, Volume 68, Issue 5, pp 1999–2013

Cell cycle arrest and mechanism of apoptosis induction in H400 oral cancer cells in response to Damnacanthal and Nordamnacanthal isolated from Morinda citrifolia

  • Gohar Shaghayegh
  • Aied M. Alabsi
  • Rola Ali-Saeed
  • Abdul Manaf Ali
  • Vui King Vincent-Chong
  • Rosnah Binti Zain
Original Article


Oral cancer is the eleventh most prevalent cancer worldwide. The most prevalent oral cancer is oral squamous cell carcinoma (OSCC). Damnacanthal (DAM) and nordamnacanthal (NDAM), the anthraquinone compounds, are isolated from the root of Morinda citrifolia L. (Noni), which has been used for the treatment of several chronic diseases including cancer. The objectives of this study were to evaluate the cytotoxicity, cell death mode, cell cycle, and the molecular mechanism of apoptosis induced by DAM and NDAM on OSCC. The cytotoxic effects of these compounds against OSCC cell lines were determined by MTT assay. The cell death mode was analysed by DNA laddering and FITC-annexin V/PI flow cytometric assays. In addition, the mechanism of apoptosis induced by DAM and NDAM was detected using mitochondrial membrane potential, Cytochrome c, and caspases assays. Finally, the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells was detected by flow cytometry. In the present study, DAM and NDAM showed cytotoxicity towards OSCC cell lines and the maximum growth inhibition for both compounds was observed in H400 cells with IC50 value of 1.9 and 6.8 μg/ml, respectively, after 72 h treatment. The results also demonstrated the inhibition of H400 OSCC cells proliferation, internucleosomal cleavage of DNA, activation of intrinsic apoptosis pathway, and cell cycle arrest caused by DAM and NDAM. Therefore, these findings suggest that DAM and NDAM can be potentially used as antitumor agents for oral cancer therapy.


Damnacanthal and nordamnacanthal Human oral squamous cell carcinoma (OSCC) Apoptosis Mitochondrial membrane potential Cytochrome c 


