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Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-κB signaling and IL-8 expression


Medulloblastoma (MB) is the most common malignant brain tumor of childhood. The transcription factor NF-κB is overexpressed in human MB and is a critical factor for MB tumor growth. NF-κB is known to regulate the expression of interleukin-8 (IL-8), the chemokine that enhances cancer cell growth and resistance to chemotherapy. We have recently shown that thymoquinone (TQ) suppresses growth of hepatocellular carcinoma cells in part by inhibiting NF-κB signaling. Here we sought to extend these studies in MB cells and show that TQ suppresses growth of MB cells in a dose- and time-dependent manner, causes G2M cell cycle arrest, and induces apoptosis. TQ significantly increased generation of reactive oxygen species (ROS), while pretreatment of MB cells with the ROS scavenger N-acetylcysteine (NAC) abrogated TQ-induced cell death and apoptosis, suggesting that TQ-induced cell death and apoptosis are oxidative stress-mediated. TQ inhibitory effects were associated with inhibition of NF-κB and altered expression of its downstream effectors IL-8 and its receptors, the anti-apoptotic Bcl-2, Bcl-xL, X-IAP, and FLIP, as well as the pro-apoptotic TRAIL-R1, caspase-8, caspase-9, Bcl-xS, and cytochrome c. TQ-triggered apoptosis was substantiated by up-regulation of the executioner caspase-3 and caspase-7, as well as cleavage of the death substrate poly(ADP-ribose)polymerase. Interestingly, pretreatment of MB cells with NAC or the pan-caspase inhibitor zVAD-fmk abrogated TQ-induced apoptosis, loss of cyclin B1 and NF-κB activity, suggesting that these TQ-mediated effects are oxidative stress- and caspase-dependent. These findings reveal that TQ induces both extrinsic and intrinsic pathways of apoptosis in MB cells, and suggest its potential usefulness in the treatment of MB.

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  1. 1.

    Gurney JG, Wall DA, Jukich PJ, Davis FG (1999) The contribution of nonmalignant tumors to CNS tumor incidence rates among children in the United States. Cancer Causes Control 10:101–105

  2. 2.

    Gilbertson RJ (2004) Medulloblastoma: signalling a change in treatment. Lancet Oncol 5:209–218. doi:10.1016/S1470-2045(04)01424-X

  3. 3.

    Research Unit, Oncology Centre, King Faisal Specialist Hospital and Research Centre Tumor Registry Annual Report. 2012. Available from: http://www.kfshrc.edu.sa/oncology/2012_Tumor_Registry_Annual_Report.pdf

  4. 4.

    Fossati P, Ricardi U, Orecchia R (2009) Pediatric medulloblastoma: toxicity of current treatment and potential role of protontherapy. Cancer Treat Rev 35:79–96. doi:10.1016/j.ctrv.2008.09.002

  5. 5.

    Jenkin D, Danjoux C, Greenberg M (1998) Subsequent quality of life for children irradiated for a brain tumor before age 4 years. Med Pediatr Oncol 31:506–511

  6. 6.

    Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108:153–164

  7. 7.

    Ozoren N, El-Deiry WS (2003) Cell surface Death Receptor signaling in normal and cancer cells. Semin Cancer Biol 13:135–147

  8. 8.

    MacFarlane M (2003) TRAIL-induced signalling and apoptosis. Toxicol Lett 139:89–97

  9. 9.

    Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811. doi:10.1038/sj.onc.1209608

  10. 10.

    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516. doi:10.1080/01926230701320337

  11. 11.

    Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193

  12. 12.

    Anon (2007) All natural (Editorial). Nat Chem Biol 3:351. http://www.nature.com/nchembio/journal/v3/n7/pdf/nchembio0707-351.pdf

  13. 13.

    Krause J and Tobin G (2013) Discovery, development, and regulation of natural products. Using old solutions to new problems-natural drug discovery in the 21st century 21:3–35

  14. 14.

    Khader M, Eckl PM (2014) Thymoquinone: an emerging natural drug with a wide range of medical applications. Iran J Basic Med Sci 17:950–957

  15. 15.

    Hajhashemi V, Ghannadi A, Jafarabadi H (2004) Black cumin seed essential oil, as a potent analgesic and antiinflammatory drug. Phytother Res 18:195–199. doi:10.1002/ptr.1390

  16. 16.

    Ahmed AM, Al-Olayan EM, Aboul-Soud MA, Al-Khedhairy AA (2010) The immune enhancer, thymoquinone, and the hope of utilizing the immune system of Aedes caspius against disease agents. Afr J Biotechnol 9:3183–3195

  17. 17.

    Entok E, Ustuner MC, Ozbayer C, Tekin N, Akyuz F, Yangi B, Kurt H, Degirmenci I, Gunes HV (2014) Anti-inflammatuar and anti-oxidative effects of Nigella sativa L.: 18FDG-PET imaging of inflammation. Mol Biol Rep 41:2827–2834. doi:10.1007/s11033-014-3137-2

  18. 18.

