Cytotoxic and antiproliferative effects of thymoquinone on rat C6 glioma cells depend on oxidative stress

  • N. G. Krylova
  • M. S. Drobysh
  • G. N. Semenkova
  • T. A. KulahavaEmail author
  • S. V. Pinchuk
  • O. I. Shadyro


Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood–brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20–50 μM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70–100 μM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.


Glioma Thymoquinone Apoptosis Reactive oxygen species Mitochondrial dysfunction 



This study was supported by the research grant of the Belarusian Republican Foundation for Fundamental Research (M17M-092) and Belarusian Ministry of Education (the national program of scientific research number 20161385).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11010_2019_3622_MOESM1_ESM.docx (54 kb)
Supplementary material 1 (DOCX 53 kb)


  1. 1.
    Delgado-Lopez PD, Corrales-Garcıa EM (2016) Survival in glioblastoma: a review on the impact of treatment modalities. Clin Transl Oncol 18(11):1062–1071. CrossRefGoogle Scholar
  2. 2.
    Jungk C, Chatziaslanidou D, Ahmadi R, Capper D, Bermejo JL (2016) Chemotherapy with BCNU in recurrent glioma: analysis of clinical outcome and side effects in chemotherapy-naive patients. BMC Cancer 16:81. CrossRefGoogle Scholar
  3. 3.
    Khosla D (2016) Concurrent therapy to enhance radiotherapeutic outcomes in glioblastoma. Ann Transl Med 4:54. CrossRefGoogle Scholar
  4. 4.
    Haar CP, Hebbar P, Wallace GC et al (2012) Drug resistance in glioblastoma: a mini review. Neurochem Res 37:1192–1200. CrossRefGoogle Scholar
  5. 5.
    Yan Y, Xu Z, Dai S (2016) Targeting autophagy to sensitive glioma to temozolomide treatment. J Exp Clin Cancer Res 35:23. CrossRefGoogle Scholar
  6. 6.
    Saify ZS, Mushtaq N, Noor F, Takween S, Arif M (1999) Role of quinone moiety as antitumour agents: a review. Pak J Pharm Sci 12(2):21–31Google Scholar
  7. 7.
    Lamson DW, Plaza SM (2003) The anticancer effects of vitamin K. Altern Med Rev 8(3):303–318Google Scholar
  8. 8.
    Darakhshan S, Bidmeshki PA, Hosseinzadeh CA, Sisakhtnezhad S (2015) Thymoquinone and its therapeutic potentials. Pharmacol Res 95–96:138–158. CrossRefGoogle Scholar
  9. 9.
    Racoma IO, Meisen WH, Wang QE, Kaur B, Wani AA (2013) Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells. PLoS ONE 8(9):e72882. CrossRefGoogle Scholar
  10. 10.
    Gurung RL, Lim SN, Khaw AK et al (2010) Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS ONE 5:e12124. CrossRefGoogle Scholar
  11. 11.
    Kolli-Bouhafs K, Boukhari A, Abusnina A, Velot E et al (2012) Thymoquinone reduces migration and invasion of human glioblastoma cells associated with FAK, MMP-2 and MMP-9 down-regulation. Invest New Drugs 30(6):2121–2131. CrossRefGoogle Scholar
  12. 12.
    Khader M, Eckl PM (2014) Thymoquinone: an emerging natural drug with a wide range of medical applications. Iran J Basic Med Sci 17:950–957Google Scholar
  13. 13.
    Krex D, Klink B, Hartmann C et al (2007) Longterm survival with glioblastoma multiforme. Brain 130:2596–2606. CrossRefGoogle Scholar
  14. 14.
    Pazhouhi M, Sariri R, Rabzia A, Khazaei M (2016) Thymoquinone synergistically potentiates temozolomide cytotoxicity through the inhibition of autophagy in U87MG cell line. Iran J Basic Med Sci 19:890–898Google Scholar
  15. 15.
    Gali-Muhtasib H, DiabAssaf M, Boltze C et al (2004) Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53dependent mechanism. Int J Oncol 25:857–866Google Scholar
  16. 16.
    Gali-Muhtasib H, Roessner A, SchneiderStock R (2006) Thymoquinone: a promising anticancer drug from natural sources. Int J Biochem Cell Biol 38:1249–1253CrossRefGoogle Scholar
