Cancer Chemotherapy and Pharmacology

, Volume 67, Issue 4, pp 867–874 | Cite as

Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin

  • Katharina Effenberger-Neidnicht
  • Rainer SchobertEmail author
Original Article



Doxorubicin is a mainstay of cancer chemotherapy despite its clinical limitations that arise from its cardiotoxicity and the high incidence of multi-drug resistance. Recent studies revealed a protective effect of thymoquinone, a non-toxic constituent of the essential oil of Nigella sativa, against doxorubicin-induced cardiotoxicity. We now investigated the influence of thymoquinone on various other effects exerted by doxorubicin in human cancer cells.


Doxorubicin, thymoquinone and equimolar mixtures of both were tested for cytotoxicity on human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, KB-V1 cervix, and MCF-7 breast carcinomas as well as multi-drug-resistant variants thereof and on non-malignant human fibroblasts (HF). Apoptosis induction was analysed via DNA fragmentation, activity studies of the caspases-3, -8 and -9, determination of changes in the mitochondrial membrane potential and in the ratio of the mRNA expressions of pro- and anti-apoptotic proteins bax and bcl-2. The generation of reactive oxygen species (ROS) was assessed by the NBT assay.


Thymoquinone improved the anti-cancer properties of doxorubicin in a cell line-specific manner. We found a significant rise of the growth inhibition by doxorubicin in HL-60 and multi-drug-resistant MCF-7/TOPO cells when thymoquinone had been added. The mode of action of both drugs and of their mixture was mainly apoptotic. In HL-60 cells, the drug mixture caused an additional concentration maximum of effector caspase-3 not observed for either of the pure drugs. The impact of the drug mixture on the mitochondria of HL-60 cells was also greater than those of the individual quinones alone. In addition, the drug mixture led to a higher concentration of reactive oxygen species in HL-60 cells.


In summary, thymoquinone is a booster for the anti-cancer effect of doxorubicin in certain cancer cell lines. Distinct improvements on efficacy, selectivity, and even breaches of multi-drug resistance were observed for equimolar mixtures of doxorubicin and thymoquinone.

Graphical abstract


Anti-tumour agents Apoptosis Conjugates Doxorubicin Multi-drug resistance Thymoquinone 



We thank the Deutsche Forschungsgemeinschaft for financial support (grant Scho 402/8-2), Ribosepharm GmbH, Gräfelfing (Germany) for a free batch of doxorubicin, and Prof. M. Ocker (Marburg) for providing the luminometric caspase kit and the facilities for measuring the bax and bcl-2 mRNA expression.


