Abstract 5664: Molecular effects of doxycycline treatment on pterygium as revealed by massive transcriptome sequencing
Authors: Ignacio M. Larrayoz, Alberto de Luis, Oscar Rua, Sara Velilla, Juan Cabello, Alfredo Martínez
Journal: Cancer Research (2012)

Abstract Pterygium is a common benign tumor growth of the ocular surface attributed to chronic ultraviolet light exposure, representing a tremendous burden, both human and financial, in many countries. At the cellular level, pterygium is characterized by proliferation of limbal cells, inflammatory infiltrates, fibrosis, angiogenesis, and extracellular matrix breakdown. Pterygia also display tumor-like features, such as propensity to invade normal tissue and high recurrence rates following resection. Currently, the only approved treatment for pterygium is surgery which, in some cases, results in high recurrence rates. In a recent article, our group has shown that doxycycline, a common oral antibiotic, was able to dramatically reduce pterygium-like lesions in a mouse model and we are nowadays conducting a clinical trial to test this ability in humans. In the present study we investigated the molecular effects of doxycycline on short-term primary cultures of pterygium cells, using ultrasequencing techniques, to better understand all the pathways affected by the antibiotic in pterygium. Use of pterygium specimens was approved by our Institutional Review Board (CEICLAR). For each patient, cells were subjected to 4 treatments: 0 (control), 50, 200, and 500 μg/mL doxycycline for 24 h. Total RNA was isolated and a library was prepared for ultrasequencing following Illumina's protocols. Gene products whose expression was significantly changed upon application of doxycycline were chosen for further confirmation by quantitative real time PCR using Taqman probes. Some secreted proteins were quantified in the supernatant of the cultures by commercially available ELISA kits Our results showed that the exposure of pterygium cells to doxycycline induced dose-dependent changes in mitochondrial-related genes as well as ER stress-related genes, VEGF signalling, and integrin related genes in pterygium. Twenty two of the differentially-induced genes identified by ultrasequencing were selected for confirmation by qRT-PCR. All of them showed a similar dose dependence on doxycycline treatment. Correlation analysis between ultrasequencing data and qRT-PCR showed a coefficient of correlation (r2) of 0.7. In addition, several cytokines were analysed by ELISA confirming the validity of the technique. The doxycycline-activated pathways identified by ultrasequencing techniques provide a plausible mechanism by which doxycycline exerts its anti-proliferative effects on pterygia and unveiled new therapeutic targets for the treatment of the disease. In addition, the high correlation between ultrasequencing and qRT-PCR data supports the use of Massive Transcriptome Sequencing as a useful and powerful tool to study the mechanism underlining complex diseases. Supported by a Grant from Spain's Ministry of Health, Social Policy, and Equality, No. DIN-021. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5664. doi:1538-7445.AM2012-5664