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Thymoquinone inhibits proliferation and invasion of human nonsmall-cell lung cancer cells via ERK pathway

Abstract

Thymoquinone (TQ) is the primary bioactive component of Nigella sativa Linn seed oil and used as anti-inflammatory, anti-oxidant, and anti-neoplastic agent. Previous studies have shown that TQ exhibits inhibitory effects on multiple cancers. However, the detailed antineoplastic effects and its molecular mechanisms of TQ on lung cancer are not entirely elucidated yet. In the present study, we aimed to investigate the effects of TQ on cell proliferation, migration, and invasion as well as its underlying anti-metastatic mechanisms in A549 cells. Lung cancer cell line A549 cells were treated with different concentration of TQ for different period of time, and the growth-inhibitory effects of TQ was measured by MTT and cell count assays; cell cycle was determined by flow cytometry; wound healing and transwell assays were used to assess the cell migration and invasion activities; Western blot and real-time quantitative RT-PCR were used to determine the expression of proliferation and invasion associated genes as well as MAPKs pathway molecules; gelatinase activity was estimated using gelatin zymography assay. The results show that TQ played a role in inhibiting the proliferation, migration, and invasion of A549 lung cancer cells, it also inhibited the expression level of PCNA, cyclin D1, MMP2, and MMP9 mRNA and protein in a dose- and time-dependent manner especially at 10, 20, 40 μmol/L concentrations. The cell cycle inhibitor P16 expression and the gelatinase activities of MMP2 and MMP9 were also inhibited by TQ dramatically. TQ reduced phosphorylation of ERK1/2; however, the proliferation and invasion inhibitory effects of TQ on A549 cells were neutralized by ERK1/2 inhibitor PD98059. In conclusion, our study confirmed that TQ could inhibit A549 cell proliferation, migration, and invasion through ERK1/2 pathway, as proposed the therapeutic potential of TQ as an anti-metastatic agent in human lung cancer treatment.

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Abbreviations

TQ:

Thymoquinone

siRNA:

Small interfering RNA

NSCLC:

Nonsmall-cell lung cancer

SCLC:

Small cell lung cancer

TIMP:

Tissue inhibitor of metalloproteinase

MMP:

Matrix metalloproteinase

ECM:

Extracellular matrix

FBS:

Fetal bovine serum

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

References

  1. 1.

    Smith RA, Cokkinides V, Brawley OW. Cancer screening in the United States, 2009: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin. 2009;59:27–41.

  2. 2.

    Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, et al. Cancer statistics, 2005. CA Cancer J Clin. 2005;55:10–30.

  3. 3.

    Chan DC, Earle KA, Zhao TL, Helfrich B, Zeng C, Baron A, et al. Exisulind in combination with docetaxel inhibits growth and metastasis of human lung cancer and prolongs survival in athymic nude rats with orthotopic lung tumors. Clin Cancer Res. 2002;8:904–12.

  4. 4.

    Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.

  5. 5.

    Woessner Jr JF. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;5:2145–54.

  6. 6.

    McCawley LJ, Matrisian LM. Matrix metalloproteinases: they’re not just for matrix anymore! Curr Opin Cell Biol. 2001;13(5):534–40.

  7. 7.

    Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev. 2007;87:1285–342.

  8. 8.

    Zhu L, Kate P, Torchilin VP. Matrix metalloprotease 2-responsive multifunctional liposomal nanocarrier for enhanced tumor targeting. ACS Nano. 2012;6:3491–8.

  9. 9.

    Olson ES, Jiang T, Aguilera TA, Nguyen QT, Ellies LG, Scadeng M, et al. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci U S A. 2010;107:4311–6.

  10. 10.

    Kajanne R, Miettinen P, Mehlem A, Leivonen SK, Birrer M, Foschi M, et al. EGF-R regulates MMP function in fibroblasts through MAPK and AP-1 pathways. J Cell Physiol. 2007;212(2):489–97.

  11. 11.

    Westermarck J, Kähäri VM. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 1999;13:781–92.

  12. 12.

    Abi Saab WF, Brown MS, Chadee DN. MLK4β functions as a negative regulator of MAPK signaling and cell invasion. Oncogenesis. 2012;1:e6.

  13. 13.

    Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov. 2005;4:988–1004.

  14. 14.

    Hawsawi ZA, Ali BA, Bamosa AO. Effect of Nigella sativa (black seed) and thymoquinone on blood glucose in albino rats. Ann Saudi Med. 2001;21:242–4.

  15. 15.

    Trang NT, Wanner MJ, Phuong le VN, Koomen GJ, Dung NX. Thymoquinone from Eupatorium ayapana. Planta Med. 1993;59:99.

  16. 16.

    Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine. 2004;11:56–64.

  17. 17.

    Woo CC, Kumar AP, Sethi G, Tan KH. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharmacol. 2012;83:443–51.

