FHL2 as a co-factor of XAF1 in the induction of apoptosis

Abstract

BACKGROUND AND AIMS: Previous studies have shown that XIAP-associated factor 1 (XAF1) plays an important role in resistance of cancer cells to apoptosis. The aim of this study is to further understand the molecular mechanisms by identifying XAFl-interacting proteins. METHODS: Yeast two-hybrid assay was used to identify Four and a Half of LIM-only protein 2 (FHL2) as an XAFl-interacting protein from an ovary cDNA library. The interaction was confirmed by co-immunoprecipitation and GST-protein pull down assays. The effects of FHL2 and XAF1 on cell viability and apoptosis were determined by transient transfection of the expression plamids into AGS gastnc cancer cells and SWill6 colon cancer cells. Transfected cells were exposed to 10-160 ng/ml of TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand). Cell viability was determined by trypan blue exclusion assay or by MTT assay. Apoptosis rate was estimated by DAPI staining. RESULTS: We confirmed the specificity of the binding between XAF1 and FHL2 by direct in vitro binding test and co-immunoprecipitation assay from cell lysates. Ectopic over-expression of XAF1 or FHL2 alone did not significantly affect cell viability at 72h after serum withdrawal. However, co-expression of XAF1 and FHL2 resulted in a significant reduction of cell viability relative to control mock transfected ceils (reduced to 47% from 81% in AGS, p < 0.001; to 52% from 82% in SW1116, p < 0.001). After exposure to 80ng/ml of TRAIL, co-expression of XAF1 and FHL2 also significantly reduced cell viability by MTT assay as compared to control cells (44% vs 90% in AGS, p < 0.001; 59% vs 91% in SW1116, p < 0.001). In addition, they also increased cellular sensitivity to TRAIL-induced apoptosis as compared to control cells (40% vs 11% in AGS, p < 0.001; 28% vs 11% in SW1116, p= 0.001). CONCLUSIONS: We identified FHL2 as a novel XAFl-interacting protein and showed that FHL2 works together with XAF1 to induce cellular apoptosis.