  17. 17.
    Chehl N, Chipitsyna G, Gong Q, Yeo CJ, Arafat HA (2009) Antiinflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB (Oxford) 11(5):373–381. CrossRefGoogle Scholar
  18. 18.
    Roepke M, Diestel A, Bajbouj K et al (2007) Lack of p53 augments thymoquinone induced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biol Ther 6:160–169CrossRefGoogle Scholar
  19. 19.
    Dastjerdi MN, Mehdiabady EM, Iranpour FG, Bahramian H (2016) Effect of thymoquinone on p53 gene expression and consequence apoptosis in breast cancer cell line. Int J Prev Med 7:66. CrossRefGoogle Scholar
  20. 20.
    Hussain AR, Uddin S, Ahmed M et al (2013) Phosphorylated IkBa predicts poor prognosis in activated B-cell lymphoma and its inhibition with thymoquinone induces apoptosis via ROS release. PLoS ONE 8(3):e60540. CrossRefGoogle Scholar
  21. 21.
    Rahmani AH, Alzohairy MA, Khan MA, Aly SM (2014) Therapeutic implications of black seed and its constituent thymoquinone in the prevention of cancer through inactivation and activation of molecular pathways. Evid Based Complement Altern Med 2014:724658. CrossRefGoogle Scholar
  22. 22.
    Kato F, Tanaka M, Nakamura K (1999) Rapid fluorometric assay for cell viability and cell growth using nucleic acid staining and cell lysis agents. Toxicol In Vitro 13:923–929CrossRefGoogle Scholar
  23. 23.
    Reers M, Smith TW, Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480–4486CrossRefGoogle Scholar
  24. 24.
    Robinson KM, Janes MS, Pehar M et al (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. PNAS 103(41):15038–15043. CrossRefGoogle Scholar
  25. 25.
    Gomes A, Fernandes E, Lima JL (2005) Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 65:45–80. CrossRefGoogle Scholar
  26. 26.
    Wrona M, Wardman P (2006) Properties of the radical intermediate obtained on oxidation of 2′,7′-dichlorodihydrofluorescein, a probe for oxidative stress. Free Rad Biol Med 41:657–667. CrossRefGoogle Scholar
  27. 27.
    Rice GC, Bump EA, Shrieve DC, Lee W, Kovacs M (1986) Quantitative analysis of cellular glutathione by flow cytometry using monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. Cancer Res 46:6105–6110Google Scholar
  28. 28.
    Laurent A, Nicco C, Chereau C et al (2005) Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 65(3):948–956Google Scholar
  29. 29.
    Wondrak GT (2009) Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal 11(12):3013–3069. CrossRefGoogle Scholar
  30. 30.
    Rinaldi M, Caffo M, Minutoli L et al (2016) ROS and brain gliomas: an overview of potential and innovative therapeutic strategies. Int J Mol Sci 17(6):E984. CrossRefGoogle Scholar
  31. 31.
    Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov. 8(7):579–591. CrossRefGoogle Scholar
  32. 32.
    Alhosin M, Abusnina A, Achour M et al (2010) Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1. Biochem Pharmacol 79(9):1251–1260. CrossRefGoogle Scholar
  33. 33.
    Arafa E-SA, Zhu Q, Shah ZI et al (2011) Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Mutat Res 706(1–2):28–35. CrossRefGoogle Scholar
  34. 34.
    Paramasivam A, Raghunandhakumar S, Priyadharsini JV, Jayaraman G (2015) In vitro anti-neuroblastoma activity of thymoquinone against neuro-2a cells via cell-cycle arrest. Asian Pac J Cancer Prev 16(18):8313–8319CrossRefGoogle Scholar
  35. 35.
    Banerjee S, Padhye S, Azmi A et al (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62(7):938–946. CrossRefGoogle Scholar
  36. 36.
