Potentiation of Bortezomib-Induced Apoptosis by TGF-β in Cultured Human Tenon's Fibroblasts: Contribution of the PI3K/Akt Signaling Pathway

PURPOSE. To investigate the effects of bortezomib on cell apoptosis and proliferation in human Tenon's capsule fibroblasts (HTFs) after cotreatment with TGF-beta. METHODS. The effect of bortezomib on the apoptosis and cell proliferation of cultured HTFs was determined with FACS analysis and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assay with or without cotreatment of TGF-beta. The apoptotic effect of cotreatment of bortezomib and TGF-beta through the PI3K/Akt pathway was determined by Western blot analysis. The mRNA level of Bcl-2 and Bax was determined by RT-PCR. p53 expression, DNA-PKcs cleavage, and c-Jun phosphorylation were determined. RESULTS. Cotreatment with bortezomib (5 mu M) and TGF-beta (10 mu M) increased the proportion of apoptotic cells in HTFs on FACS analysis, whereas either bortezomib or TGF-beta treatment alone did not. The MTT assay also showed that when bortezomib was cotreated with TGF-beta, the cell proliferation of HTFs induced by TGF-beta treatment was significantly decreased at 72-hour incubation. The cotreatment of bortezomib and TGF-beta specifically decreased the Akt phosphorylation induced by TGF-beta, indicating on Western blot analysis that these changes are mediated by the PI3K/Akt pathway. The mRNA level of an apoptosis-related factor, Bcl-2, was significantly reduced, and p53 expression, DNA-PKcs cleavage, and c-Jun phosphorylation were increased after cotreatment. CONCLUSIONS. Bortezomib-induced apoptosis is potentiated by TGF-beta cotreatment in cultured HTFs by inhibition of the PI3K/Akt pathway, indicating that the effect of bortezomib may be potentiated when the level of TGF-beta is elevated, as is observed in the postoperative period. (Invest Ophthalmol Vis Sci. 2010;51:6232-6237) DOI:10.1167/iovs.10-5747