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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

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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Institution of Excellence, Vijnana Bhavana, University of Mysore, Mysore, 570006, India

    S. Shailasree

  2. Department of Studies in Biotechnology, University of Mysore, Mysore, 570006, India

    S. R. Niranjana & H. S. Prakash

  3. Defence Research Development Organization, Bharathiar University, Coimbatore, Tamil Nadu, India

    M. Venkataramana

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  1. S. Shailasree
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  3. S. R. Niranjana
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Correspondence to H. S. Prakash.

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Highlights

1. We have used in vitro model to investigate the cytotoxic effects of p-coumaric acid (p-CA) on neuroblastoma N2a cells.

2. p-Coumaric acid-induced cytotoxicity by 72 h at 150 μmol/L stimulated 12.06 % of cells to early apoptosis and 81.23 % of cells to apoptotic or necrotic cells, as evidenced by flow cytometer studies.

3. Cytotoxic effect was mediated by excessive production of reactive oxygen species (7.5-fold over control) causing mitochondrial dysfunction and followed by release of cytochrome c.

4. Sensitizing N2a cells for induction of apoptosis by p-CA identified p53-mediated upregulation of caspase-8 messenger RNA (2.8-fold) as target gene.

5. 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.

6. Detection of lactate dehydrogenase in supernatants of p-CA-treated N2a cells relative to control cells leads to interpretation that a few cells died by necrosis.

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Shailasree, S., Venkataramana, M., Niranjana, S.R. et al. 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. Mol Neurobiol 51, 119–130 (2015). https://doi.org/10.1007/s12035-014-8700-2

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