Breast Cancer Research and Treatment

, Volume 171, Issue 3, pp 593–605 | Cite as

Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer

  • Nashwa Kabil
  • Recep Bayraktar
  • Nermin Kahraman
  • Hamada A. Mokhlis
  • George A. Calin
  • Gabriel Lopez-Berestein
  • Bulent OzpolatEmail author
Preclinical study



Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target.


In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice.


Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.


Thymoquinone eEF-2K MiR-603 Triple-negative breast cancer 



N.N.K. and B.O. conceived and coordinated the study and wrote the paper. N.N.K. performed in vitro experiments. R.B. assisted in experimental design and performed the overexpression of genes and assisted in the preparation of the figures. B.O. and N.K. prepared nanoliposomal particles incorporating TQ and performed in vivo studies. H.A.M. provided technical assistance for the animal study. G.C. and G.L.B. contributed to writing the manuscript. All authors analyzed the results and approved the final version of the manuscript.


This study was supported by the funding from Non-Coding RNA Center at M.D. Anderson Cancer Center and U54 NIH/NCI.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supplementary material

10549_2018_4847_MOESM1_ESM.docx (39 kb)
Supplementary material 1 (DOCX 38 KB)


  1. 1.
    Siegel RL, Miller KD, Jemal A (2017) Cancer statistics 2017 CA Cancer J Clin 67(1):7–30CrossRefPubMedGoogle Scholar
  2. 2.
    Hammond ME et al (2010) American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 28(16):2784–2795CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Isakoff SJ (2010) Triple-negative breast cancer: role of specific chemotherapy agents. Cancer J 16(1):53–61CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hamurcu Z et al (2016) FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells. Oncotarget 7(13):16619–16635CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Liu JC et al (2014) Combined deletion of Pten and p53 in mammary epithelium accelerates triple-negative breast cancer with dependency on eEF2K. EMBO Mol Med 6(12):1542–1560CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ashour AA et al (2014) Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells. Apoptosis 19(1):241–258CrossRefPubMedGoogle Scholar
  7. 7.
    Liu XY et al (2013) Inhibition of elongation factor-2 kinase augments the antitumor activity of Temozolomide against glioma. PLoS ONE 8(11):e81345CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kenney JW et al (2014) Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles. Adv Biol Regul 55:15–27CrossRefPubMedGoogle Scholar
  9. 9.
    Proud CG (2015) Regulation and roles of elongation factor 2 kinase. Biochem Soc Trans 43(3):328–332CrossRefPubMedGoogle Scholar
  10. 10.
    Leprivier G et al (2013) The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. Cell 153(5):1064–1079CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bayraktar R et al (2017) MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase. Oncotarget 8(7):11641–11658CrossRefPubMedGoogle Scholar
  12. 12.
    Tekedereli I et al (2012) Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer. PLoS ONE 7(7):e41171CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mansour MA et al (2002) Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem Funct 20(2):143–151CrossRefPubMedGoogle Scholar
  14. 14.
    Umar S et al (2012) Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats. Chem Biol Interact 197(1):40–46CrossRefPubMedGoogle Scholar
  15. 15.
    Pari L, Sankaranarayanan C (2009) Beneficial effects of thymoquinone on hepatic key enzymes in streptozotocin-nicotinamide induced diabetic rats. Life Sci 85(23–26):830–834CrossRefPubMedGoogle Scholar
  16. 16.
    El-Mahmoudy A et al (2002) Thymoquinone suppresses expression of inducible nitric oxide synthase in rat macrophages. Int Immunopharmacol 2(11):1603–1611CrossRefPubMedGoogle Scholar
  17. 17.
    Banerjee S et al (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62(7):938–946CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Li F, Rajendran P, Sethi G (2010) Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway. Br J Pharmacol 161(3):541–554CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Gali-Muhtasib H et al (2004) Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. Int J Oncol 25(4):857–866PubMedGoogle Scholar
  20. 20.
    Gali-Muhtasib H et al (2008) Thymoquinone triggers inactivation of the stress response pathway sensor CHEK1 and contributes to apoptosis in colorectal cancer cells. Cancer Res 68(14):5609–5618CrossRefPubMedGoogle Scholar
  21. 21.
    Woo CC et al (2011) Anticancer activity of thymoquinone in breast cancer cells: possible involvement of PPAR-gamma pathway. Biochem Pharmacol 82(5):464–475CrossRefPubMedGoogle Scholar
  22. 22.
    Hait WN et al (2006) Elongation factor-2 kinase: its role in protein synthesis and autophagy. Autophagy 2(4):294–296CrossRefPubMedGoogle Scholar
  23. 23.
    Velho-Pereira R et al (2011) Radiosensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis. Cell Biol Int 35(10):1025–1029CrossRefPubMedGoogle Scholar
  24. 24.
    Akar U et al (2008) Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells. Autophagy 4(5):669–679CrossRefPubMedGoogle Scholar
  25. 25.