    Rajput S, Kumar BN, Dey KK, Pal I, Parekh A, Mandal M (2013) Molecular targeting of Akt by thymoquinone promotes G(1) arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells. Life Sci 93(21):783–790. CrossRefGoogle Scholar
  37. 37.
    Woo CC, Hsu A, Kumar AP, Sethi G, Tan KH (2013) Thymoquinone inhibits tumor growth and induces apoptosis in a breast cancer xenograft mouse model: the role of p38 MAPK and ROS. PLoS ONE 8(10):e75356. CrossRefGoogle Scholar
  38. 38.
    Yi T, Cho S-G, Yi Z et al (2008) Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and ERK signaling pathways. Mol Cancer Ther 7(7):1789–1796. CrossRefGoogle Scholar
  39. 39.
    Salim LZ, Mohan S, Othman R et al (2013) Thymoquinone induces mitochondria-mediated apoptosis in acute lymphoblastic leukaemia in vitro. Molecules 18(9):11219–11240. CrossRefGoogle Scholar
  40. 40.
    Hussain AR, Ahmed M, Ahmed S et al (2011) Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma. Free Radic Biol Med 50:978–987. CrossRefGoogle Scholar
  41. 41.
    Yu SM, Kim SJ (2013) Thymoquinone-induced reactive oxygen species causes apoptosis of chondrocytes via PI3K/Akt and p38kinase pathway. Exp Biol Med (Maywood) 238(7):811–820. CrossRefGoogle Scholar
  42. 42.
    El-Najjar N, Chatila M, Moukadem H et al (2010) Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling. Apoptosis 15:183–195. CrossRefGoogle Scholar
  43. 43.
    Yu SM, Kim SJ (2015) The thymoquinone-induced production of reactive oxygen species promotes dedifferentiation through the ERK pathway and inflammation through the p38 and PI3K pathways in rabbit articular chondrocytes. Int J Mol Med 35(2):325–332. CrossRefGoogle Scholar
  44. 44.
    Al-Shabanah OA, Badary OA, Nagi MN, Al-Gharably NM, Al-Rikabi AC, Al-Bekairi AM (1998) Thymoquinone protects against doxorubicin-induced cardiotoxicity without compromising its antitumor activity. J Exp Clin Cancer Res 17(2):193–198Google Scholar
  45. 45.
    Deller S, Macheroux P, Sollner S (2008) Flavin-dependent quinone reductases. Cell Mol Life Sci 65:141–160. CrossRefGoogle Scholar
  46. 46.
    Siegel D, Reigan P, Ross D (2008) One- and two-electron-mediated reduction of quinones: enzymology and toxicological implications. In: Elfarra A (ed) Advances in bioactivation research. Springer-Verlag, New York, pp 169–199Google Scholar
  47. 47.
    Buffinton GD, Ollinger K, Brunmark A, Cadenas E (1989) DT-diaphorase-catalyzed reduction of 1,4-naphthoquinone derivatives and glutathionyl-quinone conjugates. Effect of substituents on autoxidation rates. Biochem J 257(2):561–571CrossRefGoogle Scholar
  48. 48.
    Watanabe N, Forman HJ (2003) Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells. Arch Biochem Biophys 411(1):145–157CrossRefGoogle Scholar
  49. 49.
    Belinsky M, Jaiswal AK (1993) NAD(P)H: quinone oxidoreductase1 (DT-diaphorase) expression in normal and tumor tissues. Cancer Metastasis Rev 12:103–117CrossRefGoogle Scholar
  50. 50.
    Kanamori M, Higa T, Sonoda Y et al (2015) Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients. Neuro-Oncology 17(4):555–565. CrossRefGoogle Scholar
  51. 51.
    Backos DS, Franklin CC, Reigan P (2012) The role of glutathione in brain tumor drug resistance. Biochem Pharmacol 83(8):1005–1012. CrossRefGoogle Scholar
  52. 52.
    Ollinger K, Kägedal K (2002) Induction of apoptosis by redox-cycling quinones. Subcell Biochem 36:151–170CrossRefGoogle Scholar
  53. 53.
    Brunmark A, Cadenas E (1989) Redox and addition chemistry of quinoid compounds and its biological implications. Free Rad Biol Med 7:435–477CrossRefGoogle Scholar
  54. 54.
    Guin PS, Das S, Mandal PC (2011) Electrochemical reduction of quinones in different media: a review. Intern J Electrochem 2011:816202. CrossRefGoogle Scholar
