Molecular Neurobiology

, Volume 51, Issue 1, pp 119–130 | Cite as

Cytotoxic Effect of p-Coumaric Acid on Neuroblastoma, N2a Cell via Generation of Reactive Oxygen Species Leading to Dysfunction of Mitochondria Inducing Apoptosis and Autophagy

  • S. Shailasree
  • M. Venkataramana
  • S. R. Niranjana
  • H. S. Prakash


p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 μmol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (~60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells.


p-Coumaric acid Neuroblastoma N2a cells Apoptosis p53 Caspase-8 Autophagy LC3-II 



The authors acknowledge the recognition of the University of Mysore as an Institution of Excellence and financial support from the Ministry of Human Resource Development, Govt. of India through UGC under UOM/IOE/RESEARCH/1/2010-11, dt 22-04-2010 project.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003) Production of reactive oxygen species by mitochondria. J Biol Chem 278:36027–36031PubMedCrossRefGoogle Scholar
  2. 2.
    Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Laura AS, Chandel NS (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cells 48:158–167CrossRefGoogle Scholar
  4. 4.
    Nakamura H, Nakamura K, Yodoi J (1997) Redox regulation of cellular activation. Annu Rev Immunol 15:351–369PubMedCrossRefGoogle Scholar
  5. 5.
    Ozben T (2007) Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci 96:2181–2196PubMedCrossRefGoogle Scholar
  6. 6.
    Xie CM, Chan WY, Yu S, Zhao J, Cheng CH (2011) Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation. Free Radic Biol Med 51:1365–1375PubMedCrossRefGoogle Scholar
  7. 7.
    Gao M, Yeh PY, Lu YS, Hsu CH, Chen KF, Lee WC, Feng WC, Chen CS, Kuo ML, Cheng AL (2008) OSU-03012, a novel celecoxib derivative, induces reactive oxygen species-related autophagy in hepatocellular carcinoma. Cancer Res 68:9348–9357PubMedCrossRefGoogle Scholar
  8. 8.
    Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811PubMedCrossRefGoogle Scholar
  9. 9.
    Mehta SS, Manhas N, Raghubir R (2007) Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Res Rev 54:34–66PubMedCrossRefGoogle Scholar
  10. 10.
    Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308PubMedCrossRefGoogle Scholar
  11. 11.
    Ehrhardt H, Hacker S, Wittmann S, Maurer M, Borkhardt A, Toloczko A, Debatin KM, Fulda S, Jeremias I (2008) Cytotoxic drug-induced, p53-mediated upregulation of caspase-8 in tumor cells. Oncogene 27:783–793PubMedCrossRefGoogle Scholar
  12. 12.
    Ehrhardt H, Wachter F, Maurer M, Stahnke K, Jeremias I (2011) Important role of caspase-8 for chemo-sensitivity of ALL cells. Clin Cancer Res 17:7605–7613PubMedCrossRefGoogle Scholar
  13. 13.
    Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ (2007) Potential therapeutic applications of autophagy. Nat Rev Drug Discov 6:304–312PubMedCrossRefGoogle Scholar
  14. 14.
    Periyasamy-Thandavan S, Jiang M, Schoenlein P, Dong Z (2009) Autophagy: molecular machinery, regulation and implications for renal pathophysiology. Am J Physiol Ren Physiol 297:F244–F256CrossRefGoogle Scholar
  15. 15.
    Tanida I, Ueno T, Kominami E (2008) LC3 and autophagy. Methods Mol Biol 445:77–88PubMedCrossRefGoogle Scholar
  16. 16.
    Gurney JG, Smith MA, Ross JA (1999) Cancer among infants. In: Gloeckler Ries LA (ed) Cancer incidence and survival among children and adolescents: United States SEER Program, 1975–1995. National Cancer Institute, Bethesda (MD), pp 149–156Google Scholar
  17. 17.
    Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS (2001) Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res 480–481:243–268PubMedCrossRefGoogle Scholar
  18. 18.
    Dragsted LO (2003) Antioxidant actions of polyphenols in humans. Int J Vitam Nutr Res 73:112–119PubMedCrossRefGoogle Scholar
