Asian Pacific Journal of Cancer Prevention

Asian Pacific Journal of Cancer Prevention (아시아태평양암예방학회)
권호 표지
DOI QR코드

DOI QR Code

In Vitro Anti-Neuroblastoma Activity of Thymoquinone Against Neuro-2a Cells via Cell-cycle Arrest

  • Paramasivam, Arumugam (Department of Genetics, Dr.ALM Post Graduate Institute of Basic Medical Sciences, Sekkizhar Campus, University of Madras) ;
  • Raghunandhakumar, Subramanian (Department of Biochemistry, Guindy Campus, University of Madras) ;
  • Priyadharsini, Jayaseelan Vijayashree (Department of Genetics, Dr.ALM Post Graduate Institute of Basic Medical Sciences, Sekkizhar Campus, University of Madras) ;
  • Jayaraman, Gopalswamy (Department of Genetics, Dr.ALM Post Graduate Institute of Basic Medical Sciences, Sekkizhar Campus, University of Madras)
  • Published : 2016.01.11
Download PDF
⟨ Previous Next ⟩

Abstract

We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose-dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

Keywords

References

  1. Aggarwal BB, Kunnumakkara AB, Harikumar KB, et al (2008). Potential of spice-derived phytochemicals for cancer prevention. Planta Med, 74, 1560-69. https://doi.org/10.1055/s-2008-1074578
  2. Alhosin M, Abusnina A, Achour M, et al (2010). Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1. Biochem Pharmacol, 79, 1251-60. https://doi.org/10.1016/j.bcp.2009.12.015
  3. Ali BH, Blunden G (2003). Pharmacological and toxicological properties of Nigella sativa. Phytother Res, 17, 299-305. https://doi.org/10.1002/ptr.1309
  4. Ando T, Kawabe T, Ohara H (2001). Involvement of the interaction between p21 and proliferation cell nuclear antigen for the maintenance of G2/M arrest after DNA damage. J Biol Chem, 276, 42971-77. https://doi.org/10.1074/jbc.M106460200
  5. Badary OA, Abd-Ellah MF, El-Mahdy MA, et al (2007). Anticlastogenic activity of thymoquinone against benzo(a) pyrene in mice. Food Chem Toxicol, 45, 88-92. https://doi.org/10.1016/j.fct.2006.08.004
  6. Badary OA, Al-Shabanah OA, Nagi MN, et al (1999). Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by TQ. Eur J Cancer Prev, 8, 435-440. https://doi.org/10.1097/00008469-199910000-00009
  7. Badary OA, Gamal El-Din A (2001). Inhibitory effects of TQ against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis. Cancer Detect Prev, 25, 362-8.
  8. Begleiter A, Leith MK (1990). Activity of quinone alkylating agents in quinone-resistant cells. Cancer Res, 50, 2872-6.
  9. Berthold F, Hero B (2000). Neuroblastoma-current drug therapy recommendations as a part of the total treatment approach. Drugs, 59, 1261-1277. https://doi.org/10.2165/00003495-200059060-00006
  10. Brodeur GM (2003). Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer, 3, 203-16. https://doi.org/10.1038/nrc1014
  11. Brown JW, Prieto LM, Perez-Stable C (2008). Estrogen and progesterone lower cyclin B1 AND D1 expression, block cell cycle in G2/M, and trigger apoptosis in human adrenal carcinoma cell cultures. Horm Metab Res, 40, 306-10. https://doi.org/10.1055/s-2008-1073140
  12. Dusre L, Covey JM, Collins C, et al (1989). DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells. Chem Biol Interact, 71, 63-78. https://doi.org/10.1016/0009-2797(89)90090-2
  13. Effenberger K, Breyer S, Schobert R (2010). Terpene conjugates of the Nigella sativa seed-oil constituent thymoquinone with enhanced efficacy in cancer cells. Chem Biodivers, 7, 129-39. https://doi.org/10.1002/cbdv.200900328
  14. Effenberger-Neidnicht K, Schobert R (2011). Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer Chemother Pharmacol, 67, 867-874. https://doi.org/10.1007/s00280-010-1386-x
  15. El-Mahdy MA, Zhu Q, Wang QE, et al (2005). Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells. Int J Cancer, 117, 409-17. https://doi.org/10.1002/ijc.21205
  16. El-Shaimaa AA, Qianzheng Z, Zubair IS, et al (2011). Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Muta Res, 706, 28-35. https://doi.org/10.1016/j.mrfmmm.2010.10.007
  17. Gali-Muhtasib H, Diab-Assaf M, Boltze C, et al (2004). Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. Int J Oncol, 25, 857-66.
