Investigating the putative therapeutic effects of sulfophane in the human lens
Purpose: Cataract (a clouding of the lens) and PCO, a lens wound-healing response that occurs following cataract surgery, are important medical problems affecting millions worldwide. Reducing the incidence of these conditions would have a marked effect on the personal well being of millions of people. The isothiocyanate, SFN is known to have dose-dependent cytoprotective and cytotoxic properties. The present study had three aims. Firstly, to determine if the isothiocyanate, SFN could yield protection to lens cells against oxidative stress. Secondly, to identify the mechanisms by which SFN can elicit cytoprotection to lens cells against oxidative stress. The final aim was to establish the ability of SFN to initiate death of lens cells and prevent PCO formation. Methods: The human lens epithelial cell line FHL 124 was used in combination with whole porcine lens culture (to assess opacity) and human capsular bags (as a PCO model) were employed as the experimental systems. Whole lens cultures were monitored using brightfield and darkfield imaging; quantification was performed with ImageJ analysis software. Capsular bags were monitored using phase-contrast microscopy. The ApoToxGlo Triplex assay was used to assess FHL 124 cell survival, cytotoxicity and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium (of FHL 124 cells and porcine lenses) were evaluated to reflect cell damage/death. To assess level of gene expression an Illumina whole genome HT-12 v4 beadchip microarray was employed. Real-time PCR was used to assess ER stress gene expression. Protein expression was validated by Western blot and immunocytochemistry (FHL 124 cells and capsular bags). Results: 30 M H2O2 exposure to FHL 124 cells caused a significant reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited (~80%) by 24 hours pre-treatment with 1 M SFN. In addition, 1 M SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole genome HT-12 v4 beadchip microarray data revealed 8 genes up-regulated following 24 hours exposure to 1 M or 2 M SFN. These included NQO1 and TXNRD1, which also demonstrated upregulation at the protein level. Nrf2 was found to actively translocate to the cell nucleus in response to a four hour exposure to 0.5, 1 and 2 M SFN. SFN was found to induce apoptosis and ER stress in FHL 124 cells at SFN concentrations 10 M and higher. Application of these concentrations of SFN to capsular bag cultures demonstrated a retardation of cell growth at 10 M and complete ablation with 100 M SFN Conclusions: The dietary component SFN, at low concentrations (≤5 M) demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract. It is likely that this protection is mediated by SFN induced Nrf2 signalling. Higher concentrations (≥10 M) of SFN are capable of inducing ER stress and cell death in human lens epithelial cells and thus provide a novel agent to be used in the prevention of PCO.
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