  19. 19.
    Dore S (2005) Unique properties of polyphenol stilbenes in the brain: more than direct antioxidant actions; gene/protein regulatory activity. Neurosignals 14:61–70Google Scholar
  20. 20.
    Sang S, Hou Z, Lambert JD, Yang CS (2005) Redox properties of tea polyphenols and related biological activities. Antioxid Redox Signal 7:1704–1714PubMedCrossRefGoogle Scholar
  21. 21.
    Grever MCB (2001) Cancer drug discovery and development. In: De Vita VHS, Rosenberg SA (eds) Cancer: principles and practice of oncology. Lippincott Raven, Philadelphia, pp 328–339Google Scholar
  22. 22.
    Janicke B, Onning G, Oredsson SM (2005) Differential effects of ferulic acid and p-coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco-2. J Agric Food Chem 53:6658–6665PubMedCrossRefGoogle Scholar
  23. 23.
    Jaganathan SK, Supriyanto E, Mandal M (2013) Events associated with apoptotic effect of p-coumaric acid in HCT-15 colon cancer cells. World J Gastroenterol 19:7726–7734PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Larrosa M, Lodovici M, Morbidelli L, Dolara P (2008) Hydrocaffeic and p-coumaric acids, natural phenolic compounds, inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo. Free Radic Res 42:903–910PubMedCrossRefGoogle Scholar
  25. 25.
    Abdel-Wahab MH, El-Mahdy MA, Abd-Ellah MF, Helal GK, Khalifa F, Hamada FM (2003) Influence of p-coumaric acid on doxorubicin-induced oxidative stress in rat’s heart. Pharmacol Res 48:461–465PubMedCrossRefGoogle Scholar
  26. 26.
    Vauzour D, Corona G, Spencer JP (2010) Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyldopamine induced neurotoxicity. Arch Biochem Biophys 501:106–111PubMedCrossRefGoogle Scholar
  27. 27.
    Ferguson LR, Zhu ST, Harris PJ (2005) Antioxidant and antigenotoxic effects of plant cell wall hydroxycinnamic acids in cultured HT-29 cells. Mol Nutr Food Res 49:585–593PubMedCrossRefGoogle Scholar
  28. 28.
    Seo YK, Kim SJ, Boo YC, Baek JH, Lee SH, Koh JS (2010) Effects of p-coumaric acid on erythema and pigmentation of human skin exposed to ultraviolet radiation. Clin Exp Dermatol 36:260–266PubMedCrossRefGoogle Scholar
  29. 29.
    Barros MP, Lemos M, Maistro EL, Leite MF, Sousa JP, Bastos JK, Andrade SF (2008) Evaluation of antiulcer activity of the main phenolic acids found in Brazilian green propolis. J Ethnopharmacol 120:372–377PubMedCrossRefGoogle Scholar
  30. 30.
    Luceri CL, Lodovici GM, Antonucci E, Abbate R, Masini E, Dolara P (2007) p-Coumaric acid, a common dietary phenol, inhibits platelet activity in vitro and in vivo. Br J Nutr 97:458–463PubMedCrossRefGoogle Scholar
  31. 31.
    Shastry P, Basu A, Rajadhyaksha MS (2001) Neuroblastoma cell lines—a versatile in vitro model in neurobiology. Int J Neurosci 108:109–126PubMedCrossRefGoogle Scholar
  32. 32.
    Radio NM, Mundy WR (2008) Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29:361–376PubMedCrossRefGoogle Scholar
  33. 33.
    Azad MB, Chen Y, Gibson SB (2009) Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal 11:777–790PubMedCrossRefGoogle Scholar
  34. 34.
    Sareen D, van Ginkel PR, Takach JC, Mohiuddin A, Darjatmoko SR, Albert DM, Polans AS (2006) Mitochondria as the primary target of resveratrol-induced apoptosis in human retinoblastoma cells. Invest Ophthalmol Vis Sci 47:3708–3716PubMedCrossRefGoogle Scholar
  35. 35.
    Yang C, Kaushal V, Shah SV, Kaushal GP (2008) Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. Am J Physiol Ren Physiol 294:777–787CrossRefGoogle Scholar
  36. 36.
    Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  37. 37.
    Shailasree S, Kini KR, Deepak S, Kumudini BS, Shetty HS (2004) Accumulation of hydroxyproline-rich glycoproteins in pearl millet seedlings in response to Sclerospora graminicola infection. Plant Sci 167:1227–1234Google Scholar