  18. Gali-Muhtasib H, Kuester D, Mawrin C, et al (2008). Thymoquinone triggers inactivation of the stress response pathway sensor CHEK1 and contributes to apoptosis in colorectal cancer cells. Cancer Res, 68, 5609-18. https://doi.org/10.1158/0008-5472.CAN-08-0884
  19. Gali-Muhtasib H, Roessner A, Schneider-Stock R (2006). Thymoquinone: a promising anti-cancer drug from natural sources. Int J Biochem Cell Biol, 38, 1249-53. https://doi.org/10.1016/j.biocel.2005.10.009
  20. Gali-Muhtasib HU, Bou Kheir WG, Kheir LA, et al (2004). Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anticancer Drugs, 15, 389-99. https://doi.org/10.1097/00001813-200404000-00012
  21. Gehen SC, Vitiello PF, Bambara RA (2007). Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia. Am J Physiol Lung Cell Mol Physiol, 292, 716-24. https://doi.org/10.1152/ajplung.00135.2006
  22. Goubin F, Ducommun B (1995). Identification of binding domains on the p21cip1 Cyclin-dependent kinase inhibitor. Oncogene, 10, 2281-7.
  23. Gurung RL, Lim SN, Khaw AK, et al (2010). Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS ONE, 12124.
  24. Harper JW, Elledge SJ, Keyomarsi K, et al (1995). Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell, 6, 387-400. https://doi.org/10.1091/mbc.6.4.387
  25. Hartwell LH, Kastan MB (1994). Cell cycle control and cancer. Science, 266, 1821-8. https://doi.org/10.1126/science.7997877
  26. Hindges R, Hubscher U (1997). DNA Polymerase ${\delta}$, an essential enzyme for DNA transactions. Biol Chem, 378, 345-62.
  27. Hosseinzadeh H, Parvardeh S (2004). Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine, 11, 56-64. https://doi.org/10.1078/0944-7113-00376
  28. Ivankovic S, Stojkovic R, Jukic M, et al (2006). The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo. Exp Oncol, 28, 220-4.
  29. Kakino S, Sasaki K, Kurose A (1996). Intracellular localization of Cyclin B1 during the cell cycle in glioma cells. Cytometry, 24, 49-54. https://doi.org/10.1002/(SICI)1097-0320(19960501)24:1<49::AID-CYTO6>3.0.CO;2-D
  30. Karp JE, Broder S (1995). Molecular foundations of cancer: new targets for intervention. Nat Med, 1, 309-20. https://doi.org/10.1038/nm0495-309
  31. Kaseb AO, Chinnakannu K, Chen D, et al (2007). Androgen receptor and E2F-1-targeted thymoquinone therapy for hormone refractory prostate cancer. Cancer Res, 67, 7782-8. https://doi.org/10.1158/0008-5472.CAN-07-1483
  32. Kim JH (2007). Homoisoflavanone inhibits retinal neovascularization through cell cycle arrest with decrease of cdc2 expression. Biochem Biophys Res Commun, 362, 848-52. https://doi.org/10.1016/j.bbrc.2007.08.100
  33. Kontopidis G, Wu SY, Zheleva DI (2005). Structural and biochemical studies of human proliferating cell nuclear antigen complexes provide a rationale for cyclin association and inhibitor design. Proc Natl Acad Sci USA, 102, 1871-6. https://doi.org/10.1073/pnas.0406540102
  34. Li R, Hannon GJ, Beach D, Stillman B (1996). Subcellular distribution of p21 and PCNA in normal and repair deficient cells following DNA damage. Curr Biol, 6, 189-99. https://doi.org/10.1016/S0960-9822(02)00452-9
  35. Li R, Waga Hannon GJ, Beach D, et al (1994). Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature, 371, 534-7 https://doi.org/10.1038/371534a0
  36. Lo FH, Mak NK, Leung KN (2007). Studies on the antitumor activities of the soy isoflavone daidzein on murine neuroblastoma cells. Biomed Pharmaco Ther, 61, 591-595. https://doi.org/10.1016/j.biopha.2007.08.021
  37. Lowry OH, Rosebrough NJ, Farr AL, et al (1951). Protein measurement with the Folin-phenol reagent. J Biol Chem, 193, 265-75.
  38. Maris JM, Matthay KK (1999). Molecular biology of neuroblastoma. J Clin Oncol, 17, 2264-79. https://doi.org/10.1200/JCO.1999.17.7.2264
  39. Morgan WA, Hartley JA (1992). Cohen GM. Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells. Biochem Pharmacol, 44, 215-21. https://doi.org/10.1016/0006-2952(92)90003-2
  40. Norwood AA, Tan M, May M, et al (2006). Comparison of potential chemotherapeutic agents, 5-fluoruracil, green tea, and thymoquinone on colon cancer cells. Biomed Sci Instrum, 42, 350-6.
  41. Obaya AJ, Sedivy JM (2002). Regulation of cyclin-Cdk activity in mammalian cells. Cell Mol Life Sci, 59, 126-142. https://doi.org/10.1007/s00018-002-8410-1
