Abstract
Purpose :
Posterior capsule opacification (PCO) commonly develops following cataract surgery and is a wound-healing response that can ultimately lead to secondary visual loss. Improved management of this problem is required. The isothiocyantae, sulforaphane (SFN) is reported to exert cytoprotective and cytotoxic actions and the latter may be exploited to treat/prevent PCO.
Methods :
The human lens epithelial cell line FHL124 and human lens capsular bag system were used as experimental models. The MTS assay was used to assess cell populations and the LDH assay was employed to assess cell damage/death. To determine migration, a scratch assay was performed. Gene expression and protein levels were determined using real-time PCR and western blot methods respectively. To investigate protein distribution and cell architecture immunofluorescence and transmission electron microscopy were utilized. Level of reactive oxidant species (ROS) was determined using a commercially available kit (Promega). In vitro capsular bags were generated from human donor eyes by simulated cataract surgery, isolated by cutting of the zonules and secured to a 35mm tissue culture dish using entomological pins. Ongoing observations were made using phase-contrast microscopy.
Results :
SFN concentrations of 10µM and above significantly impaired wound-healing in a human lens capsular bag model. A similar pattern of response was also seen with a human lens cell line, FHL124. SFN treatment promoted increased expression of ER stress genes, which also corresponded with protein expression. Evidence of autophagy was observed in response to SFN as determined by increased LC3-II levels and detection of autophagic vesicles. This response was disrupted by established autophagy inhibitors chloroquine and 3-MA. SFN was found to promote MAPK signaling and inhibition of ERK activation, using U0126, prevented SFN induced LC3-II elevation and vesicle formation. SFN also significantly increased levels of ROS.
Conclusions :
Taken together, our findings suggest that SFN is capable of inducing ER stress and autophagy, which could contribute to reduced lens cell growth and viability. SFN could thus serve as a putative therapeutic agent for PCO.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.