July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Reactive Oxygen Species Play a Key Role in Endoplasmic Reticulum Stress Triggered by Sulforaphane in Human Epithelial Lens Cells
Author Affiliations & Notes
  • Ngoc Phuong Thao Huynh
    University of East Anglia, Norwich, United Kingdom
  • Michael Wormstone
    University of East Anglia, Norwich, United Kingdom
  • Footnotes
    Commercial Relationships   Ngoc Phuong Thao Huynh, None; Michael Wormstone, None
  • Footnotes
    Support  The Humane Research Trust
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2077. doi:
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      Ngoc Phuong Thao Huynh, Michael Wormstone; Reactive Oxygen Species Play a Key Role in Endoplasmic Reticulum Stress Triggered by Sulforaphane in Human Epithelial Lens Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2077.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Posterior capsule opacification (PCO) is a fibrotic condition occurring upon cataract surgery that can cause visual deterioration. The isothiocyanate, sulforaphane (SFN) is reported to exert cytoprotective and cytotoxic actions and the latter may be exploited to treat/prevent PCO. Previous work has shown that SFN can induce endoplasmic reticulum stress (ERS) and increase reactive oxygen species (ROS). This study aimed to examine the role of ROS in three ERS pathways (IRE1, PERK and ATF6) associated with SFN in human epithelial lens cells.

Methods : The human lens epithelial cell line FHL124 was used for the study. Cells were treated with 50μM SFN for different durations depending on the experiment type. The gene expression of ERN1 and DDIT3 were detected by real time (RT)-PCR (n=4). Levels of two proteins, p-eIF2α (n=4) and XBP-1 (n=3), were determined by Western blot (WB). ATF6 transcriptional activity was measured by the dual luciferase reporter assay (n=4). 1mM N-acetyl cysteine (NAC) was added to cells, to scavenge ROS, 1 hour prior to SFN treatment. Untreated cells served as controls. One-way ANOVA with Tukey’s post hoc analysis was used for statistical analysis.

Results : We initially examined two ERS pathways, IRE1 and PERK. In response to SFN after 18 hours, the expression of endoplasmic reticulum stress genes ERN1, encoding IRE1, and DDIT3, encoding CHOP – a downstream signal of PERK, were upregulated significantly, such that levels relative to control were 465 ± 105% and 864 ± 179% respectively. The protein levels of two ERS markers: p-eIF2α and XBP-1 were elevated 443 ± 88% and 672 ± 285% respectively, in cells treated with SFN for 18 hours relative to control. To investigate the ATF6 pathway, a reporter assay was employed, which revealed a significant 891 ± 348% increase in ATF6 activity following a 6 hour SFN treatment, relative to control. SFN induced promotion of gene expression, elevated protein associated with the IRE1 and PERK pathways and increased ATF6 activity were significantly inhibited by pre-treatment with NAC.

Conclusions : Our findings show SFN can trigger ERS in lens epithelial cells and that ROS plays a key role in SFN mediated responses. SFN is a potential therapeutic agent for PCO, in particular, and a promising reagent to study the relationship between oxidative stress and ERS, in general.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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