The apoptotic and anti-proliferative effect of Lysyl oxidase propeptide in Y79 human retinoblastoma cells.

Purpose: Retinoblastoma (RB) caused by the mutation of the RB1 gene is one of the most common ocular malignancies in children The propeptide region of lysyl oxidase (LOX), the enzyme involved in the cross-linking of collagen and elastin, has been identified to be anti-tumorigenic in various cancers. However, this role of lysyl oxidase propeptide (LOX-PP) in RB is still elusive. This study aims to identify the anti-tumorigenic effect of LOX-PP in human Y79 RB cells. Methods: LOX-PP was overexpressed in Y79 RB cells, and differential gene expression was assessed by microarray followed by pathway analysis using transcriptome analysis console (TAC) software. Additionally, cell proliferation was studied by PrestoBlue assay, and DNA content was evaluated by cell cycle and apoptosis assays. The pro-apoptotic and anti-proliferative mechanisms induced by the overexpression of/exogenously added LOX-PP was evaluated by western blotting and real-time PCR. Results: The expression of the LOX-PP transcript was significantly decreased in Y79 RB cells compared to human retinal endothelial cells. Gene expression analysis in LOX-PP overexpressed Y79 RB cells showed deregulation of pathways involved in apoptosis, cell cycle, focal adhesion-PI3K-AKT signaling, and DNA repair mechanisms. Interestingly, LOX-PP overexpressed Y79 RB cells showed significantly increased apoptosis, decreased proliferation, and cell cycle arrest at S-phase with a concordant reduction of proliferative cell nuclear antigen and Cyclin D1 protein expressions. Moreover, pAKT (S473) was significantly downregulated in Y79 RB cells, which decreased NFκB leading to significantly reduced BCL2 expression. Conclusions: Our results demonstrate the anti-tumorigenic effect of LOX-PP in Y79 RB cells by inducing apoptosis and decreasing proliferation. This effect was mediated by the downregulation of AKT signaling. These results suggest that LOX-PP can be explored as a therapeutic molecule in RB.

Epigenetic alterations associated with dexamethasone sodium phosphate through DNMT and TET in RPE cells.

Purpose: To elucidate the mechanism behind epigenetic alteration associated with dexamethasone (DEX) sodium phosphate treatment. Methods: We performed enzyme-linked immunosorbent assay to quantify changes in global DNA methylation and hydroxymethylation, quantitative real-time PCR (qRT-PCR) of the DNA methylation- and hydroxymethylation-related gene, in vitro DNA methyltransferase (DNMT) enzymatic activity assays with purified DNMTs, and DNA hydroxymethylation pattern with super-resolution imaging. Results: We identified global DNA hypomethylation and hyper-hydroxymethylation upon DEX treatment, associated with aberrant mRNA expression levels of DNMT and ten-eleven translocation (TET) proteins. Additionally, DEX exposure could directly hinder DNMT activities. Conclusions: We showed that DEX-induced epigenetic alterations are linked to aberrant DNMT and TET expression, potentially through an essential role of DNMT.