Skip to main content

The combination of thymoquinone and paclitaxel shows anti-tumor activity through the interplay with apoptosis network in triple-negative breast cancer

Tumor Biology volume 37, pages 4467–4477 (2016)Cite this article

Abstract

Thymoquinone (TQ) is the active ingredient of Nigella sativa which has a therapeutic potential in cancer therapy and prevention. In this study, TQ has been shown to induce specific cytotoxicity and apoptosis and to inhibit wound healing in triple-negative breast cancer cell line. TQ also inhibited cancer growth in a mouse tumor model. Moreover, TQ and paclitaxel (Pac) combination inhibited cancer growth in cell culture and in mice. Genes involved in TQ and TQ-Pac-mediated cytotoxicity were studied using focused real-time PCR arrays. After bioinformatic analysis, genes in apoptosis, cytokine, and p53 signaling categories were found to be modulated with a high significance in TQ-treated cells (p < 10−28, p < 10−8, and p < 10−6, respectively). Important to note, TQ has been found to regulate the genes involved in the induction of apoptosis through death receptors (p = 5.5 × 10−5). Additionally, tumor suppressor genes such as p21, Brca1, and Hic1 were highly upregulated by TQ and TQ-Pac combination. Interestingly, when cells were treated with high dose TQ, several growth factors such as Vegf and Egf were upregulated and several pro-apoptotic factors such as caspases were downregulated possibly pointing out key pathways manipulated by cancer cells to resist against TQ. In cells treated with the combination of TQ and Pac, genes in apoptosis cascade (p < 10−12), p53 signaling (p = 10−5), and JAK-STAT signaling (p < 10−3) were differentially expressed. TQ has also been shown to induce protein levels of cleaved Caspase-3, Caspase-7, and Caspase-12 and PARP and to reduce phosphorylated p65 and Akt1. The in vivo therapeutic potential of TQ-Pac combination and the genetic network involved in this synergy have been shown for the first time to the best of our knowledge.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. El-Dakhakhany M. Studies on the chemical constitution of Egyptian N. sativa L. seeds. Planta Med. 1963;11:465–70.

    Article  Google Scholar 

  2. Rahmani AH, Alzohairy MA, Khan MA, Aly SM. Therapeutic implications of black seed and its constituent thymoquinone in the prevention of cancer through inactivation and activation of molecular pathways. Evid Based Complement Alternat Med. 2014. doi:10.1155/2014/724658.

    PubMed  PubMed Central  Google Scholar 

  3. El-Mahdy MA, Zhu QZ, Wang QE, Wani G, Wani AA. Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells. Int J Cancer. 2005;117(3):409–17. doi:10.1002/Ijc.21205.

    CAS  Article  PubMed  Google Scholar 

  4. Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A, et al. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. Int J Oncol. 2004;25(4):857–66.

    CAS  PubMed  Google Scholar 

  5. el Arafa SA, Zhu Q, Shah ZI, Wani G, Barakat BM, Racoma I, et al. Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Mutat Res. 2011;706(1-2):28–35. doi:10.1016/j.mrfmmm.2010.10.007.

    CAS  Article  Google Scholar 

  6. Banerjee S, Kaseb AO, Wang ZW, Kong DJ, Mohammad M, Padhye S, et al. Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Res. 2009;69(13):5575–83. doi:10.1158/0008-5472.Can-08-4235.

    CAS  Article  PubMed  Google Scholar 

  7. Sethi G, Ahn KS, Aggarwal BB. Targeting nuclear factor-kappa B activation pathway by thymoquinone: role in suppression of antiapoptotic gene products and enhancement of apoptosis. Mol Cancer Res. 2008;6(6):1059–70. doi:10.1158/1541-7786.MCR-07-2088.

    CAS  Article  PubMed  Google Scholar 

  8. Gali-Muhtasib HU, Abou Kheir WG, Kheir LA, Darwiche N, Crooks PA. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anti-Cancer Drugs. 2004;15(4):389–99. doi:10.1097/01.cad.0000125054.43188.56.

