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Planta Med 2016; 82(04): 312-321
DOI: 10.1055/s-0035-1558289
Biological and Pharmacological Activity
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Thymoquinone from Nigella sativa Seeds Promotes the Antitumor Activity of Noncytotoxic Doses of Topotecan in Human Colorectal Cancer Cells in Vitro

Rana Khalife
1   Department of Natural Sciences, Lebanese American University of Beirut, Beirut, Lebanon
2   Faculty of Science, Beirut Arab University, Debbieh, Lebanon
,
Mohammad Hassan Hodroj
1   Department of Natural Sciences, Lebanese American University of Beirut, Beirut, Lebanon
,
Rajaa Fakhoury
2   Faculty of Science, Beirut Arab University, Debbieh, Lebanon
,
Sandra Rizk
1   Department of Natural Sciences, Lebanese American University of Beirut, Beirut, Lebanon
› Author Affiliations
Further Information

Publication History

received 16 March 2015
revised 01 October 2015

accepted 20 October 2015

Publication Date:
05 February 2016 (online)

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Abstract

Topotecan, a topoisomerase I inhibitor, is an anticancer drug widely used in the therapy of lung, ovarian, colorectal, and breast adenocarcinoma. Due to the primary dose-limiting toxicity of topotecan, which is myelosuppressive, it is necessary to identify other chemotherapeutic agents that can work synergistically with topotecan to increase its efficacy and limit its toxicity. Many studies have shown synergism upon the combination of topotecan with other chemotherapeutic agents such as gemcitabine. Other studies have demonstrated that pre-exposing cells to naturally occurring compounds such as thymoquinone, followed by gemcitabine or oxaliplatin, resulted in higher growth inhibition compared to treatment with gemcitabine or oxaliplatin alone. Our aim was to elucidate the underlying mechanism of action of topotecan in the survival and apoptotic pathways in human colon cancer cell lines in comparison to thymoquinone, to study the proapoptotic and antiproliferative effects of thymoquinone on the effectiveness of the chemotherapeutic agent topotecan, and to investigate the potential synergistic effect of thymoquinone with topotecan. Cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours in order to determine the IC50 for each drug. Combined therapy was then tested with ± 2 values for the IC50 of each drug. The reduction in proliferation was significantly dose- and time-dependent. After determining the best combination (40 µM thymoquinone and 0.6 µM topotecan), cell proteins were extracted after treatment, and the expression levels of B-cell lymphoma 2 and of its associated X protein, proteins p53 and p21, and caspase-9, caspase-3, and caspase-8 were studied by Western blot. In addition, cell cycle analysis and annexin/propidium iodide staining were performed. Both drugs induced apoptosis through a p53-independent mechanism, whereas the expression of p21 was only seen in thymoquinone treatment. Cell cycle arrest in the S phase was detected with each compound separately, while combined treatment only increased the production of fragmented DNA. Both compounds induced apoptosis through the extrinsic pathway after 24 hours; however, after 48 hours, the intrinsic pathway was activated by topotecan treatment only. In conclusion, thymoquinone increased the effectiveness of the chemotherapeutic reagent topotecan by inhibiting proliferation and lowering toxicity through p53- and Bax/Bcl2-independent mechanisms.

Key words

Nigella sativa - Ranunculaceae - topotecan - thymoquinone - colorectal cancer - apoptosis - caspases
 