  18. 18.

    Jafri SH, Glass J, Shi R, Zhang S, Prince M, Kleiner-Hancock H. Thymoquinone and cisplatin as a therapeutic combination in lung cancer: in vitro and in vivo. J Exp Clin Cancer Res. 2010;29:87.

  19. 19.

    Li F, Rajendran P, Sethi G. Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway. Br J Pharmacol. 2010;161:541–54.

  20. 20.

    Hua H, Li M, Luo T, Yin Y, Jiang Y. Matrix metalloproteinases in tumorigenesis: an evolving paradigm. Cell Mol Life Sci. 2011;68:3853–68.

  21. 21.

    Arafa e-SA, Zhu Q, Shah ZI, Wani G, Barakat BM, Racoma I. Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Mutat Res. 2011;706:28–35.

  22. 22.

    Ivankovic S, Stojkovic R, Jukic M, Milos M, Milos M, Jurin M. The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo. Exp Oncol. 2006;28:220–4.

  23. 23.

    Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, et al. Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Ther. 2008;7:1789–96.

  24. 24.

    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. Fundam Clin Pharmacol. 2013;27:557–69.

  25. 25.

    Banerjee S, Azmi AS, Padhye S, Singh MW, Baruah JB, Philip PA, et al. Structure-activity studies on therapeutic potential of thymoquinone analogs in pancreatic cancer. Pharm Res. 2010;27:1146–58.

  26. 26.

    Friedl P, Alexander S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell. 2011;147:992–1009.

  27. 27.

    Sahai E. Mechanisms of cancer cell invasion. Curr Opin Genet Dev. 2005;15:87–96.

  28. 28.

    Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst. 1997;89:1260–70.

  29. 29.

    González-Avila G, Iturria C, Vadillo F, Terán L, Selman M, Pérez-Tamayo R. 72-kD (MMP-2) and 92-kD (MMP-9) type IV collagenase production and activity in different histologic types of lung cancer cells. Pathobiology. 1998;66:5–16.

  30. 30.

    Ylisirniö S, Höyhtyä M, Turpeenniemi-Hujanen T. Serum matrix metalloproteinases-2, -9 and tissue inhibitors of metalloproteinases-1, -2 in lung cancer–TIMP-1 as a prognostic marker. Anticancer Res. 2000;20:1311–6.

  31. 31.

    Kim J, Hwan KS. CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells. PLoS One. 2013;8:e74342.

  32. 32.

    Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog. 2004;40:143–9.

  33. 33.

    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;30:2121–31.

  34. 34.

    Chen WP, Tang JL, Bao JP, Wu LD. Thymoquinone inhibits matrix metalloproteinase expression in rabbit chondrocytes and cartilage in experimental osteoarthritis. Exp Biol Med (Maywood). 2010;235:1425–31.

  35. 35.

    Amith SR, Jayanth P, Finlay T, Franchuk S, Gilmour A, Abdulkhalek S, et al. Detection of Neu1 sialidase activity in regulating Toll-like receptor activation. J Vis Exp. 2010; 2142.

  36. 36.

    Tang SW, Yang TC, Lin WC, Chang WH, Wang CC, Lai MK, et al. Nicotinamide N-methyltransferase induces cellular invasion through activating matrix metalloproteinase-2 expression in clear cell renal cell carcinoma cells. Carcinogenesis. 2011;32:138–45.

  37. 37.

    Murthy SR, Dupart E, Al-Sweel N, Chen A, Cawley NX, Loh YP. Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion. Cancer Lett. 2013;341:204–13.

  38. 38.

    Tao X, Hill KS, Gaziova I, Sastry SK, Qui S, Szaniszlo P, et al. Silencing Met receptor tyrosine kinase signaling decreased oral tumor growth and increased survival of nude mice. Oral Oncol. 2014;50:104–12.

  39. 39.

    Lin CW, Chen PN, Chen MK, Yang WE, Tang CH, Yang SF, et al. Kaempferol reduces matrix metalloproteinase-2 expression by down-regulating ERK1/2 and the activator protein-1 signaling pathways in oral cancer cells. PLoS One. 2013;81:e80883.

  40. 40.

    Lee SH, Jaganath IB, Manikam R, Sekaran SD. Inhibition of Raf-MEK-ERK and hypoxia pathways by phyllanthus prevents metastasis in human lung (A549) cancer cell line. BMC Complement Altern Med. 2013;13:271.

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Correspondence to Xi-xin Yan.

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Yang, J., Kuang, X., Lv, P. et al. Thymoquinone inhibits proliferation and invasion of human nonsmall-cell lung cancer cells via ERK pathway. Tumor Biol. 36, 259–269 (2015). https://doi.org/10.1007/s13277-014-2628-z

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Keywords

  • Nonsmall-cell lung cancer
  • Thymoquinone
  • Proliferation
  • Invasion
  • Signal transduction pathway