    Khader M, Bresgen N, Eckl PM (2010) Antimutagenic effects of ethanolic extracts from selected Palestinian medicinal plants. J Ethnopharmacol 127:319–324. doi:10.1016/j.jep.2009.11.001

  19. 19.

    Linjawi SA, Khalil WK, Hassanane MM, Ahmed ES (2015) Evaluation of the protective effect of Nigella sativa extract and its primary active component thymoquinone against DMBA-induced breast cancer in female rats. Arch Med Sci 11:220–229. doi:10.5114/aoms.2013.33329

  20. 20.

    Banerjee S, Padhye S, Azmi A, Wang Z, Philip PA, Kucuk O, Sarkar FH, Mohammad RM (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62:938–946. doi:10.1080/01635581.2010.509832

  21. 21.

    Al-Salahi R, Marzouk M, Ashour AE, Alswaidan I (2014) Antitumor activity of 1,2,4-triazolo[1,5-a]quinazolines. Asian J Chem 26:2173–2176

  22. 22.

    van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP (1996) A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24:131–139. doi:10.1002/(SICI)1097-0320(19960601)24:2<131:AID-CYTO5>3.0.CO;2-M

  23. 23.

    Clarke RG, Lund EK, Johnson IT, Pinder AC (2000) Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content. Cytometry 39:141–150. doi:10.1002/(SICI)1097-0320(20000201)39:2<141:AID-CYTO7

  24. 24.

    Ricci JE, Waterhouse N, Green DR (2003) Mitochondrial functions during cell death, a complex (I–V) dilemma. Cell Death Differ 10:488–492. doi:10.1038/sj.cdd.4401225

  25. 25.

    Bueno-da-Silva AE, Brumatti G, Russo FO, Green DR, Amarante-Mendes GP (2003) Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity. Cell Death Differ 10:592–598. doi:10.1038/sj.cdd.4401210

  26. 26.

    Ashour AE, Abd-Allah AR, Korashy HM, Attia SM, Alzahrani AZ, Saquib Q, Bakheet SA, Abdel-Hamied HE, Jamal S, Rishi AK (2014) Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression, elevated levels of TRAIL receptors, oxidative stress and apoptosis. Mol Cell Biochem 389:85–98. doi:10.1007/s11010-013-1930-1

  27. 27.

    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 6:1059–1070. doi:10.1158/1541-7786.MCR-07-2088

  28. 28.

    Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr (1998) NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680–1683

  29. 29.

    Murray AW (1992) Creative blocks: cell-cycle checkpoints and feedback controls. Nature 359:599–604. doi:10.1038/359599a0

  30. 30.

    Shoieb AM, Elgayyar M, Dudrick PS, Bell JL, Tithof PK (2003) In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. Int J Oncol 22:107–113

  31. 31.

    Hussain AR, Ahmed M, Ahmed S, Manogaran P, Platanias LC, Alvi SN, Al-Kuraya KS, Uddin S (2011) Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma. Free Radic Biol Med 50:978–987. doi:10.1016/j.freeradbiomed.2010.12.034

  32. 32.

    Gurung RL, Lim SN, Khaw AK, Soon JF, Shenoy K, Mohamed Ali S, Jayapal M, Sethu S, Baskar R, Hande MP (2010) Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS One 5:e12124. doi:10.1371/journal.pone.0012124

  33. 33.

    Dergarabetian EM, Ghattass KI, El-Sitt SB, Al-Mismar RM, El-Baba CO, Itani WS, Melhem NM, El-Hajj HA, Bazarbachi AA, Schneider-Stock R, Gali-Muhtasib HU (2013) Thymoquinone induces apoptosis in malignant T-cells via generation of ROS. Front Biosci (Elite Ed) 5:706–719

  34. 34.

    Verstrepen L, Carpentier I, Verhelst K, Beyaert R (2009) ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling. Biochem Pharmacol 78:105–114. doi:10.1016/j.bcp.2009.02.009

  35. 35.

    Peng L, Liu A, Shen Y, Xu HZ, Yang SZ, Ying XZ, Liao W, Liu HX, Lin ZQ, Chen QY, Cheng SW, Shen WD (2013) Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-κB pathway. Oncol Rep 29:571–578. doi:10.3892/or.2012.2165

  36. 36.

    Spiller SE, Logsdon NJ, Deckard LA, Sontheimer H (2011) Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo. BMC Cancer 11:136. doi:10.1186/1471-2407-11-136

  37. 37.