  • References

  • 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69-90
    CrossrefPubMedGoogle Scholar
  • 2 Shamseddine A, Musallam K. Cancer epidemiology in Lebanon. Middle East J Cancer 2010; 1: 41-44
    PubMedGoogle Scholar
  • 3 Ulukan H, Swaan PW. Camptothecins: a review of their chemotherapeutic potential. Drugs 2002; 62: 2039-2057
    CrossrefPubMedGoogle Scholar
  • 4 Tsunetoh S, Teraj Y, Sasaki H, Tanabe A, Tanaka Y, Sekijima T, Fujiwara S, Kawaguchi H, Kanemura M, Yamashita Y, Ohmichi M. Topotecan as a molecular targeting agent which blocks the Akt and VEGF cascade in platinum-resistant ovarian cancers. Cancer Biol Ther 2010; 10: 1137-1146
    CrossrefPubMedGoogle Scholar
  • 5 Paik PK, Rudin CM, Brown A, Rizvi NA, Takebe N, Travis W, Naiyer A, Travis W, James L, Ginsberg MS, Juergens R, Markus S, Tyson L, Subzwari S, Kris MG, Krug LM. A phase I study of obatoclax mesylate, a Bcl-2 antagonist, plus topotecan in solid tumor malignancies. Cancer Chemother Pharmacol 2010; 66: 1079-1085
    CrossrefPubMedGoogle Scholar
  • 6 Hertzberg RP, Caranfa MJ, Hecht SM. On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. Biochemistry 1989; 28: 4629-4638
    CrossrefPubMedGoogle Scholar
  • 7 Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878
    PubMedGoogle Scholar
  • 8 Tsang PS, Cheuk AT, Chen QR, Song YK, Badgett TC, Wei JS, Khan J. Synthetic lethal screen identifies NF-κB as a Target for combination therapy with topotecan for patients with neuroblastoma. BMC Cancer 2012; 12: 101-111
    CrossrefPubMedGoogle Scholar
  • 9 Kollmannsberger C, Mross K, Jakob A, Kanz L, Bokemeyer C. Topotecan, a novel topoisomerase I inhibitor: pharmacology and clinical experience. Oncology 1999; 56: 1-12
    CrossrefPubMedGoogle Scholar
  • 10 Ten Bokkel Huinink W, Carmichael J, Armstrong D, Gordon A, Malfetano J. Efficacy and safety of topotecan in the treatment of advanced ovarian carcinoma. Semin Oncol 1997; 24: S5-19-S5-25
    PubMedGoogle Scholar
  • 11 Hackl C, Man S, Francia G, Milsom C, Xu P, Kerbel RS. Metronomic oral topotecan prolongs survival and reduces liver metastasis in improved preclinical orthotopic and adjuvant therapy colon cancer models. Gut 2013; 62: 259-271
    CrossrefPubMedGoogle Scholar
  • 12 Legarza K, Yang LX. Novel camptothecin derivatives. In Vivo 2005; 19: 283-292
    PubMedGoogle Scholar
  • 13 Chintagumpala MM, Friedman HS, Stewart CF, Kepner J, McLendon RE, Modrich PL, McCluggage C, Burger P, Holmes E, Thompson S. A phase II window trial of procarbazine and topotecan in children with high-grade glioma: a report from the childrenʼs oncology group. J Neurooncol 2006; 77: 193-198
    CrossrefPubMedGoogle Scholar
  • 14 Fassberg J, Stella VI. A kinetic and mechanistic study of the hydrolysis of camptothecin and some analogues. J Pharm Sci 1992; 81: 676-684
    CrossrefPubMedGoogle Scholar
  • 15 Hawkins DS, Bradfield S, Whitlock JA, Krailo M, Franklin J, Blaney SM, Adamson PC, Reaman G. Topotecan by 21-day continuous infusion in children with relapsed or refractory solid tumors: a childrenʼs oncology group study. Pediatr Blood Cancer 2006; 47: 790-794
    CrossrefPubMedGoogle Scholar
  • 16 Klautke G, Schutze M, Bombor I, Benecke R, Piek J, Fietkau R. Concurrent chemoradiotherapy and adjuvant chemotherapy with topotecan for patients with glioblastoma multiforme. J Neurooncol 2006; 77: 199-205
    CrossrefPubMedGoogle Scholar
  • 17 Adams DJ, Sandvold M, Myhren F, Jacobsen T, Giles F, Rizzieri DD. Anti proliferative activity of ELACYTTM (CP-4055) in combination with cloretazine (VNP40101 M), idarubicin, gemcitabine, irinotecan and topotecan in human leukemia and lymphoma cells. Leuk Lymphoma 2008; 49: 786-797