    Chiang AC, Massague J (2008) Molecular basis of metastasis. N Engl J Med 359(26):2814–2823CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Safdari Y et al (2015) Natural inhibitors of PI3K/AKT signaling in breast cancer: emphasis on newly-discovered molecular mechanisms of action. Pharmacol Res 93:1–10CrossRefPubMedGoogle Scholar
  27. 27.
    Sethi S, Li Y, Sarkar FH (2013) Regulating miRNA by natural agents as a new strategy for cancer treatment. Curr Drug Targets 14(10):1167–1174CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lin Q et al (2017) Targeting microRNAs: a new action mechanism of natural compounds. Oncotarget 8(9):15961–15970PubMedGoogle Scholar
  29. 29.
    Sethi G, Ahn KS, Aggarwal BB (2008) Targeting nuclear factor-kappa B activation pathway by thymoquinone: role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol Cancer Res 6(6):1059–1070CrossRefPubMedGoogle Scholar
  30. 30.
    Andre F, Zielinski CC (2012) Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents. Ann Oncol 23(Suppl 6):vi46–vi51CrossRefGoogle Scholar
  31. 31.
    Brown M et al (2008) NF-kappaB in carcinoma therapy and prevention. Expert Opin Ther Targets 12(9):1109–1122CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Jeong W-S et al (2004) Modulatory properties of various natural chemopreventive agents on the activation of NF-κB signaling pathway. Pharm Res 21(4):661–670CrossRefPubMedGoogle Scholar
  33. 33.
    Sutton KM, Greenshields AL, Hoskin DW (2014) Thymoquinone, a bioactive component of black caraway seeds, causes G1 phase cell cycle arrest and apoptosis in triple-negative breast cancer cells with mutant p53. Nutr Cancer 66(3):408–418CrossRefPubMedGoogle Scholar
  34. 34.
    Al-Malki AL, Sayed AA (2014) Thymoquinone attenuates cisplatin-induced hepatotoxicity via nuclear factor kappa-beta. BMC Complement Altern Med 14:282CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wilson AJ et al (2015) Thymoquinone enhances cisplatin-response through direct tumor effects in a syngeneic mouse model of ovarian cancer. J Ovarian Res 8:46CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Baldwin AS (2001) Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest 107(3):241–246CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Shen HM, Tergaonkar V (2009) NFkappaB signaling in carcinogenesis and as a potential molecular target for cancer therapy. Apoptosis 14(4):348–363CrossRefPubMedGoogle Scholar
  38. 38.
    Connelly L et al (2011) Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden. Oncogene 30(12):1402–1412CrossRefPubMedGoogle Scholar
  39. 39.
    Zhang L, Bai Y, Yang Y (2016) Thymoquinone chemosensitizes colon cancer cells through inhibition of NF-kappaB. Oncol Lett 12(4):2840–2845CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Chavez KJ, Garimella SV, Lipkowitz S (2010) Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 32(1–2):35–48PubMedPubMedCentralGoogle Scholar
  41. 41.
    Lehmann BD et al (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121(7):2750–2767CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Asaduzzaman Khan M et al (2017) Thymoquinone, as an anticancer molecule: from basic research to clinical investigation. Oncotarget 8(31):51907–51919PubMedPubMedCentralGoogle Scholar
  43. 43.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefPubMedGoogle Scholar
  44. 44.
    Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6(11):857 –866CrossRefPubMedGoogle Scholar
  45. 45.
    Lu J et al (2005) MicroRNA expression profiles classify human cancers. Nature 435(7043):834–838CrossRefPubMedGoogle Scholar
  46. 46.
    Iorio MV, Croce CM (2009) MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 27(34):5848–5856CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Shah MY et al (2016) microRNA therapeutics in cancer—an emerging concept. EBioMedicine 12:34–42CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Schmidt MF (2014) Drug target miRNAs: chances and challenges. Trends Biotechnol 32(11):578–585CrossRefPubMedGoogle Scholar
  49. 49.
    Phuah NH, Nagoor NH (2014) Regulation of microRNAs by natural agents: new strategies in cancer therapies. Biomed Res Int 2014:804510CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Shin VY et al (2011) NF-kappaB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors. Carcinogenesis 32(2):240–245CrossRefPubMedGoogle Scholar
  51. 51.
    Duan Q et al (2012) ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer. PLoS ONE 7(2):e31518CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Li Q et al (2010) MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion. J Gastroenterol Hepatol 25(1):164–171CrossRefPubMedGoogle Scholar
  53. 53.
    Yuan Y, Tong L, Wu S (2015) microRNA and NF-kappa B, in microRNA: basic science: from molecular biology to clinical practice. In: Santulli G (ed). Springer International Publishing, Cham, pp 157–170Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Nashwa Kabil
    • 1
  • Recep Bayraktar
    • 1
  • Nermin Kahraman
    • 1
  • Hamada A. Mokhlis
    • 1
    • 2
  • George A. Calin
    • 1
    • 3
  • Gabriel Lopez-Berestein
    • 1
    • 3
  • Bulent Ozpolat
    • 1
    • 3
    Email author
  1. 1.Department of Experimental TherapeuticsThe University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Department of Pharmacology and Toxicology, Faculty of PharmacyAl-Azhar UniversityCairoEgypt
  3. 3.Center for RNA Interference and Non-Coding RNAsThe University of Texas MD Anderson Cancer CenterHoustonUSA

Personalised recommendations