    CAS  Article  PubMed  Google Scholar 

  9. Guicciardi ME, Gores GJ. Life and death by death receptors. Faseb J. 2009;23(6):1625–37. doi:10.1096/Fj.08.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Mohamed A, Afridi DM, Garani O, Tucci M. Thymoquinone inhibits the activation of NF-kappaB in the brain and spinal cord of experimental autoimmune encephalomyelitis. Biomed Sci Instrum. 2005;41:388–93.

    CAS  PubMed  Google Scholar 

  11. Connelly L, Barham W, Onishko HM, Sherrill T, Chodosh LA, Blackwell TS, et al. Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden. Oncogene. 2011;30(12):1402–12. doi:10.1038/onc.2010.521.

    CAS  Article  PubMed  Google Scholar 

  12. Tekeoglu I, Dogan A, Ediz L, Budancamanak M, Demirel A. Effects of thymoquinone (volatile oil of black cumin) on rheumatoid arthritis in rat models. Phytother Res. 2007;21(9):895–7. doi:10.1002/ptr.2143.

    CAS  Article  PubMed  Google Scholar 

  13. Chehl N, Chipitsyna G, Gong Q, Yeo CJ, Arafat HA. Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB (Oxford). 2009;11(5):373–81. doi:10.1111/j.1477-2574.2009.00059.x.

    Article  Google Scholar 

  14. Ravindran J, Nair HB, Sung B, Prasad S, Tekmal RR, Aggarwal BB. Thymoquinone poly(lactide-co-glycolide) nanoparticles exhibit enhanced anti-proliferative, anti-inflammatory, and chemosensitization potential. Biochem Pharmacol. 2010;79(11):1640–7. doi:10.1016/j.bcp.2010.01.023.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Sakalar C, Yuruk M, Kaya T, Aytekin M, Kuk S, Canatan H. Pronounced transcriptional regulation of apoptotic and TNF-NF-kappa-B signaling genes during the course of thymoquinone mediated apoptosis in HeLa cells. Mol Cell Biochem. 2013;383(1-2):243–51. doi:10.1007/s11010-013-1772-x.

    CAS  Article  PubMed  Google Scholar 

  16. Rajput S, Kumar BNP, Dey KK, Pal I, Parekh A, Mandal M. 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. 2013;93(21):783–90. doi:10.1016/j.lfs.2013.09.009.

    CAS  Article  PubMed  Google Scholar 

  17. Sutton KM, Greenshields AL, Hoskin DW, 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 Int J. 2014;66(3):408–18. doi:10.1080/01635581.2013.878739.

    CAS  Article  Google Scholar 

  18. Woo CC, Hsu A, Kumar AP, Sethi G, Tan KHB. Thymoquinone inhibits tumor growth and induces apoptosis in a breast cancer xenograft mouse model: the role of p38 MAPK and ROS. PLoS One. 2013;8(10):e75356. doi:10.1371/journal.pone.0075356.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Attoub S, Sperandio O, Raza H, Arafat K, Al-Salam S, Al Sultan MA, et al. Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro and tumor growth in vivo. Fund Clin Pharmacol. 2013;27(5):557–69. doi:10.1111/j.1472-8206.2012.01056.x.

    CAS  Article  Google Scholar 

  20. Velho-Pereira R, Kumar A, Pandey BN, Jagtap AG, Mishra KP. Radiosensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis. Cell Biol Int. 2011;35(10):1025–9. doi:10.1042/Cbi20100701.

    Article  PubMed  Google Scholar 

  21. Effenberger-Neidnicht K, Schobert R. Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer Chemother Pharmacol. 2011;67(4):867–74. doi:10.1007/s00280-010-1386-x.

    CAS  Article  PubMed  Google Scholar 

  22. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc. 1971;93(9):2325–7.