    Sun S, Wang Q, Giang A, Cheng C, Soo C, Wang CY, Liau LM, Chiu R (2011) Knockdown of CypA inhibits interleukin-8 (IL-8) and IL-8-mediated proliferation and tumor growth of glioblastoma cells through down-regulated NF-κB. J Neurooncol 101:1–14. doi:10.1007/s11060-010-0220-y

  38. 38.

    Patel PS, Varney ML, Dave BJ, Singh RK (2002) Regulation of constitutive and induced NF-κB activation in malignant melanoma cells by capsaicin modulates interleukin-8 production and cell proliferation. J Interferon Cytokine Res 22:427–435. doi:10.1089/10799900252952217

  39. 39.

    Huang S, Pettaway CA, Uehara H, Bucana CD, Fidler IJ (2001) Blockade of NF-κB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 20:4188–4197. doi:10.1038/sj.onc.1204535

  40. 40.

    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. Molecular Cancer Res 6:1059–1070. doi:10.1158/1541-7786.MCR-07-2088

  41. 41.

    Singh RK, Lokeshwar BL (2009) Depletion of intrinsic expression of Interleukin-8 in prostate cancer cells causes cell cycle arrest, spontaneous apoptosis and increases the efficacy of chemotherapeutic drugs. Mol Cancer 8:57. doi:10.1186/1476-4598-8-57

  42. 42.

    Luppi F, Longo AM, de Boer WI, Rabe KF, Hiemstra PS (2007) Interleukin-8 stimulates cell proliferation in non-small cell lung cancer through epidermal growth factor receptor transactivation. Lung Cancer 56:25–33. doi:10.1016/j.lungcan.2006.11.014

  43. 43.

    Brew R, Erikson JS, West DC, Kinsella AR, Slavin J, Christmas SE (2000) Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine 12:78–85. doi:10.1006/cyto.1999.0518

  44. 44.

    Martinou JC, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 21:92–101. doi:10.1016/j.devcel.2011.06.017

  45. 45.

    El-Mahdy MA, Zhu Q, Wang QE, Wani G, Wani AA (2005) Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells. Int J Cancer 117:409–417. doi:10.1002/ijc.21205

  46. 46.

    Karin M, Lin A (2002) NF-κB at the crossroads of life and death. Nat Immunol 3:221–227. doi:10.1038/ni0302-221

  47. 47.

    Holcik M, Korneluk RG (2001) XIAP, the guardian angel. Nat Rev Mol Cell Biol 2:550–556. doi:10.1038/35080103

  48. 48.

    Keating J, Tsoli M, Hallahan AR, Ingram WJ, Haber M, Ziegler DS (2012) Targeting the inhibitor of apoptosis proteins as a novel therapeutic strategy in medulloblastoma. Mol Cancer Ther 11:2654–2663. doi:10.1158/1535-7163.MCT-12-0352

  49. 49.

    Pingoud-Meier C, Lang D, Janss AJ, Rorke LB, Phillips PC, Shalaby T, Grotzer MA (2003) Loss of caspase-8 protein expression correlates with unfavorable survival outcome in childhood medulloblastoma. Clin Cancer Res 9:6401–6409

  50. 50.

    Han DK, Chaudhary PM, Wright ME, Friedman C, Trask BJ, Riedel RT, Baskin DG, Schwartz SM, Hood L (1997) MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death. Proc Natl Acad Sci USA 94:11333–11338

  51. 51.

    Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Richardson PG, Hideshima T, Munshi N, Treon SP, Anderson KC (2002) Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications. Blood 99:4079–4086

  52. 52.

    Aguilera DG, Das CM, Sinnappah-Kang ND, Joyce C, Taylor PH, Wen S, Hasselblatt M, Paulus W, Fuller G, Wolff JE, Gopalakrishnan V (2009) Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL. J Neurooncol 93:303–318. doi:10.1007/s11060-008-9788-x

  53. 53.

    Zuzak TJ, Steinhoff DF, Sutton LN, Phillips PC, Eggert A, Grotzer MA (2002) Loss of caspase-8 mRNA expression is common in childhood primitive neuroectodermal brain tumour/medulloblastoma. Eur J Cancer 38:83–91

  54. 54.

    Grotzer MA, Eggert A, Zuzak TJ, Janss AJ, Marwaha S, Wiewrodt BR, Ikegaki N, Brodeur GM, Phillips PC (2000) Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase-8 expression. Oncogene 19:4604–4610. doi:10.1038/sj.onc.1203816

  55. 55.

    Ulasli SS, Celik S, Gunay E, Ozdemir M, Hazman O, Ozyurek A, Koyuncu T, Unlu M (2013) Anticancer effects of thymoquinone, caffeic acid phenethyl ester and resveratrol on A549 non-small cell lung cancer cells exposed to benzo(a)pyrene. Asian Pac J Cancer Prev 14:6159–6164

  56. 56.