  1. Alabsi AM, Ali R, Ali AM, Al-Dubai SAR, Harun H, Abu Kasim NH, Alsalahi A (2012) Apoptosis induction, cell cycle arrest and in vitro anticancer activity of gonothalamin in a cancer cell lines. Asian Pac J Cancer Prev 13:5131–5136CrossRefGoogle Scholar
  2. Ali A, Ismail N, Mackeen M, Yazan L, Mohamed S, Ho A, Lajis N (2000) Antiviral, cyototoxic and antimicrobial activities of anthraquinones isolated from the roots of Morinda elliptica. Pharm Biol 38:298–301CrossRefGoogle Scholar
  3. Ali R, Alabsi AM, Ali AM, Ideris A, Omar R, Yusoff K, Ali RS (2011) Cytolytic effects and apoptosis induction of Newcastle disease virus strain AF2240 on anaplastic astrocytoma brain tumor cell line. Neurochem Res 36:2051–2062CrossRefGoogle Scholar
  4. Alitheen N, Mashitoh A, Yeap S, Shuhaimi M, Manaf AA, Nordin L (2010) Cytotoxic effect of damnacanthal, nordamnacanthal, zerumbone and betulinic acid isolated from Malaysian plant sources. Int Food Res J 17:711–719Google Scholar
  5. Antonsson B, Martinou J-C (2000) The Bcl-2 protein family. Exp Cell Res 256:50–57CrossRefGoogle Scholar
  6. Aubry JP, Blaecke A, Lecoanet-Henchoz S, Jeannin P, Herbault N, Caron G, Moine V, Bonnefoy JY (1999) Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry 37:197–204CrossRefGoogle Scholar
  7. Balaram P, Sridhar H, Rajkumar T, Vaccarella S, Herrero R, Nandakumar A, Ravichandran K, Ramdas K, Sankaranarayanan R, Gajalakshmi V, Muñoz N, Franceschi S (2002) Oral cancer in southern India: the influence of smoking, drinking, paan-chewing and oral hygiene. Int J Cancer 98:440–445CrossRefGoogle Scholar
  8. Czerski L, Nuñez G (2004) Apoptosome formation and caspase activation: is it different in the heart? J Mol Cell Cardiol 37:643–652CrossRefGoogle Scholar
  9. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefGoogle Scholar
  10. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2014) Globocan 2012, Cancer incidence and mortality worldwide. International Agency for Research on Cancer, Lyon.
  11. Ghani WM, Razak IA, Yang Y-H, Talib NA, Ikeda N, Axell T, Gupta PC, Handa Y, Abdullah N, Zain RB (2011) Factors affecting commencement and cessation of betel quid chewing behaviour in Malaysian adults. BMC Public Health 11:82CrossRefGoogle Scholar
  12. Gordaliza M (2007) Natural products as leads to anticancer drugs. Clin Transl Oncol 9:767–776CrossRefGoogle Scholar
  13. Haghiac M, Walle T (2005) Quercetin induces necrosis and apoptosis in SCC-9 oral cancer cells. Nutr Cancer 53:220–231CrossRefGoogle Scholar
  14. Hiramatsu T, Imoto M, Koyano T, Umezawa K (1993) Induction of normal phenotypes in ras-transformed cells by damnacanthal from Morinda citrifolia. Cancer Lett 73:161–166CrossRefGoogle Scholar
  15. Ismail NH, Ali AM, Aimi N, Kitajima M, Takayama H, Lajis NH (1997) Anthraquinones from Morinda citrifolia. Phytochemistry 14:1723–1725CrossRefGoogle Scholar
  16. Jakubikova J, Bao Y, Sedlak J (2005) Isothiocyanates induce cell cycle arrest, apoptosis and mitochondrial potential depolarization in HL-60 and multidrug-resistant cell lines. Anticancer Res 25:3375–3386Google Scholar
  17. Jasril LN, Mooi LY, Abdullah MA, Sukari MA, Ali AM (2003) Antitumor promoting and antioxidant activities of anthraquinones isolated from the cell suspension culture of Morinda elliptica. Asia Pac J Mol Biol Biotechnol 11:3–7Google Scholar
  18. Johnson NW, Warnakulasuriya S, Gupta P, Dimba E, Chindia M, Otoh EC, Sankaranarayanan R, Califano J, Kowalski L (2011) Global oral health inequalities in incidence and outcomes for oral cancer causes and solutions. Adv Dent Res 23:237–246CrossRefGoogle Scholar
  19. Kamei H, Koide T, Kojima T, Hashimoto Y, Hasegawa M (1998) Inhibition of cell growth in culture by quinones. Cancer Biother Radiopharm 13:185–188CrossRefGoogle Scholar
  20. Kanokmedhakul K, Kanokmedhakul S, Phatchana R (2005) Biological activity of Anthraquinones and Triterpenoids from Prismatomeris fragrans. J Ethnopharmacol 100:284–288CrossRefGoogle Scholar
  21. Konoshima T, Kozuka M, Koyama J, Tagahara T, Okatani T, Tagahara K, Tokud AH (1989) Studies on inhibitors of skin tumor promotion, VI. Inhibitory effects of quinones on Epsterin-Barr virus activation. J Nat Prod 52:987–995CrossRefGoogle Scholar
  22. Krithika R, Mohankumar R, Verma RJ, Shrivastav PS, Mohamad IL, Gunasekaran P, Narasimhan S (2009) Isolation, characterization and antioxidative effect of phyllanthin against CCl4-induced toxicity in HepG2 cell line. Chem Biol Interact 181:351–358CrossRefGoogle Scholar
  23. Kumar B, Kumar A, Pandey B, Mishra K, Hazra B (2009) Role of mitochondrial oxidative stress in the apoptosis induced by diospyrin diethylether in human breast carcinoma (MCF-7) cells. Mol Cell Biochem 320:185–195CrossRefGoogle Scholar
  24. Lin F-L, Hsu J-L, Chou CH, Wu WJ, Chang CI, Liu HJ (2011) Activation of p38 MAPK by damnacanthal mediates apoptosis in SKHep 1 cells through the DR5/TRAIL and TNFR1/TNF-α and p53 pathways. Eur J Pharmacol 650:120–129CrossRefGoogle Scholar
  25. Machida YJ, Hamlin JL, Dutta A (2005) Right place, right time, and only once: replication initiation in metazoans. Cell 123:13–24CrossRefGoogle Scholar
  26. Neville BW, Day TA (2002) Oral cancer and precancerous lesions. CA: A Cancer J Clin 52:195–215Google Scholar
  27. Nhan TQ, Liles WC, Schwartz SM (2006) Physiological functions of caspases beyond cell death. Am J Pathol 169:729–737CrossRefGoogle Scholar
  28. Nicholson D (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6:1028–1042CrossRefGoogle Scholar
  29. Nishida K, Yamaguchi O, Otsu K (2008) Crosstalk between autophagy and apoptosis in heart disease. Circ Res 103:343–351CrossRefGoogle Scholar
  30. Pavia M, Pileggi C, Nobile CG, Angelillo IF (2006) Association between fruit and vegetable consumption and oral cancer: a meta-analysis of observational studies. Am J Clin Nutr 83:1126–1134Google Scholar
  31. Pietenpol J, Stewart Z (2002) Cell cycle checkpoint signalling: cell cycle arrest versus apoptosis. Toxicology 181:475–481CrossRefGoogle Scholar
  32. Sato D, Kondo S, Yazawa K, Mukudai Y, Li C, Kamatani T, Katsuta H, Yoshihama Y, Shirota T, Shintani S (2013) The potential anticancer activity of extracts derived from the roots of Scutellaria baicalensis on human oral squamous cell carcinoma cells. Mol Clin Oncol 1:105–111Google Scholar
  33. Schwartz GK, Shah MA (2005) Targeting the cell cycle: a new approach to cancer therapy. J Clin Oncol 23:9408–9421CrossRefGoogle Scholar
  34. Shier WT (1991) Mammalian cell culture on $5 a day: a laboratory manual of low cost methods. Los Banos Univ Philipp 64:9–16Google Scholar
  35. Stewart B, Kleihues P (2003) Head and neck cancer. World cancer report. International Agency for Research on Cancer, LyonGoogle Scholar
  36. Torre LA, Bray F, Siegel RL, Ferlay J, Ward E, Forman D (2015) Global cancer statistics, 2002. CA: A Cancer J Clin 65:87–108Google Scholar
  37. Wang Z-B, Liu Y-Q, Cui Y-F (2005) Pathways to caspase activation. Cell Biol Int 29:489–496CrossRefGoogle Scholar
  38. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555–556CrossRefGoogle Scholar
  39. Zhang J, Xu M (2002) Apoptotic DNA fragmentation and tissue homeostasis. Trends Cell Biol 12:84–89CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Gohar Shaghayegh
    • 1
  • Aied M. Alabsi
    • 1
    • 2
  • Rola Ali-Saeed
    • 3
  • Abdul Manaf Ali
    • 3
  • Vui King Vincent-Chong
    • 2
  • Rosnah Binti Zain
    • 2
  1. 1.Department of Oral Biology and Biomedical Sciences, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia
  2. 2.Oral Cancer Research and Coordinating Centre, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia
  3. 3.School of Biotechnology, Faculty of Bioresource and Food IndustryUniversity Sultan Zainal AbidinKuala TerengganuMalaysia

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