RPE Cell Apoptosis by the Combination Treatment of SAHA and Lactacystin

ABSTRACT

PURPOSE: To establish a new therapeutic strategy for proliferative vitreoretinopathhy (PVR), we examined the effect of combined treatment with HDAC inhibitor SAHA and proteasome inhibitor lactacystin in human retinal pigment epithelial (RPE) cells, ARPE-19. METHODS: Viability was determined by trypan blue exclusion assay. Mitochondrial membrane potential (MMP) was measured by flow cytometry. Proteasome activity was measured by fluorophotometry. The expression and degradation of apoptosis-related proteins were assesssed by Western blotting. Subcellular location of apoptosis-related factors was monitored by confocal miscroscopy. RESULTS: A single treatment with 5 micro M SAHA or 10 micro M lactacystin did not reduce cell viability. However, combination treatment with 5 micro M SAHA and 10 micro M lactacystin substantially reduced the viability, because the mixture induced the reduction of MMP and nuclear condensation or fragmentation. Moreover, the combination treatment triggered the activation of caspase-3 and the production of PARP cleavage products. These data indicate that the combination treatment efficiently induces apoptosis in ARPE-19 cells. However, co-treatment of SAHA did not augment the proteasome inhibitory activity of lactacystin, nor did co-treatment of lactacystin augment acetylation of histones. It is notable that while p53 and CAD were observed in the mitochondria of cells treated with SAHA, they were translocated into the nucleus after the combination treatment. CONCLUSIONS: These results suggest that the combination treatment of SAHA and lactacystin effectively induced apoptosis in ARPE-19 cells. Further work is warranted to develop this combination therapy as a novel therapeutic strategy for PVR.