    CrossrefPubMedGoogle Scholar
  • 18 Holcombe RF, Kong KM, Wimmer D. Combined topoisomerase I inhibition for the treatment of metastatic colon cancer. Anticancer Drugs 2004; 15: 569-574
    CrossrefPubMedGoogle Scholar
  • 19 Padhye S, Banerjee S, Ahmad A, Mohammad R, Sarkar FH. From here to eternity – the secret of Pharaohs: Therapeutic potential of black cumin seeds and beyond. Cancer Ther 2008; 6: 495-510
    PubMedGoogle Scholar
  • 20 El-Dakhakhny M. Studies on the chemical constitution of the Egyptian Nigella sativa L. seeds. II) The essential oil. Planta Med 1963; 11: 465-470
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Zedlitz S, Kaufmann R, Boehncke WH. Allergic contact dermatitis from black cumin (Nigella sativa) oil-containing ointment. Contact Dermatitis 2002; 46: 188-189
    CrossrefPubMedGoogle Scholar
  • 22 Al-Hader A, Aqel A, Hassan Z. Hypogycemic effects of the volatile oil of Nigella sativa seeds. Int J Pharmacol 1993; 31: 96-100
    CrossrefPubMedGoogle Scholar
  • 23 Al-Ghamdi MS. The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa . J Ethnopharmacol 2001; 76: 45-48
    CrossrefPubMedGoogle Scholar
  • 24 Chakravorty N. Inhibition of histamine release from mast cells by nigellone. Ann Allergy 1993; 70: 237-242
    PubMedGoogle Scholar
  • 25 Khan MA, Ashfaq MK, Zuberi HS, Mahmood MS, Gilani AH. The in vivo antifungal activity of the aqueous extract from Nigella sativa seeds. Phytother Res 2003; 17: 183-186
    CrossrefPubMedGoogle Scholar
  • 26 Worthen DR, Ghosheh OA, Crooks PA. The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L. Anticancer Res 1998; 18: 1527-1532
    PubMedGoogle Scholar
  • 27 Zaoui A, Cherrah Y, Lacaille-Dubois MA, Settaf A, Amarouch H, Hassar M. Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Therapie 2000; 55: 379-382
    PubMedGoogle Scholar
  • 28 Shoieb AM, Elgayyar M, Dudrick PS, Bell JL, Tithof PK. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. Int J Oncol 2003; 22: 107-113
    PubMedGoogle Scholar
  • 29 Banerjee S, Kaseb AO, Wang Z, Kong D, Mohammad M, Padhye S, Sarkar FH, Mohammad RM. Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Res 2009; 69: 5575-5583
    CrossrefPubMedGoogle Scholar
  • 30 Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A, Schneider-Stock R. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p 53-dependent mechanism. Int J Oncol 2004; 25: 857-866
    PubMedGoogle Scholar
  • 31 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. Anticancer Drugs 2004; 15: 389-399
    CrossrefPubMedGoogle Scholar
  • 32 Jafri SH, Glass J, Shi R, Zhang O, Prince M, Hancock HK. Thymoquinone and cisplatin as a therapeutic combination in lung cancer: In vitro and in vivo . J Exp Clin Cancer Res 2010; 29: 87-98
    CrossrefPubMedGoogle Scholar
  • 33 Norsharina I, Maznah I, Aied A, Ghanya A. Thymoquinone rich fraction from Nigella sativa and thymoquinone are cytotoxic towards colon and leukemic carcinoma cell lines. J Med Plants Res 2011; 5: 3359-3366
    PubMedGoogle Scholar
  • 34 Roepke M, Diestel A, Bajbouj K, Walluscheck D, Schonfeld P, Roessner A, Schneider-Stock R, Gali-Muhtasib H. Lack of p 53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biol Ther 2007; 6: 160-169
    CrossrefPubMedGoogle Scholar
  • 35 Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 2006; 25: 4798-4811
    CrossrefPubMedGoogle Scholar
