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Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression, elevated levels of TRAIL receptors, oxidative stress and apoptosis

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Abstract

Hepatocellular carcinoma (HCC) is the fourth most common solid tumor worldwide. The chemokine interleukin-8 (IL-8) is overexpressed in HCC and is a potential target for therapy. Although the transcription factor NF-κB regulates IL-8 expression, and while thymoquinone (TQ; the most bioactive constituent of black seed oil) inhibits NF-κB activity, the precise mechanisms by which TQ regulates IL-8 and cancer cell growth remain to be clarified. Here, we report that TQ inhibited growth of HCC cells in a dose- and time-dependent manner, caused G2M cell cycle arrest, and stimulated apoptosis. Apoptosis was substantiated by activation of caspase-3 and -9, as well as cleavage of poly(ADP-ribose)polymerase. TQ treatments inhibited expression of NF-κB and suppressed IL-8 and its receptors. TQ treatments caused increased levels of reactive oxygen species (ROS) and mRNAs of oxidative stress-related genes, NQO1 and HO-1. Pretreatment of HepG2 cells with N-acetylcysteine, a scavenger of ROS, prevented TQ-induced cell death. TQ treatment stimulated mRNA expression of pro-apoptotic Bcl-xS and TRAIL death receptors, and inhibited expression of the anti-apoptotic gene Bcl-2. TQ enhanced TRAIL-induced death of HepG2 cells, in part by up-regulating TRAIL death receptors, inhibiting NF-κB and IL-8 and stimulating apoptosis. Altogether, these findings provide insights into the pleiotropic molecular mechanisms of TQ-dependent suppression of HCC cell growth and underscore potential of this compound as anti-HCC drug.

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References

  1. Badary OA, Gamal El-Din AM (2001) Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis. Cancer Detect Prev 25(4):362–368

    CAS  PubMed  Google Scholar 

  2. Badary OA, Nagi MN, al-Shabanah OA, al-Sawaf HA, al-Sohaibani MO, al-Bekairi AM (1997) Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can J Physiol Pharmacol 75(12):1356–1361

    Article  CAS  PubMed  Google Scholar 

  3. Banerjee S, Padhye S, Azmi A, Wang Z, Philip PA, Kucuk O, Sarkar FH, Mohammad RM (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62(7):938–946. doi:10.1080/01635581.2010.509832

    Article  CAS  PubMed  Google Scholar 

  4. Banerjee S, Padhye S, Azmi A, Wang Z, Philip PA, Kucuk O, Sarkar FH, Mohammad RM (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62(7):938–946. doi:10.1080/01635581.2010.509832

    Article  CAS  PubMed  Google Scholar 

  5. 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(5):435–440

    Article  CAS  PubMed  Google Scholar 

  6. Hoofnagle JH (2004) Hepatocellular carcinoma: summary and recommendations. Gastroenterology 127(5 Suppl 1):S319–S323

    Article  PubMed  Google Scholar 

  7. McGlynn KA, London WT (2005) Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol 19(1):3–23. doi:10.1016/j.bpg.2004.10.004

    Article  PubMed  Google Scholar 

  8. Abdo AA, Karim HA, Al Fuhaid T, Sanai FM, Kabbani M, Al Jumah A, Burak K (2006) Saudi Gastroenterology Association guidelines for the diagnosis and management of hepatocellular carcinoma: summary of recommendations. Ann Saudi Med 26(4):261–265

    PubMed  Google Scholar 

  9. Tannapfel A, Wittekind C (2002) Genes involved in hepatocellular carcinoma: deregulation in cell cycling and apoptosis. Virchows Arch 440(4):345–352. doi:10.1007/s00428-002-0617-x

    Article  CAS  PubMed  Google Scholar 

  10. Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM (1992) Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 258(5089):1798–1801

    Article  CAS  PubMed  Google Scholar 

  11. Ren Y, Poon RT, Tsui HT, Chen WH, Li Z, Lau C, Yu WC, Fan ST (2003) Interleukin-8 serum levels in patients with hepatocellular carcinoma: correlations with clinicopathological features and prognosis. Clin Cancer Res 9(16 Pt 1):5996–6001

    CAS  PubMed  Google Scholar 

  12. Akiba J, Yano H, Ogasawara S, Higaki K, Kojiro M (2001) Expression and function of interleukin-8 in human hepatocellular carcinoma. Int J Oncol 18(2):257–264

    CAS  PubMed  Google Scholar 

  13. Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76(24):13001–13014

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Korashy HM, El-Kadi AO (2004) Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)-regulated genes in cultured hepatoma Hepa 1c1c7 cells. Toxicology 201(1–3):153–172. doi:10.1016/j.tox.2004.04.011

    Article  CAS  PubMed  Google Scholar 

  15. Bueno-da-Silva AE, Brumatti G, Russo FO, Green DR, Amarante-Mendes GP (2003) Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity. Cell Death Differ 10(5):592–598. doi:10.1038/sj.cdd.4401210

