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Involvement of Mitogen-Activated Protein Kinase Pathway in T-2 Toxin-Induced Cell Cycle Alteration and Apoptosis in Human Neuroblastoma Cells

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Abstract

T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50 % inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.

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Abbreviations

CVDE:

Crystal violet dye exclusion

CHAPS:

3[(3-Cholamidopropyl) dimethlylammonio]-1-propanesulfate

PARP:

Poly(ADP-ribose) polymerase

PMSF:

Phenylmethylsulfonyl fluoride

DTT:

Dithiothreitol

PBS:

Phosphate-buffered saline

DCF-DA:

2,7-dichlorofluorescein diacetate

LDH:

Lactate dehydrogenase

MAPK:

Mitogen-activated protein kinase

ROS:

Reactive oxygen species

PI:

Propidium iodide

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Acknowledgments

The authors thank Dr. M. P. Kaushik, Director, DRDE, for providing the necessary facilities and encouragement, and Ms. Mona Agrawal is thankful to CSIR for the award of Senior Research Fellowship.

Conflict of Interest

The authors report no conflicts of interest.

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Author notes

  1. P. V. Lakshmana Rao

    Present address: DRDO-BU Center for Life Sciences, Bharathiar University Campus, Coimbatore, India

Authors and Affiliations

  1. Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior, 474002, India

    Mona Agrawal, A. S. B. Bhaskar & P. V. Lakshmana Rao

Authors
  1. Mona Agrawal
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  2. A. S. B. Bhaskar
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Correspondence to P. V. Lakshmana Rao.

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Agrawal, M., Bhaskar, A.S.B. & Rao, P.V.L. Involvement of Mitogen-Activated Protein Kinase Pathway in T-2 Toxin-Induced Cell Cycle Alteration and Apoptosis in Human Neuroblastoma Cells. Mol Neurobiol 51, 1379–1394 (2015). https://doi.org/10.1007/s12035-014-8816-4

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  • DOI: https://doi.org/10.1007/s12035-014-8816-4

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