(8):1697–1705CrossRefGoogle Scholar
  16. 16.
    Kolgiri V, Patil VW (2017) Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients. Braz J Infect Dis 21:35–41. CrossRefPubMedGoogle Scholar
  17. 17.
    Si L, Liu W, Hayashi T, Ji Y, Fu J, Nie Y, Mizuno K, Hattori S, Onodera S, Ikejima T (2019) Silibinin-induced apoptosis of breast cancer cells involves mitochondrial impairment. Arch Biochem Biophys 671:42–51. CrossRefPubMedGoogle Scholar
  18. 18.
    Dong X, Guan X, Chen K, Jin S, Wang C, Yan L, Shi Z, Zhang X, Chen L, Wan Q (2017) Abnormal mitochondrial dynamics and impaired mitochondrial biogenesis in trigeminal ganglion neurons in a rat model of migraine. Neurosci Lett 636:127–133. CrossRefPubMedGoogle Scholar
  19. 19.
    Fonseca TB, Sanchez-Guerrero A, Milosevic I, Raimundo N (2019) Mitochondrial fission requires DRP1 but not dynamins. Nature 570:E34–E42. CrossRefPubMedGoogle Scholar
  20. 20.
    Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, Malech HL, Ledbetter JA, Elkon KB, Kaplan MJ (2016) Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med 22:146–153. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bordon Y (2018) mtDNA synthesis ignites the inflammasome. Nat Rev Immunol 18:539. CrossRefPubMedGoogle Scholar
  22. 22.
    Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, Ramanujan VK, Wolf AJ, Vergnes L, Ojcius DM, Rentsendorj A, Vargas M, Guerrero C, Wang Y, Fitzgerald KA, Underhill DM, Town T, Arditi M (2012) Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36:401–414. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Liu X, Zhao W, Wang W, Lin S, Yang L (2017) Puerarin suppresses LPS-induced breast cancer cell migration, invasion and adhesion by blockage NF-kappaB and Erk pathway. Biomed Pharmacother 92:429–436. CrossRefPubMedGoogle Scholar
  24. 24.
    Li H, Zhang X, Chen M, Chen J, Gao T, Yao S (2018) Dexmedetomidine inhibits inflammation in microglia cells under stimulation of LPS and ATP by c-Fos/NLRP3/caspase-1 cascades. EXCLI J 17:302–311. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Si L, Liu W, Hayashi T, Ji Y, Fu J, Nie Y, Mizuno K, Hattori S, Onodera S, Ikejima T (2019) Silibinin-induced apoptosis of breast cancer cells involves mitochondrial impairment. Arch Biochem Biophys. CrossRefPubMedGoogle Scholar
  26. 26.
    Liu B, Yang P, Ye Y, Zhou Y, Li L, Tashiro S, Onodera S, Ikejima T (2011) Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells. Free Radic Res 45:835–847. CrossRefPubMedGoogle Scholar
  27. 27.
    Ham J, Lim W, Bazer FW, Song G (2018) Silibinin stimluates apoptosis by inducing generation of ROS and ER stress in human choriocarcinoma cells. J Cell Physiol 233:1638–1649. CrossRefPubMedGoogle Scholar
  28. 28.
    Kim KW, Choi CH, Kim TH, Kwon CH, Woo JS, Kim YK (2009) Silibinin inhibits glioma cell proliferation via Ca2+/ROS/MAPK-dependent mechanism in vitro and glioma tumor growth in vivo. Neurochem Res 34:1479–1490. CrossRefPubMedGoogle Scholar
  29. 29.
    Song X, Zhou B, Zhang P, Lei D, Wang Y, Yao G, Hayashi T, Xia M, Tashiro S, Onodera S, Ikejima T (2016) Protective effect of silibinin on learning and memory impairment in LPS-treated rats via ROS-BDNF-TrkB pathway. Neurochem Res 41:1662–1672. CrossRefPubMedGoogle Scholar
  30. 30.
    Kalemci S, Topal Y, Celik SY, Yilmaz N, Beydilli H, Kosar MI, Dirican N, Altuntas I (2015) Silibinin attenuates methotrexate-induced pulmonary injury by targeting oxidative stress. Exp Ther Med 10:503–507. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kim TH, Woo JS, Kim YK, Kim KH (2014) Silibinin induces cell death through reactive oxygen species-dependent downregulation of notch-1/ERK/Akt signaling in human breast cancer cells. J Pharmacol Exp Ther 349:268–278. CrossRefPubMedGoogle Scholar
  32. 32.
    Bahat A, Goldman A, Zaltsman Y, Khan DH, Halperin C, Amzallag E, Krupalnik V, Mullokandov M, Silberman A, Erez A, Schimmer AD, Hanna JH, Gross A (2018) MTCH2-mediated mitochondrial fusion drives exit from naive pluripotency in embryonic stem cells. Nat Commun 9:5132. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Li H, He F, Zhao X, Zhang Y, Chu X, Hua C, Qu Y, Duan Y, Ming L (2017) YAP inhibits the apoptosis and migration of human rectal cancer cells via suppression of JNK-Drp1-mitochondrial fission-HtrA2/Omi pathways. Cell Physiol Biochem 44:2073–2089. CrossRefPubMedGoogle Scholar
  34. 34.
