Abstract
Purpose:
In cataract surgery, after lens extraction an artificial ocular lens is implanted to the remaining capsular bag. Remaining lens epithelial cells (HLEC) migrate and proliferate on the lens implant, resulting in posterior capsule opacification (PCO). The up-regulation of different Growth factors, changes in the actin cytosceleton and migration have been identified before as key factors. In this study, the effect of Erlotinib, a selective EGFR inhibitor approved for cancer therapy, was investigated in vitro on HLEC. The expression of EGF, TGF-β, FGF, EGF-R and changes in the actin cytoskeleton were observed, followed by a thorough computational analysis of cellular migration.
Methods:
The effect of Erlotinib on the actin-cytoskeleton was evaluated using Alexa Fluor 488 Phalloidin and epifluorescence microscopy. Expression of EGF, FGF, TGF-β and EGFR was measured using rt-PCR and ELISA. Computational data of an Time Lapse Microscopy assay with HLEC was used for analysis of cell migration with a particular focus on cell-cell interaction, cell velocity, displacement before and after mitosis and random and directed movement.
Results:
EGF, TGFβ, FGF and EGFR mRNA expression and protein is reduced with increasing concentrations of Erlotinib. Erlotinib reduces the amount of Actin stress fibers and stress fiber diameter. Erlotinib decreases the cells ability to engage into and to lose interaction to other cells. Distributions of velocities show that the change in velocity is not uniform across the cell cluster with increasing concentrations of Erlotinib. Cells moved significantly faster after mitosis, without EGFR Inhibition. Erlotinib reduced this effect to no significance. Cells within the cell cluster moved less random with increasing EGFR Blockade.
Conclusions:
As a novel effect of Erlotinib on HLEC, we described the down-regulation of EGF, FGF, TGF-β and EGFR expression, all of them known to play a major role in PCO. In this study, different novel aspects of cell migration in vitro under EGFR inhibition have been highlighted. The influence of Erlotinib on Actin assembly is illustrated and partly explains the change in cellular migration. This suggests a possible future role of Erlotinib as a pharmacologic prophylaxis for PCO.
Keywords: 652 posterior capsular opacification (PCO) •
445 cataract •
533 gene/expression