  • 36 Cain K, Bratton SB, Langlais C, Walker G, Brown DG, Sun XM, Cohen GM. Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes. J Biol Chem 2000; 275: 6067-6070
    CrossrefPubMedGoogle Scholar
  • 37 Wirries A, Breyer S, Quint K, Schobert R, Ocker M. Thymoquinone hydrazone derivatives cause cell cycle arrest in p 53-competent colorectal cancer cells. Exp Ther Med 2010; 1: 369-375
    PubMedGoogle Scholar
  • 38 Kaseb AO, Chinnakannu K, Chen D, Sivanandam A, Tejwani S, Menon M, Dou QP, Prem-Veer Reddy G. Androgen receptor and E2 F-1 targeted thymoquinone therapy for hormone-refractory prostate cancer. Cancer Res 2007; 67: 7782-7788
    CrossrefPubMedGoogle Scholar
  • 39 Petak I, Tillman DM, Harwood FG, Mihalik R, Houghton JA. Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells. Cancer Res 2000; 60: 2643-2650
    PubMedGoogle Scholar
  • 40 DʼArpa P, Beardmore C, Liu LF. Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res 1990; 50: 6919-6924
    PubMedGoogle Scholar
  • 41 Goldwasser F, Shimizu T, Jackman J, Hoki Y, OʼConnor PM, Kohn KW, Pommier Y. Correlations between S and G2 arrest and the cytotoxicity of camptothecin in human colon carcinoma cells. Cancer Res 1996; 56: 4430-4437
    PubMedGoogle Scholar
  • 42 Shao RG, Cao CX, Zhang H, Kohn KW, Wold MS, Pommier Y. Replication mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA: DNA-PK complexes. EMBO J 1990; 18: 1397-1406
    PubMedGoogle Scholar
  • 43 Morris EJ, Geller HM. Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase- I: evidence for cell cycle-independent toxicity. J Cell Biol 1996; 134: 757-770
    CrossrefPubMedGoogle Scholar
  • 44 Nakashio A, Fujita N, Rokudai S, Sato S, Tsuruo T. Signaling pathway during topotecan-induced apoptosis prevention of phosphatidylinositol 3′-kinase-Akt survival. Cancer Res 2000; 60: 5303-5309
    PubMedGoogle Scholar
  • 45 Tomicic MT, Christmann M, Kaina B. Topotecan-triggered degradation of topoisomerase I is p 53-dependent and impacts cell survival. Cancer Res 2005; 65: 8920-8926
    CrossrefPubMedGoogle Scholar
  • 46 Miyashita T, Reed JC. Tumor suppressor p 53 is a direct transcriptional activator of the human Bax gene. Cell 1995; 80: 293-299
    CrossrefPubMedGoogle Scholar
  • 47 Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, Krammer PH. P53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med 1998; 188: 2033-2045
    CrossrefPubMedGoogle Scholar
  • 48 Lackinger D, Eichhorn U, Kaina B. Effect of ultraviolet light, methyl methanesulfonate and ionizing radiation on the genotoxic response and apoptosis of mouse fibroblasts lacking c-Fos, p 53 or both. Mutagenesis 2001; 16: 233-241
    CrossrefPubMedGoogle Scholar
  • 49 Lackinger D, Kaina B. Primary mouse fibroblasts deficient for c-Fos, p 53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis. Mutat Res 2000; 457: 113-123
    CrossrefPubMedGoogle Scholar
  • 50 Smith ML, Ford JM, Hollander MC, Bortnick RA, Amundson SA, Seo YR, Deng C, Hanawalt PC, Fornace jr. AJ. P53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p 53, p 21, and/or gadd45 genes. Mol Cell Biol 2000; 20: 3705-3714
    CrossrefPubMedGoogle Scholar
  • 51 Abbas T, Dutta A. P21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 2009; 9: 400-414
    CrossrefPubMedGoogle Scholar
  • 52 Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res 2000; 60: 7133-7141
    PubMedGoogle Scholar
  • 53 Devy J, Wargnier R, Pluot M, Nabiev I, Sukhanova A. Topotecan-induced alterations in the amount and stability of human DNA topoisomerase I in solid tumor cell lines. Anticancer Res 2004; 24: 1745-1752
    PubMedGoogle Scholar