    Article  CAS  PubMed  Google Scholar 

  16. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139(2):271–279

    Google Scholar 

  17. van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP (1996) A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24(2):131–139. doi:10.1002/(SICI)1097-0320(19960601)24:2<131:AID-CYTO5>3.0.CO;2-M

    Article  PubMed  Google Scholar 

  18. Clarke RG, Lund EK, Johnson IT, Pinder AC (2000) Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content. Cytometry 39(2):141–150. doi:10.1002/(SICI)1097-0320(20000201)39:2<141:AID-CYTO7>3.0.CO;2-O

    Article  CAS  PubMed  Google Scholar 

  19. Nagi MN, Alam K, Badary OA, al-Shabanah OA, al-Sawaf HA, al-Bekairi AM (1999) Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism. Biochem Mol Biol Int 47(1):153–159

    CAS  PubMed  Google Scholar 

  20. 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 MCR 6(6):1059–1070. doi:10.1158/1541-7786.MCR-07-2088

    Article  CAS  Google Scholar 

  21. Aruoma OI, Halliwell B, Hoey BM, Butler J (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 6(6):593–597

    Article  CAS  PubMed  Google Scholar 

  22. Murray AW (1992) Creative blocks: cell-cycle checkpoints and feedback controls. Nature 359(6396):599–604. doi:10.1038/359599a0

    Article  CAS  PubMed  Google Scholar 

  23. 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(3):409–417. doi:10.1002/ijc.21205

    Article  CAS  PubMed  Google Scholar 

  24. Gurung RL, Lim SN, Khaw AK, Soon JF, Shenoy K, Mohamed Ali S, Jayapal M, Sethu S, Baskar R, Hande MP (2010) Thymoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. PLoS ONE 5(8):e12124. doi:10.1371/journal.pone.0012124

    Article  PubMed Central  PubMed  Google Scholar 

  25. Gilmore TD (1999) The Rel/NF-kappaB signal transduction pathway: introduction. Oncogene 18(49):6842–6844. doi:10.1038/sj.onc.1203237

    Article  CAS  PubMed  Google Scholar 

  26. Campbell LM, Maxwell PJ, Waugh DJ (2013) Rationale and means to target pro-inflammatory interleukin-8 (CXCL8) signaling in cancer. Pharmaceuticals (Basel) 6(8):929–959. doi:10.3390/ph6080929

    Article  Google Scholar 

  27. Kim YS, Schwabe RF, Qian T, Lemasters JJ, Brenner DA (2002) TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells. Hepatology 36(6):1498–1508. doi:10.1053/jhep.2002.36942

    CAS  PubMed  Google Scholar 

  28. Verstrepen L, Carpentier I, Verhelst K, Beyaert R (2009) ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling. Biochem Pharmacol 78(2):105–114. doi:10.1016/j.bcp.2009.02.009

    Article  CAS  PubMed  Google Scholar 

  29. Schneider-Stock R, Fakhoury IH, Zaki AM, El-Baba CO, Gali-Muhtasib HU (2013) Thymoquinone: fifty years of success in the battle against cancer models. Drug Discov Today. doi:10.1016/j.drudis.2013.08.021

    PubMed  Google Scholar 

  30. Abdollahi T, Robertson NM, Abdollahi A, Litwack G (2003) Identification of interleukin 8 as an inhibitor of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in the ovarian carcinoma cell line OVCAR3. Cancer Res 63(15):4521–4526

    CAS  PubMed  Google Scholar 

  31. Hussain AR, Ahmed M, Ahmed S, Manogaran P, Platanias LC, Alvi SN, Al-Kuraya KS, Uddin S (2011) Thymoquinone suppresses growth and induces apoptosis via generation of reactive oxygen species in primary effusion lymphoma. Free Radical Biol Med 50(8):978–987. doi:10.1016/j.freeradbiomed.2010.12.034

    Article  CAS  Google Scholar 

  32. Choi C, Kutsch O, Park J, Zhou T, Seol DW, Benveniste EN (2002) Tumor necrosis factor-related apoptosis-inducing ligand induces caspase-dependent interleukin-8 expression and apoptosis in human astroglioma cells. Mol Cell Biol 22(3):724–736

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. 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 a p53-dependent mechanism. Int J Oncol 25(4):857–866

    CAS  PubMed  Google Scholar 

  34. Li Q, Zhao LY, Zheng Z, Yang H, Santiago A, Liao D (2011) Inhibition of p53 by adenovirus type 12 E1B-55K deregulates cell cycle control and sensitizes tumor cells to genotoxic agents. J Virol. doi:10.1128/JVI.00492-11

    Google Scholar 

  35. Vikhanskaya F, Lee MK, Mazzoletti M, Broggini M, Sabapathy K (2007) Cancer-derived p53 mutants suppress p53-target gene expression–potential mechanism for gain of function of mutant p53. Nucleic Acids Res 35(6):2093–2104. doi:10.1093/nar/gkm099