  42. Padhye S, Banerjee S, Ahmad A, et al (2008). From here to eternity the secret of pharaohs: therapeutic potential of black cumin seeds and beyond. Cancer Ther, 6, 495-510.
  43. Paramasivam A, Kalaimangai M, Sambantham S, et al (2012a). Anti-angiogenic activity of thymoquinone by the downregulation of VEGF using zebrafish (Danio rerio) model. Biomed Prev Nutr, 2, 169-73. https://doi.org/10.1016/j.bionut.2012.03.011
  44. Paramasivam A, Sambantham S, Shabnam J, et al (2012b). Anticancer effects of thymoquinone in mouse neuroblastoma (Neuro-2a) cells through caspase-3 activation with downregulation of XIAP. Toxicol Lett, 213, 151-9. https://doi.org/10.1016/j.toxlet.2012.06.011
  45. Prasad S, Kaur J, Roy P (2007). Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells. Life Sci, 81, 1323-31. https://doi.org/10.1016/j.lfs.2007.07.033
  46. Prosperi E (1997). Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog Cell Cycle Res, 3, 193-210.
  47. Qiu X, Forman HJ, Schonthal AH, et al (1996). Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinyl benzoquinones by HCT116 cells. J Biol Chem, 271, 31915-21. https://doi.org/10.1074/jbc.271.50.31915
  48. Ravindran J, Nair HB, Sung B, et al (2010). Thymoquinone poly (lactide-co-glycolide) nanoparticles exhibit enhanced antiproliferative, anti-inflammatory, and chemosensitization potential. Biochem Pharmacol, 79, 1640-7. https://doi.org/10.1016/j.bcp.2010.01.023
  49. Roepke M, Diestel A, Bajbouj K, et al (2007). Lack of p53 augments thymoquinoneinduced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biol Ther, 6, 160-9. https://doi.org/10.4161/cbt.6.2.3575
  50. 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, 1059-70. https://doi.org/10.1158/1541-7786.MCR-07-2088
  51. Shoieb AM, Elgayyar M, Dudrick PS, et al (2003). Tithof PK. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. Internat J Oncol, 22, 107-113.
  52. Singh RP, Dhanalakshmi S, Agarwal R (2002). Phytochemicals as cell cycle modulators. Cell Cycle, 1, 156-61.
  53. Szepesi A, Gelfand EW, Lucas JJ (1994). Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes. Blood, 84, 3413-21.
  54. Trang NT, Wanner MJ, Phuong L, et al (1993). Thymoquinone from Eupatorium ayapana. Planta Med, 59, 99. https://doi.org/10.1055/s-2006-959619
  55. Velho-Pereira R, Amit Kumar K, Pandey BN, et al (2011). Radio sensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis. Cell Biol Int, 35, 1025-9. https://doi.org/10.1042/CBI20100701
  56. Vogelstein B, Lane D, Levine AJ (2000). Surfing the p53 network. Nature, 408, 307-10. https://doi.org/10.1038/35042675
  57. Vuorelaa P, Leinonenb M, Saikkuc P, et al (2004). Natural products in the process of finding new drug candidates. Curr Med Chem, 11, 1375-89. https://doi.org/10.2174/0929867043365116
  58. Wilson-Simpson F, Vance S, Benghuzzi H (2007). Physiological responses of ES-2 ovarian cell line following administration of epigallocatechin-3-gallate (EGCG), thymoquinone (TQ) and selenium (SE). Biomed Sci Instrum, 43, 378-383.
  59. Worthen DR, Ghosheh OA, Crooks PA (1998). The in vitro anti-tumor activity of some crude and purified components of blackseed. Nigella sativa L. Anticancer Res, 18, 1527-32.
  60. Wu RC, Hohenstein A, Park JM, et al (1998). Role of p53 in aziridinylbenzoquinone induced p21waf1 expression. Oncogene, 17, 357-65. https://doi.org/10.1038/sj.onc.1201930
  61. Xiong Y, Zhang H, Beach D (1992). D type cyclin associated with multiple protein kinases and the DNA replication and repair factor PCNA. Cell, 71, 505-14. https://doi.org/10.1016/0092-8674(92)90518-H
  62. Yi T, Cho SG, Yi Z, et al (2008). Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Ther, 7, 1789-96. https://doi.org/10.1158/1535-7163.MCT-08-0124
  63. Zhang H, Hannon GJ, Beach D (1994). p21-containing cyclin kinases exist in both active and inactive states. Genes Dev, 8, 1750-8. https://doi.org/10.1101/gad.8.15.1750
  64. Zhang H, Xiong Y, Beach D (1993). Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol Biol Cell, 4, 897-906. https://doi.org/10.1091/mbc.4.9.897

Cited by

  1. vol.57, pp.18, 2017, https://doi.org/10.1080/10408398.2016.1277971