    CAS  Article  PubMed  Google Scholar 

  23. Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature. 1979;277(5698):665–7.

    CAS  Article  PubMed  Google Scholar 

  24. Erdemoğlu N, Bilge S. The antitumor effects of the taxane class compounds. Ankara Ecz Fak Derg. 2000;29(1):77–90.

    Google Scholar 

  25. Dirican A, Atmaca H, Bozkurt E, Erten C, Karaca B, Uslu R. Novel combination of docetaxel and thymoquinone induces synergistic cytotoxicity and apoptosis in DU-145 human prostate cancer cells by modulating PI3K-AKT pathway. Clin Transl Oncol. 2015;17(2):145–51. doi:10.1007/s12094-014-1206-6.

    CAS  Article  PubMed  Google Scholar 

  26. Lettre R, Paweletz N, Werner D, Granzow C. Sublines of the Ehrlich-Lettre mouse ascites tumour. A new tool for experimental cell research. Naturwissenschaften. 1972;59(2):59–63.

    CAS  Article  PubMed  Google Scholar 

  27. Dexter DL, Kowalski HM, Blazar BA, Fligiel Z, Vogel R, Heppner GH. Heterogeneity of tumor cells from a single mouse mammary tumor. Cancer Res. 1978;38(10):3174–81.

    CAS  PubMed  Google Scholar 

  28. Heppner GH, Miller FR, Shekhar PM. Nontransgenic models of breast cancer. Breast Cancer Res. 2000;2(5):331–4.

    CAS  Article  PubMed  Google Scholar 

  29. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57. doi:10.1038/nprot.2008.211.

    CAS  Article  Google Scholar 

  30. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1–13. doi:10.1093/Nar/Gkn923.

    Article  Google Scholar 

  31. Bai T, Yang Y, Wu YL, Jiang S, Lee JJ, Lian LH, et al. Thymoquinone alleviates thioacetamide-induced hepatic fibrosis and inflammation by activating LKB1-AMPK signaling pathway in mice. Int Immunopharmacol. 2014;19(2):351–7. doi:10.1016/j.intimp.2014.02.006.

    CAS  Article  PubMed  Google Scholar 

  32. Ghazwani M, Zhang YF, Gao X, Fan J, Li J, Li S. Anti-fibrotic effect of thymoquinone on hepatic stellate cells. Phytomedicine. 2014;21(3):254–60. doi:10.1016/j.phymed.2013.09.014.

    CAS  Article  PubMed  Google Scholar 

  33. Ashour AE, Abd-Allah AR, Korashy HM, Attia SM, Alzahrani AZ, Saquib Q, et al. 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. 2014;389(1-2):85–98. doi:10.1007/s11010-013-1930-1.

    CAS  Article  PubMed  Google Scholar 

  34. Badr G, Mohany M, Abu-Tarboush F. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-(XL) expression. Lipids Health Dis. 2011. doi:10.1186/1476-511x-10-236.

    Google Scholar 

  35. Badr G, Lefevre EA, Mohany M. Thymoquinone inhibits the CXCL12-induced chemotaxis of multiple myeloma cells and increases their susceptibility to Fas-mediated apoptosis. PLoS One. 2011;6(9):e23741. doi:10.1371/journal.pone.0023741.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Li F, Rajendran P, Sethi G. Thymoquinone. Br J Pharmacol. 2010;161(3):541–54. doi:10.1111/j.1476-5381.2010.00874.x.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Rajput S, Kumar BNP, Banik P, Parida S, Mandal M. Thymoquinone restores radiation-induced TGF-beta expression and abrogates EMT in chemoradiotherapy of breast cancer cells. J Cell Physiol. 2015;230(3):620–9. doi:10.1002/Jcp.24780.