  38. 38.
    Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Hao JK (2012) Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif 45:487–498PubMedCrossRefGoogle Scholar
  39. 39.
    Kerr JF, Winterford CM, Harmon BV (1994) Apoptosis, its significance in cancer and cancer therapy. Cancer 73:2013–2026PubMedCrossRefGoogle Scholar
  40. 40.
    Petrosillo G, Matera M, Moro N, Ruggiero FM, Paradies G (2009) Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin. Free Radic Biol Med 46:88–94PubMedCrossRefGoogle Scholar
  41. 41.
    Li PF, Dietz R, von Harsdorf R (1999) p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-1. EMBO J 18:6027–6036PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Smith RAJ, Porteous CM, Gane AM, Murphy MP (2003) Delivery of bioactive molecules to mitochondria in vivo. Proc Natl Acad Sci U S A 100:5407–5412PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Von Harsdorf R, Li P, Dietz R (1999) Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation 99:2934–2941CrossRefGoogle Scholar
  44. 44.
    Pingoud-Meier C, Lang D, Janss AJ, Rorke LB, Phillips PC, Shalaby T (2003) Downregulation of caspase-8 protein expression correlates with unfavorable survival outcome in childhood medulloblastoma. Clin Cancer Res 9:6401–6409PubMedGoogle Scholar
  45. 45.
    Blagosklonny MV (2002) p53: an ubiquitous target of anticancer drugs. Int J Cancer 98:161–166PubMedCrossRefGoogle Scholar
  46. 46.
    Fuster JJ, Sanz-Gonzalez SM, Moll UM, Andres V (2007) Classical and novel roles of p53: prospects for anticancer therapy. Trends Mol Med 13:192–199PubMedCrossRefGoogle Scholar
  47. 47.
    Hug H, Enari M, Nagata S (1994) No requirement of reactive oxygen intermediates in Fas-mediated apoptosis. FEBS Lett 351:311–313PubMedCrossRefGoogle Scholar
  48. 48.
    Langer C, Jurgensmeier JM, Bauer G (1996) Reactive oxygen species act at both TGF-beta-dependent and -independent steps during induction of apoptosis of transformed cells by normal cells. Exp Cell Res 222:117–124PubMedCrossRefGoogle Scholar
  49. 49.
    Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, Birrell GW, Peehl DM, Knox SJ (2002) Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ 9:252–263PubMedCrossRefGoogle Scholar
  50. 50.
    Ling LU, Tan KB, Lin H, Chiu GN (2011) The role of reactive oxygen species and autophagy in safingol-induced cell death. Cell Death Dis 2:e129PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Mi YL, Zhang CQ (2005) Protective effect of quercetin on aroclor 1254-induced oxidative damage in cultured chicken spermatogonial cells. Toxicol Sci 88:545–550CrossRefGoogle Scholar
  52. 52.
    Amelio I, Melino G, Knight RA (2011) Cell death pathology: cross-talk with autophagy and its clinical implications. Biochem Biophys Res Commun 414:277–281PubMedCrossRefGoogle Scholar
  53. 53.
    Cho YS, Park SY, Shin HS, Chan FK (2010) Physiological consequences of programmed necrosis, an alternative form of cell demise. Mol Cells 29:327–332PubMedCrossRefGoogle Scholar
  54. 54.
    Han W, Xie J, Li L, Liu Z, Hu X (2009) Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis. Apoptosis 14:674–686PubMedCrossRefGoogle Scholar
  55. 55.
    Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, Joanna R-W, Los MJ (2013) Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp 61:43–58CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • S. Shailasree
    • 1
  • M. Venkataramana
    • 3
  • S. R. Niranjana
    • 2
  • H. S. Prakash
    • 2
  1. 1.Institution of Excellence, Vijnana BhavanaUniversity of MysoreMysoreIndia
  2. 2.Department of Studies in BiotechnologyUniversity of MysoreMysoreIndia
  3. 3.Defence Research Development OrganizationBharathiar UniversityCoimbatoreIndia

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