Potentiation of bortezomib-induced apoptosis by TGF-beta in cultured human Tenon's fibroblasts: contribution of the PI3K/Akt signaling pathway.
OBJECTIVE
To investigate the effects of bortezomib on cell apoptosis and proliferation in human Tenon's capsule fibroblasts (HTFs) after cotreatment with TGF-β.


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-β. The apoptotic effect of cotreatment of bortezomib and TGF-β 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 μM) and TGF-β (10 μM) increased the proportion of apoptotic cells in HTFs on FACS analysis, whereas either bortezomib or TGF-β treatment alone did not. The MTT assay also showed that when bortezomib was cotreated with TGF-β, the cell proliferation of HTFs induced by TGF-β treatment was significantly decreased at 72-hour incubation. The cotreatment of bortezomib and TGF-β specifically decreased the Akt phosphorylation induced by TGF-β, 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-β 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-β is elevated, as is observed in the postoperative period.
Chung
Eun Jee
EJ
Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyounggi-do, Korea.
Moon
Sang Woong
SW
Jung
Sun-Ah
SA
Cho
Young Jae
YJ
Kim
Si Wouk
SW
Lee
Joon H
JH
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D019254:Genes, bcl-2 / Q000235:genetics; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D058475:Tenon Capsule / Q000473:pathology; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000235:genetics
C494826:BAX protein, human; D001897:Boronic Acids; D011480:Protease Inhibitors; D011719:Pyrazines; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D000069286:Bortezomib; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; C022616:thiazolyl blue