Thymoquinone synergistically potentiates temozolomide cytotoxicity through the inhibition of autophagy in U87MG cell line

Document Type : Original Article


1 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

2 Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran


Objective(s): Glioblastoma multiforme (GBM) is one of the most lethal forms of human cancer and temozolomide (TMZ) is currently part of the standard treatment for this disease. Combination therapy using natural substances can enhance the anti-cancer activity of TMZ. The purpose of this study was to evaluate the effect of TMZ in combination with thymoquinone (TQ) on human GBM cell line (U87MG).
Materials and Methods: The cell line was treated with TMZ and/or TQ. Cell viability was assessed using trypan blue and MTT assay. The effect of TMZ and/or TQ on colony-forming ability of the cells was investigated. Apoptosis and autophagy were quantified by fluorescent dye staining. The expression level of two autophagy related genes (ATG) were assessed using RT-PCR. Furthermore, nitric oxide (NO) production was detected by Griess reaction.
Results: After treatment with TMZ and/or TQ, the cell viability was reduced in a time- and dose-dependent manner, and the cell survival fraction (SF) was significantly decreased (P=0.000). Apoptosis index of U87MG cells was also significantly increased (P=0.000). Autophagy was significantly increased by TMZ (P=0.000) and decreased by TQ (P=0.018). Also TMZ and/or TQ significantly decreased NO production by U87MG cell (P=0.000).
Conclusion: TQ enhanced the anti-cancer activity of TMZ by inhibition of autophagy at the transcriptional level and decreased the colony-forming ability and NO production of U87MG cell line.


