Abstract
Purpose :
Pathological neovascularization is a common condition associated with human ocular diseases including the cornea. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) (PAM) pathway is known to regulate numerous cellular functions including angiogenesis. The use of PAM pathway inhibitors (PAMI) could be an alternative approach for corneal neovascularization treatment. However, this pathway has not yet been studied in corneal structures.
Methods :
Human corneal epithelial (HCE) cells were grown in DMEM/F12 medium supplemented with FBS, EGF, insulin, penicillin and streptomycin. After 1 h in medium without supplements, cells were exposed to 5, 10 or 100 nM PAMI (NVP-BEZ235, PI103, LY294002) in serum-free medium for 24 h. Unexposed and 0.001% DMSO-exposed cells were used as controls. Message and protein levels of human PIK3CA, PIK3R1, Akt, mTOR, RPS6KB1, 4EBP1, and ACTB genes were studied by RT-PCR and Western blotting, respectively. Changes in expression of p70 and Akt phosphorylated (P) forms was measured as an indicator of PAMI efficacy. XTT and TUNEL assays were used to determine possible toxicity of PAMI on HCE cells in terms of cell viability or apoptosis, respectively. Data are expressed as mean ± SD, and were analyzed by the Student’s t-test.
Results :
HCE cells expressed detectable levels of PAM target genes as well as total and phosphorylated forms of Akt and p70. Exposure to PAMI affected P-Akt and P-p70 expression in a differential manner depending on the specific PAMI. A decreasing dose-dependency effect was observed for the expression of P-p70 in HCE cells after NVP-BEZ235 exposure. However, exposure to PI103 and LY294002 leaded to erratic expressions of phosphorylated forms. HCE cell viability after PAMI exposure was always higher than 80% for all PAMI concentrations. Apoptotic cells were detected in similar numbers as those detected in control unexposed cells.
Conclusions :
Corneal epithelial cells expressed target PAM genes as well as total and phosphorylated forms of Akt and p70. The PAMI NVP-BEZ235 reduced P-Akt and P-p70 levels without affecting cell viability. These findings indicate that PAM pathway could be a new target for antiangiogenic drugs to treat corneal neovascularization.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.