  1. 1.
    Cragg GM, Grothaus PG, Newman DJ (2009) Impact of natural products on developing new anti-cancer agents. Chem Rev 109:3012–3043PubMedCrossRefGoogle Scholar
  2. 2.
    Colucci MA, Moody CJ, Couch GD (2008) Natural and synthetic quinones and their reduction by the quinone reductase enzyme NQO1: from synthetic organic chemistry to compounds with anticancer potential. Org Biomol Chem 6:637–656PubMedCrossRefGoogle Scholar
  3. 3.
    Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ (2000) Role of quinones in toxicology. Chem Res Toxicol 13:135–160PubMedCrossRefGoogle Scholar
  4. 4.
    Robert J (1998) Anthracyclines. In: Grochow LB, Ames MM (eds) A clinician′s guide to chemotherapy. pharmacokinetic and pharmacodynamics, Williams & Wilkins, Baltimore, pp 93–173Google Scholar
  5. 5.
    Badary OA, Gamal El-Din AM (2001) Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis. Cancer Detect Prev 25:362–368PubMedGoogle Scholar
  6. 6.
    Gali-Muhtasib H, Roessner A, Schneider-Stock R (2006) Thymoquinone: A promising anti-cancer drug from natural sources. Int J Biochem Cell Biol 38:1249–1253PubMedCrossRefGoogle Scholar
  7. 7.
    Gali-Muhtasib H, Ocker M, Kuester D, Krueger S, El-Hajj Z, Diestel A, Evert M, El-Najjar N, Peters B, Jurjus A, Roessner A, Schneider-Stock R (2008) Thymoquinone reduces mouse colon tumour cell invasion and inhibits tumour growth in murine colon cancer models. J Cell Mol Med 12:330–342PubMedCrossRefGoogle Scholar
  8. 8.
    Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A, Schneider-Stock R (2004) Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via p53-dependent mechanism. Int J Oncol 25:857–866PubMedGoogle Scholar
  9. 9.
    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 117:409–417PubMedCrossRefGoogle Scholar
  10. 10.
    Breyer S, Effenberger K, Schobert R (2009) Effects of thymoquinone-fatty acid conjugates on cancer cells. Chem Med Chem 4:761–768PubMedGoogle Scholar
  11. 11.
    Effenberger K, Breyer S, Schobert R (2010) Terpene conjugates of the nigella sativa seed oil constituent thymoquinone with enhanced efficacy in cancer cells. Chem Biodiv 7:129–139CrossRefGoogle Scholar
  12. 12.
    Effenberger K, Breyer S, Schobert R (2010) Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones. Eur J Med Chem 45:1947–1954PubMedCrossRefGoogle Scholar
  13. 13.
    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 17:193–198PubMedGoogle Scholar
  14. 14.
    Nagi MN, Mansour MA (2000) Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection. Pharmacol Res 41:283–289PubMedCrossRefGoogle Scholar
  15. 15.
    Badary OA, Al-Shabanah OA, Nagi MN, Al-Rikabi AC, Elmazar MM (1999) Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone. Eur J Cancer Prev 8:435–440PubMedCrossRefGoogle Scholar
  16. 16.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63CrossRefGoogle Scholar
  17. 17.
    Earnshaw WC, Martins LM, Kaufmann SC (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383–424PubMedCrossRefGoogle Scholar
  18. 18.
    Bas A, Forsberg G, Hammarström S, Hammarström ML (2004) Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T-lymphocytes. Scand J Immunol 59:560–573CrossRefGoogle Scholar
  19. 19.
    Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144:891–901PubMedCrossRefGoogle Scholar
  20. 20.
    Rook GAW, Steele J, Umar S, Dockrell HM (1985) A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for the activation of human macrophages by gamma interferon. J Immunol Methods 82:161–167PubMedCrossRefGoogle Scholar
  21. 21.
    Chou TC, Talalay P (1981) Generalized equations for the analysis of inhibitors of Michaelis-Menten and higher order kinetic systems with two or more mutually exclusive and non-exclusive inhibitors. Eur J Biochem 115:207–216PubMedCrossRefGoogle Scholar
  22. 22.
    Chou TC, Talalay P (1984) Quantitative analysis of dose effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzymol Regul 22:27–55CrossRefGoogle Scholar
  23. 23.
    Wang S, Konorev EA, Kotamraju S, Joseph J, Kalivendi S, Kalyanarama B (2004) Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. J Biol Chem 279:25535–25543PubMedCrossRefGoogle Scholar
  24. 24.
    Mizutani H, Tada-Oikawa S, Hiraku Y, Kojima M, Kawanishi S (2005) Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide. Life Sci 76:1439–1453PubMedCrossRefGoogle Scholar
  25. 25.
    Gewirtz DA (1999) A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol 55:727–741CrossRefGoogle Scholar
  26. 26.
    Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in anti-tumour activity and cardiotoxicity. Pharmacol Rev 56:185–229PubMedCrossRefGoogle Scholar
  27. 27.
    El-Najjar N, Chatila M, Moukadem H, Vuorela H, Ocker M, Gandesiri M, Schneider-Stock R, Gala-Muhtasib H (2010) Reactive oxygen species mediated thymoquinone-induced apoptosis and activate ERK and JNK signalling. Apoptosis 15:183–195PubMedCrossRefGoogle Scholar
  28. 28.
    Badary OA, Taha RA, El-Din AMG, Abdel-Wahab MH (2003) Thymoquinone is a potent superoxide anion scavenger. Drug Chem Toxicol 26:87–98PubMedCrossRefGoogle Scholar
  29. 29.
    Benimetskaya L, Lai JC, Khvorova A, Wu S, Hua E, Miller P, Zhang LM, Stein CA (2004) Relative bcl-2 independence of drug-induced cytotoxicity and resistance in 518A2 melanoma cells. Clin Cancer Res 10:8371–8379PubMedCrossRefGoogle Scholar
  30. 30.
    Budihardjo I, Oliver H, Lutter M, Luo X, Wang X (1999) Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15:269–290PubMedCrossRefGoogle Scholar
  31. 31.
    Bossy-Wetzel E, Newmeyer DD, Green DR (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independent of mitochondrial transmembrane depolarization. EMBO J 17:37–49PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Katharina Effenberger-Neidnicht
    • 1
  • Rainer Schobert
    • 1
    Email author
  1. 1.Organisch-chemisches Laboratorium der Universität BayreuthBayreuthGermany

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