    Woo CC, Loo SY, Gee V, Yap CW, Sethi G, Kumar AP, Tan KH (2011) Anticancer activity of thymoquinone in breast cancer cells: possible involvement of PPAR-gamma pathway. Biochem Pharmacol 82:464–475. doi:10.1016/j.bcp.2011.05.030

  57. 57.

    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 66:408–418. doi:10.1080/01635581.2013.878739

  58. 58.

    Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB (1993) bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597–608

  59. 59.

    Lindenboim L, Schlipf S, Kaufmann T, Borner C, Stein R (2004) Bcl-x(S) induces an NGF-inhibitable cytochrome c release. Exp Cell Res 297:392–403. doi:10.1016/j.yexcr.2004.03.001

  60. 60.

    Lindenboim L, Yuan J, Stein R (2000) Bcl-xS and Bax induce different apoptotic pathways in PC12 cells. Oncogene 19:1783–1793. doi:10.1038/sj.onc.1203495

  61. 61.

    Sumantran VN, Ealovega MW, Nunez G, Clarke MF, Wicha MS (1995) Overexpression of Bcl-XS sensitizes MCF-7 cells to chemotherapy-induced apoptosis. Cancer Res 55:2507–2510

  62. 62.

    Clarke MF, Apel IJ, Benedict MA, Eipers PG, Sumantran V, Gonzalez-Garcia M, Doedens M, Fukunaga N, Davidson B, Dick JE, Minn AJ, Boise LH, Thompson CB, Wicha M, Nunez G (1995) A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. Proc Natl Acad Sci USA 92:11024–11028

  63. 63.

    Schneider-Stock R, Fakhoury IH, Zaki AM, El-Baba CO, Gali-Muhtasib HU (2013) Thymoquinone: fifty years of success in the battle against cancer models. Drug Discov Today. doi:10.1016/j.drudis.2013.08.021

  64. 64.

    Rajput S, Kumar BN, Sarkar S, Das S, Azab B, Santhekadur PK, Das SK, Emdad L, Sarkar D, Fisher PB, Mandal M (2013) Targeted apoptotic effects of thymoquinone and tamoxifen on XIAP mediated Akt regulation in breast cancer. PLoS One 8:e61342. doi:10.1371/journal.pone.0061342

  65. 65.

    Tewari M, Quan LT, O’Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801–809

  66. 66.

    D’Amours D, Sallmann FR, Dixit VM, Poirier GG (2001) Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci 114:3771–3778

  67. 67.

    Zhang R, Humphreys I, Sahu RP, Shi Y, Srivastava SK (2008) In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway. Apoptosis 13:1465–1478. doi:10.1007/s10495-008-0278-6

  68. 68.

    Ohshima-Hosoyama S, Davare MA, Hosoyama T, Nelon LD, Keller C (2011) Bortezomib stabilizes NOXA and triggers ROS-associated apoptosis in medulloblastoma. J Neurooncol 105:475–483. doi:10.1007/s11060-011-0619-0

  69. 69.

    Aruoma OI, Halliwell B, Hoey BM, Butler J (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 6:593–597

  70. 70.

    Gilmore TD (1999) The Rel/NF-κB signal transduction pathway: introduction. Oncogene 18:6842–6844. doi:10.1038/sj.onc.1203237

  71. 71.

    Jin S, Tong T, Fan W, Fan F, Antinore MJ, Zhu X, Mazzacurati L, Li X, Petrik KL, Rajasekaran B, Wu M, Zhan Q (2002) GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21:8696–8704. doi:10.1038/sj.onc.1206034

  72. 72.

    Gali-Muhtasib HU, Abou Kheir WG, Kheir LA, Darwiche N, Crooks PA (2004) Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anticancer Drugs 15:389–399

  73. 73.

    Hussain AR, Uddin S, Ahmed M, Al-Dayel F, Bavi PP, Al-Kuraya KS (2013) Phosphorylated IkappaBalpha predicts poor prognosis in activated B-cell lymphoma and its inhibition with thymoquinone induces apoptosis via ROS release. PLoS One 8:e60540. doi:10.1371/journal.pone.0060540

  74. 74.

    Al-Majed AA, Al-Omar FA, Nagi MN (2006) Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus. Eur J Pharmacol 543:40–47. doi:10.1016/j.ejphar.2006.05.046

  75. 75.

    Hosseinzadeh H, Parvardeh S (2004) Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine 11:56–64. doi:10.1078/0944-7113-00376

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The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No RGP-VPP-272.

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Correspondence to Abdelkader E. Ashour.

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Ashour, A.E., Ahmed, A.F., Kumar, A. et al. Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-κB signaling and IL-8 expression. Mol Cell Biochem 416, 141–155 (2016). https://doi.org/10.1007/s11010-016-2703-4

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  • Medulloblastoma
  • Daoy cells
  • Thymoquinone
  • Apoptosis
  • NF-κB
  • Cancer