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Iron-Induced Apoptotic Cell Death and Autophagy Dysfunction in Human Neuroblastoma Cell Line SH-SY5Y

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Abstract

Iron accumulation plays a major role in neuronal cell death which has severe effects on mental health like neurodegenerative disorders. The present work aims to explore the involvement of molecular pathways involved in iron-mediated neuronal cell death using Ferric Ammonium Citrate (FAC) as a source of iron to treat neuroblastoma SH-SY5Y cells. In this study, it was found that cytotoxicity induced by iron treatment is highly correlated with enhanced intracellular reactive oxygen species (ROS) generation and loss of mitochondrial integrity. Appearance of early and late apoptotic cells with altered nuclear morphology and increased expression of effector proteins, i.e., cleaved Caspase 3 and cleaved PARP (Poly-ADP-ribose Polymerase), clearly confirmed iron-induced apoptotic cell deaths. Furthermore, excess accumulation of acidic vesicles and microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3II/I expressions were observed. Simultaneously, ultrastructural studies of SH-SY5Y cells demonstrated the accumulation of a large number of autophagosomes, autophagic vacuolization, and swollen mitochondria which further confirmed the induction of autophagy concomitant with mitochondrial damage. Furthermore, increased incorporation of lysosome-specific dye, LysoTracker Deep Red, and the red fluorescence retention of LC3-GFP-RFP constructs indicates the incomplete autophagy or autophagy dysfunction due to altered lysosomal activity. Hence, the present work unveiled the interruption in autophagy progression caused by the plausible suppression of lysosomal activity due to iron treatment resulting in autophagic cell death in SH-SY5Y cell lines. In general, both apoptotic and autophagic pathways were prominent and each of the pathways played their prospective roles, in iron-mediated neuronal cell death.

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Acknowledgments

JB gratefully acknowledges the financial support from the University Grants Commission (Major Research Project) No. 41-539/2012 (SR) and West Bengal University of Technology TEQIP II program for providing infrastructural facilities. JR is further thankful to ICMR SRF (No. 46/68/2015/BMS/TRM) for providing Senior Research Fellowship. SR acknowledges SERB for awarding the National Post Doctoral Fellowship (PDF/2017/001443). The authors are also thankful to Bose Institute, Kolkata, for providing the flow cytometry facilities and National Institute of Cholera and Enteric Diseases, Kolkata, for providing the TEM facilities.

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Authors and Affiliations

  1. Department of Biotechnology, Maulana Abul Kalam Azad University of Technology, West Bengal, NH 12, Haringhata, West Bengal, 741249, India

    Jyotirmoy Rakshit, Arijit Mallick, Susmita Roy & Jaya Bandyopadhyay

  2. Division of Electron Microscopy, ICMR-National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, West Bengal, 700010, India

    Arpita Sarbajna & Moumita Dutta

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  1. Jyotirmoy Rakshit
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Rakshit, J., Mallick, A., Roy, S. et al. Iron-Induced Apoptotic Cell Death and Autophagy Dysfunction in Human Neuroblastoma Cell Line SH-SY5Y. Biol Trace Elem Res 193, 138–151 (2020). https://doi.org/10.1007/s12011-019-01679-6

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