    CAS  Article  PubMed  Google Scholar 

  38. Kundu J, Choi BY, Jeong CH, Kundu JK, Chun KS. Thymoquinone induces apoptosis in human colon cancer HCT116 cells through inactivation of STAT3 by blocking JAK2- and Src-mediated phosphorylation of EGF receptor tyrosine kinase. Oncol Rep. 2014;32(2):821–8. doi:10.3892/Or.2014.3223.

    PubMed  Google Scholar 

  39. Chou J, Provot S, Werb Z. GATA3 in development and cancer differentiation: cells GATA have it! J Cell Physiol. 2010;222(1):42–9. doi:10.1002/Jcp.21943.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Radpour R, Kohler C, Haghighi MM, Fan AXC, Holzgreve W, Zhong XY. Methylation profiles of 22 candidate genes in breast cancer using high-throughput MALDI-TOF mass array. Oncogene. 2009;28(33):2969–78. doi:10.1038/Onc.2009.149.

    CAS  Article  PubMed  Google Scholar 

  41. Fujii H, Biel MA, Zhou W, Weitzman SA, Baylin SB, Gabrielson E. Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer. Oncogene. 1998;16(16):2159–64. doi:10.1038/sj.onc.1201976.

    CAS  Article  PubMed  Google Scholar 

  42. Yusuf RZ, Duan Z, Lamendola DE, Penson RT, Seiden MV. Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation. Curr Cancer Drug Targets. 2003;3(1):1–19.

    CAS  Article  PubMed  Google Scholar 

  43. Ajabnoor GMA, Crook T, Coley HM. Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells. Cell Death Dis. 2012;3:e260. doi:10.1038/cddis.2011.139.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by The Scientific and Technological Research Council of Turkey (TUBİTAK), Grant Number: 113S322 (C. SAKALAR) and Erciyes University Scientific Research Fund (EU-BAP), Grant Number: TOA-2014-4877 (C. SAKALAR). Authors would like to thank to Prof. Dr. Nedime Serakinci, Near East University, Cyprus and Prof. Dr. Feridoun Karimi-Busheri University of Alberta, Canada for critically reading the manuscript.

Author information

Affiliations

  1. Department of Medical Biology, School of Medicine, Erciyes University, 38039, Melikgazi, Kayseri, Turkey

    Çağrı Şakalar, Banu İskender, Sedat Sezen, Huriye Aksu, Mustafa Çakır, Büşra Kurt, Ali Turan & Halit Canatan

  2. Genome and Stem Cell Center, Erciyes University, Melikgazi, Kayseri, Turkey

    Çağrı Şakalar, Kenan İzgi, Banu İskender, Sedat Sezen, Huriye Aksu, Mustafa Çakır, Büşra Kurt, Ali Turan & Halit Canatan

  3. Department of Medical Biochemistry, School of Medicine, Erciyes University, Melikgazi, Kayseri, Turkey

    Kenan İzgi

Authors
  1. Çağrı Şakalar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenan İzgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Banu İskender
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sedat Sezen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huriye Aksu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mustafa Çakır
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Büşra Kurt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ali Turan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Halit Canatan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Çağrı Şakalar.

Ethics declarations

Conflicts of interest

None

Ethics approval

Ethical approval for the study was obtained from Erciyes University Animal Researche Local Ethics Committee and the ethic regulations have been followed in accordance with the international, national, and institutional guidelines.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 2
figure 6

(JPEG 693 kb)

Full size image
ESM 3
figure 7

(JPEG 1805 kb)

Full size image

ESM 1

(DOC 34 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Şakalar, Ç., İzgi, K., İskender, B. et al. The combination of thymoquinone and paclitaxel shows anti-tumor activity through the interplay with apoptosis network in triple-negative breast cancer. Tumor Biol. 37, 4467–4477 (2016). https://doi.org/10.1007/s13277-015-4307-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-015-4307-0

Keywords

  • Thymoquinone
  • Paclitaxel
  • Breast cancer
  • In vivo
  • Synergy
  • Gene expression profile
  • Intrinsic and extrinsic apoptosis axis
  • p53 signaling