Microarray Analysis and Biochemical Correlations of Oxidative Stress Responsive Genes in Retinoblastoma

Abstract

Purpose: Oxidative stress, which refers to the biological damage caused by free radicals produced in excess of innate antioxidant defenses, is indicated in the ocular cancer retinoblastoma (RB). Here we have analysed the differential expression of oxidative stress responsive genes in oxidant-induced RB cells, and in RB tumor tissues.

Methods: The study included cultured RB cells, and four RB tumor tissues. The reactive oxygen species (ROS) levels in Y79 cells and the RB tumor induced by hydrogen peroxide were quantified by Dichlorofluorescein (DCF) fluorescence assay. We then analysed the gene expression profile of cultured RB cells induced with hydrogen peroxide (400 µM H₂O₂ for 8 h) by microarray analysis, and the expression of select genes were validated in Y79 cells and RB tumor tissues by real-time PCR analysis.

Results: The oxidant-induced RB tumors showed an average increase in ROS levels of 44-fold compared to induced non-neoplastic donor retina. H₂O₂-induced RB cell line showed a 3-fold increase in ROS levels. Microarray analysis on RB cell line induced with H₂O₂ showed differentially regulated genes involved in cellular processes such as: oxidative stress, angiogenesis, lipid metabolism, cell proliferation, and cell signaling pathways. Several up-regulated genes such as SOD, GPX, CAT, CDC25A, CREBBP, JUN, MMP-2, iNOS, CRYAA, RXRA, ACACB and HMGCR were validated by real-time PCR. These results corroborated with the gene expression analysis in RB tumor tissues. Relating the antioxidant gene expression with the clinico-pathologic features of the tumor tissues, we found that the tumor with invasion of choroid, optic nerve and retinal pigment epithelium, had relatively higher ROS levels and minimal antioxidant gene expression, when compared with the tumor with only choroidal invasion.

Conclusions: The study suggests active involvement of redox signaling pathways in the pathogenesis of RB. Consideration of oxidative stress components in the clinical management of RB patients is emphasized.

KEYWORDS::

• Retinoblastoma
• Oxidative stress
• Reactive oxygen species
• Hydrogen peroxide
• Antioxidant
• Microarray

ACKNOWLEDGMENTS

We thank Ms Kalaivani and Ms Aiyaz (Genotypic Technology Pvt. Ltd.) for microarray analysis and Mr Hitesh, Bionivid Technology for pathway analysis.

Declaration of interest: The authors report no conflicts of interest. We thank the Dept. of Biotechnology (DBT), Ministry of Science and Technology, New Delhi, India for the research grant, Project no: BT/PR13091/GBD/27/180/2009.