1. Grossman SA, Batara JF. Current management of glioblastoma multiforme. Semin Oncol 2004; 31: 635-644.
2. Central Brain Tumor Registry of the United States (CTBRUS) Report. 2010.
3. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJB, et al Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. New Engl J Med 2005; 352: 987-996.
4. Marchesi F, Turriziani M, Tortorelli G, Avvisati G, Torino F, De Vecchis L. Triazene compounds: mechanism of action and related DNA repair systems. Pharmacol Res 2007; 56: 275-287.
5. FDA Approval for Temozolomide. 2010. National Cancer Institute treatment/drugs/fda-temozolomide.
6. Serwer LP, James CD. Challenges in drug delivery to tumors of the central nervous system: an overview of pharmacological and surgical considerations. Adv Drug Deliv Rev 2012; 64: 590-597.
7. Wheelhouse RT, Stevens MFG. Decomposition of the antitumour drug temozolomide in deuteriated phosphate buffer: methyl group transfer is accompanied by deuterium exchange. J Chem Soc Chem Commun 1993; 1993: 1177-1178.
8. Jhaveri N, Cho H, Torres S, Wang W, Schonthal AH, Petasis NA, et al Noscapine inhibits tumor growth in TMZ-resistant gliomas. Cancer Lett 2011; 312: 245-252.
9. Gali-Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: A promising anticancer drug from natural sources. Int J Biochem Cell Biol 2006; 38: 1249-1253.
10. Banerjee S, Padhye S, Azmi A, Wang Z, Philip PA, Kucuk O, et al Review on molecular and therapeutic potential of thymoquinone in cancer. Nut Cancer 2010; 62: 938-946.
11. Worthen DR, Ghosheh OA, Crooks PA. The in vitro antitumor activity of some crude and purified components of blackseed, Nigella sativa L. Anticancer Res 1998; 18: 1527-1532.
12. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et al Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000; 343: 1350-1354.
13. Lena A, Rechichi M, Salvetti A, Bartoli B, Vecchio D, Scarcelli V, et al Drugs targeting the mitochondrial pore act as cytotoxic and cytostatic agents in temozolomide-resistant glioma cells. J Transl Med 2009; 5: 7-13.
14. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M,            et al Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 2013 Oct 10;4:e838.
15. Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 2011; 18: 571-580.
16. Weidberg H, Shvets E, Elazar Z. Biogenesis and cargo selectivity of autophagosomes. Annu Rev Biochem 2011; 80: 125-156.
17. Miller MR, Megson IL. Recent developments in nitric oxide donor drugs. Br J Pharmacol 2007; 151: 305-321.
18. Fukumura D, Kashiwagi S, Jain RK. The role of nitric oxide in tumour progression. Nat Rev Cancer 2006; 6: 521-534.
19. Ladas EJ, Jacobson JS, Kennedy DD, Teel K, Fleischauer A, Kelly KM. Antioxidants and cancer therapy: a systematic review. J Clin Oncol 2004; 22: 517-528.
20. Racoma IO, Meisen WH, Wang QE, Kaur B, Wani AA. Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells. PLoS One 2013; 8: 8e72882.
21. Renata MŻ, Maria HB, Anna F, Sylwia KN, Diana S, Halina C. Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line. BMC Complement Altern Med 2013; 13: 50.
22. Valentina C, Luana Q, Alessia DB, Laura B, Massimo B, Giulio L, et al Effects of thymoquinone on isolated and cellular proteasomes. FEBS Journal 2010; 277: 2128-2141.
23. Rezakhani L, Rashidi Z, Mirzapur P, Khazaei M. Antiproliferatory effects of crab shell extract on breast cancer cell line (MCF7). J Breast Cancer 2014; 17: 219-225.
24. Mirzapur P, Rashidi Z, Rezakhani L, Khazaei M. In vitro inhibitory effect of crab shell extract on human umbilical vein endothelial cell. In Vitro Cell Dev Biol Anim 2015; 51: 36-41.
25. Chou TC. Drug combination studies and their synergy quantificationusing the Chou‑Talalay method. Cancer Res 2010;70:440-446.
26. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc 2006; 1: 2315-2319.
27. Hale AN, Ledbetter DJ, Gawriluk TR, Rucker EB. 3rd. 2013. Autophagy: regulation and role in development. Autophagy. 9, 951–972.
28. Bądziul D, Jakubowicz-Gil J, Paduch R, Głowniak K, Gawron A. Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep-2 cells. Mol Cell Biochem 2014; 392: 213-227.
29. Sun J, Zhang X, Broderick M, Fein H. Measurement of nitric oxide production in biological systems by using Griess reaction assay. Sensors 2003; 3: 276-284.
30. Khazaei M, Roshankhah S, Ghorbani R, Chobsaz F. Sildenafil effect on nitric oxide secretion by normal human endometrial epithelial cells cultured In vitro. IJFS 2011; 5: 142-147.
31. Portnow J, Badie B, Chen M, Liu A, Blanchard S, Synold TW. The neuropharmacokinetics of temozolomide in patients with resectable brain tumors: potential implications for the current approach to chemoradiation. Clin Cancer Res 2009; 15: 7092-7098.
32. Rosso L, Brock CS, Gallo JM, Saleem A, Price PM, Turkheimer FE, et al A new model for prediction of drug distribution in tumor and normal tissues: pharmacokinetics of temozolomide in glioma patients. Cancer Res 2009; 69: 120-127.
33. Krex D, Klink B, Hartmann C, von Deimling A, Pietsch T, Simon M, et al Longterm survival with glioblastoma multiforme. Brain 2007; 130: 2596-2606.
34. Gurung RL, Lim SN, Khaw AK, Soon JF, Shenoy K, Mohamed Ali S, et al Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS One 2010; 5: e12124.
35. Kolli-Bouhafs K, Boukhari A, Abusnina A, Velot E, Gies JP, Lugnier C, et al Thymoquinone reduces migration and invasion of human glioblastoma cells associated with FAK, MMP-2 and MMP-9 down-regulation. Invest New Drugs 2012; 30: 2121-2131.
36. Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005; 5: 726-734.
37. Wang Z, Han W, Sui X, Fang Y, Pan H. Autophagy: A novel therapeutic target for hepatocarcinoma (Review). Oncol Lett 2014; 7: 1345-1351.
38. Shen W, Hu JA, Zheng JS. Mechanism of temozolomide-induced antitumour effects on glioma  cells. J Int Med Res 2014; 42: 164-172.
39. Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M. et al Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One 2013; 8: e55665.
40. Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun 2001; 282: 1075-1079.
41. Badn W, Siesjö P. The dual role of nitric oxide in glioma. Curr Pharm Des 2010; 16: 428-430.