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

  37. Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74(4):609–619

    Article  CAS  PubMed  Google Scholar 

  38. LaCasse EC, Baird S, Korneluk RG, MacKenzie AE (1998) The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17(25):3247–3259. doi:10.1038/sj.onc.1202569

    Article  PubMed  Google Scholar 

  39. Zhang R, Humphreys I, Sahu RP, Shi Y, Srivastava SK (2008) In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway. Apoptosis 13(12):1465–1478. doi:10.1007/s10495-008-0278-6

    Article  CAS  PubMed  Google Scholar 

  40. Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM (2002) Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem Funct 20(2):143–151. doi:10.1002/cbf.968

    Article  CAS  PubMed  Google Scholar 

  41. Bianchet MA, Faig M, Amzel LM (2004) Structure and mechanism of NAD[P]H: quinone acceptor oxidoreductases (NQO). Methods Enzymol 382:144–174. doi:10.1016/S0076-6879(04)82009-3

    Article  CAS  PubMed  Google Scholar 

  42. Mayer B, Oberbauer R (2003) Mitochondrial regulation of apoptosis. News Physiol Sci 18:89–94

    CAS  PubMed  Google Scholar 

  43. Lee MW, Park SC, Kim JH, Kim IK, Han KS, Kim KY, Lee WB, Jung YK, Kim SS (2002) The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa cells. Cancer Lett 182(1):75–82

    Article  CAS  PubMed  Google Scholar 

  44. Nioi P, Hayes JD (2004) Contribution of NAD(P)H: quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors. Mutat Res 555(1–2):149–171. doi:10.1016/j.mrfmmm.2004.05.023

    Article  CAS  PubMed  Google Scholar 

  45. Talalay P, Dinkova-Kostova AT (2004) Role of nicotinamide quinone oxidoreductase 1 (NQO1) in protection against toxicity of electrophiles and reactive oxygen intermediates. Methods Enzymol 382:355–364. doi:10.1016/S0076-6879(04)82019-6

    Article  CAS  PubMed  Google Scholar 

  46. Kohle C, Badary OA, Nill K, Bock-Hennig BS, Bock KW (2005) Serotonin glucuronidation by Ah receptor- and oxidative stress-inducible human UDP-glucuronosyltransferase (UGT) 1A6 in Caco-2 cells. Biochem Pharmacol 69(9):1397–1402. doi:10.1016/j.bcp.2005.02.010

    Article  PubMed  Google Scholar 

  47. Riley RJ, Workman P (1992) DT-diaphorase and cancer chemotherapy. Biochem Pharmacol 43(8):1657–1669

    Article  CAS  PubMed  Google Scholar 

  48. Wilkening S, Stahl F, Bader A (2003) Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties. Drug Metab Dispos 31(8):1035–1042. doi:10.1124/dmd.31.8.1035

    Article  CAS  PubMed  Google Scholar 

  49. De Haan LH, Boerboom AM, Rietjens IM, van Capelle D, De Ruijter AJ, Jaiswal AK, Aarts JM (2002) A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells. Biochem Pharmacol 64(11):1597–1603

    Article  PubMed  Google Scholar 

  50. Applegate LA, Luscher P, Tyrrell RM (1991) Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 51(3):974–978

    CAS  PubMed  Google Scholar 

  51. Gong P, Cederbaum AI, Nieto N (2003) Increased expression of cytochrome P450 2E1 induces heme oxygenase-1 through ERK MAPK pathway. J Biol Chem 278(32):29693–29700. doi:10.1074/jbc.M304728200

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by a grant from King Abdulaziz city for science and technology (KACST; Grant No.: ARP-29-265) and the Department of Veterans Affairs Merit Review grant (AKR).

Conflict of interest

We have no personal or financial conflict of interest and have not entered into any agreement that could interfere with our access to the data on the research, or upon our ability to analyze the data independently, to prepare manuscripts, and to publish them.

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  1. Shazia Jamal

    Present address: Crescent School of Life Science, BS Abdur Rahman University, Vandalur, Chennai, 600048, India

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia

    Abdelkader E. Ashour, Adel R. Abd-Allah, Hesham M. Korashy, Sabry M. Attia, Abdelrahman Z. Alzahrani & Saleh A. Bakheet

  2. Department of Pharmacology and Toxicology, College of Pharmacy, Al-Azhar University, Cairo, Egypt

    Adel R. Abd-Allah & Sabry M. Attia

  3. College of Science, King Saud University, Riyadh, Saudi Arabia

    Quaiser Saquib

  4. Department of Pathology, College of Medicine for Girls, Al-Azhar University, Cairo, Egypt

    Hala E. Abdel-Hamied

  5. Department of Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

    Shazia Jamal & Arun K. Rishi

  6. John D. Dingell Veterans Affairs Medical Center, Detroit, MI, USA

    Arun K. Rishi

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  1. Abdelkader E. Ashour
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Correspondence to Abdelkader E. Ashour.

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Ashour, A.E., Abd-Allah, A.R., Korashy, H.M. 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 389, 85–98 (2014). https://doi.org/10.1007/s11010-013-1930-1

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