    Jin X, Zhu L, Cui Z, Tang J, Xie M, Ren G (2019) Elevated expression of GNAS promotes breast cancer cell proliferation and migration via the PI3 K/AKT/Snail1/E-cadherin axis. Clin Transl Oncol. CrossRefPubMedGoogle Scholar
  35. 35.
    Caino MC, Ghosh JC, Chae YC, Vaira V, Rivadeneira DB, Faversani A, Rampini P, Kossenkov AV, Aird KM, Zhang R, Webster MR, Weeraratna AT, Bosari S, Languino LR, Altieri DC (2015) PI3 K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion. Proc Natl Acad Sci USA 112:8638–8643. CrossRefPubMedGoogle Scholar
  36. 36.
    Zhou Q, Gensch C, Keller C, Schmitt H, Esser J, Moser M, Liao JK (2015) MnTBAP stimulates angiogenic functions in endothelial cells through mitofusin-1. Vascul Pharmacol 72:163–171. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lugus JJ, Ngoh GA, Bachschmid MM, Walsh K (2011) Mitofusins are required for angiogenic function and modulate different signaling pathways in cultured endothelial cells. J Mol Cell Cardiol 51:885–893. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhang J, Wang J, Luan T, Zuo Y, Chen J, Zhang H, Ye Z, Wang H, Hai B (2019) Deubiquitinase USP9X regulates the invasion of prostate cancer cells by regulating the ERK pathway and mitochondrial dynamics. Oncol Rep 41:3292–3304. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Lee JY, Kapur M, Li M, Choi MC, Choi S, Kim HJ, Kim I, Lee E, Taylor JP, Yao TP (2014) MFN1 deacetylation activates adaptive mitochondrial fusion and protects metabolically challenged mitochondria. J Cell Sci 127:4954–4963. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Zhang H, Wang P, Bisetto S, Yoon Y, Chen Q, Sheu SS, Wang W (2017) A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration. Cardiovasc Res 113:160–170. CrossRefPubMedGoogle Scholar
  41. 41.
    Wang Z, Li Y, Sarkar FH (2010) Signaling mechanism(s) of reactive oxygen species in epithelial–mesenchymal transition reminiscent of cancer stem cells in tumor progression. Curr Stem Cell Res Ther 5:74–80CrossRefGoogle Scholar
  42. 42.
    Wu Y, Antony S, Meitzler JL, Doroshow JH (2014) Molecular mechanisms underlying chronic inflammation-associated cancers. Cancer Lett 345:164–173. CrossRefPubMedGoogle Scholar
  43. 43.
    West AP, Shadel GS (2017) Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat Rev Immunol 17:363–375. CrossRefPubMedGoogle Scholar
  44. 44.
    Gao Q, Zhao YJ, Wang XY, Qiu SJ, Shi YH, Sun J, Yi Y, Shi JY, Shi GM, Ding ZB, Xiao YS, Zhao ZH, Zhou J, He XH, Fan J (2012) CXCR44 upregulation contributes to a proinflammatory tumor microenvironment that drives metastasis and poor patient outcomes in hepatocellular carcinoma. Cancer Res 72:3546–3556. CrossRefPubMedGoogle Scholar
  45. 45.
    El Hasasna H, Saleh A, Al Samri H, Athamneh K, Attoub S, Arafat K, Benhalilou N, Alyan S, Viallet J, Al Dhaheri Y, Eid A, Iratni R (2016) Rhus coriaria suppresses angiogenesis, metastasis and tumor growth of breast cancer through inhibition of STAT3, NFkappaB and nitric oxide pathways. Sci Rep 6:21144. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Cohen EN, Gao H, Anfossi S, Mego M, Reddy NG, Debeb B, Giordano A, Tin S, Wu Q, Garza RJ, Cristofanilli M, Mani SA, Croix DA, Ueno NT, Woodward WA, Luthra R, Krishnamurthy S, Reuben JM (2015) Inflammation mediated metastasis: immune induced epithelial-to-mesenchymal transition in inflammatory breast cancer cells. PLoS ONE 10:e0132710. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Lingling Si
    • 1
  • Jianing Fu
    • 1
  • Weiwei Liu
    • 1
  • Toshihiko Hayashi
    • 1
    • 2
  • Yuheng Nie
    • 1
  • Kazunori Mizuno
    • 3
  • Shunji Hattori
    • 3
  • Hitomi Fujisaki
    • 3
  • Satoshi Onodera
    • 4
  • Takashi Ikejima
    • 1
    • 5
    Email author
  1. 1.Wuya College of InnovationShenyang Pharmaceutical UniversityShenyangPeople’s Republic of China
  2. 2.Department of Chemistry and Life ScienceSchool of Advanced Engineering, Kogakuin UniversityHachiojiJapan
  3. 3.Nippi Research Institute of BiomatrixTorideJapan
  4. 4.Medical Research Institute of Curing MibyoMachidaJapan
  5. 5.Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & DevelopmentShenyang Pharmaceutical UniversityShenyangPeople’s Republic of China

Personalised recommendations