  55. 55.
    Khalife KH, Lupidi G (2007) Nonenzymatic reduction of thymoquinone in physiological conditions. Free Radic Res 41(2):153–161. CrossRefGoogle Scholar
  56. 56.
    Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12–15. CrossRefGoogle Scholar
  57. 57.
    Neuzil J, Dong LF, Rohlena J, Truksa J, Ralph SJ (2013) Classification of mitocans, anti-cancer drugs acting on mitochondria. Mitochondrion 13(3):199–208. CrossRefGoogle Scholar
  58. 58.
    Weinberg SE, Chandel NS (2015) Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol 11(1):9–15. CrossRefGoogle Scholar
  59. 59.
    Katsetos CD, Anni H, Dráber P (2013) Mitochondrial dysfunction in gliomas. Semin Pediatr Neurol 20(3):216–227. CrossRefGoogle Scholar
  60. 60.
    Sethi G, Ahn KS, Aggarwal BB (2008) Targeting nuclear factor-κB activation pathway by thymoquinone: role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol Cancer Res 6(6):1059–1070. CrossRefGoogle Scholar
  61. 61.
    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 161:541–554CrossRefGoogle Scholar
  62. 62.
    Trachootham D, Lu W, Ogasawara MA, Nilsa RD, Huang P (2008) Redox regulation of cell survival. Antioxid Redox Signal 10(8):1343–1374. CrossRefGoogle Scholar
  63. 63.
    Connelly L, Barham W, Onishko HM et al (2011) Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden. Oncogene 30:1402–1412. CrossRefGoogle Scholar
  64. 64.
    Xu D, Ma Y, Zhao B et al (2014) Thymoquinone induces G2/M arrest, inactivates PI3K/Akt and nuclear factor-κB pathways in human cholangiocarcinomas both in vitro and in vivo. Oncol Rep 31(5):2063–2070. CrossRefGoogle Scholar
  65. 65.
    Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, González-Barón M (2004) PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 30(2):193–204. CrossRefGoogle Scholar
  66. 66.
    Li X, Wu C, Chen N et al (2016) PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget 7(22):33440–33450. Google Scholar
  67. 67.
    Jiang BH, Liu LZ (2008) Role of mTOR in anticancer drug resistance: perspectives for improved drug treatment. Drug Resist Updates 11(3):63–76. CrossRefGoogle Scholar
  68. 68.
    Chakravarti A, Zhai G, Suzuki Y et al (2004) The prognostic significance of phosphatidylinositol 3-kinase pathway activation in human gliomas. J Clin Oncol 22:1926–1933. CrossRefGoogle Scholar
  69. 69.
    Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103(2):239–252CrossRefGoogle Scholar
  70. 70.
    Wada T, Penninger JM (2004) Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23(16):2838–2849. CrossRefGoogle Scholar
  71. 71.
    Du L, Lyle CS, Obey TB et al (2004) Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent. J Biol Chem 279(12):11957–11966CrossRefGoogle Scholar
  72. 72.
    Krylova NG, Kulahava TA, Koran SV, Semenkova GN (2017) Proliferation of cultured glioma cells mediated by coenzyme Q10 under conditions of serum deprivation. Cell Tissue Biol 11(3):220–226. CrossRefGoogle Scholar

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

Authors and Affiliations

  • N. G. Krylova
    • 1
  • M. S. Drobysh
    • 2
  • G. N. Semenkova
    • 2
  • T. A. Kulahava
    • 1
    Email author
  • S. V. Pinchuk
    • 3
  • O. I. Shadyro
    • 2
  1. 1.Department of Biophysics, Faculty of PhysicsBelarusian State UniversityMinskBelarus
  2. 2.Department of Radiation Chemistry and Pharmaceutical Technologies, Faculty of ChemistryBelarusian State UniversityMinskBelarus
  3. 3.Institute of Biophysics and Cell Engineering of National Academy of Sciences of BelarusMinskBelarus

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