The Effect Of XAF1 On Cell Growth And The Underlying Mechanism In Gastric Cancer Cell Line, AGS

BackgroundInhibitor of apoptosis (IAPs) proteins belong to an important anti-apoptotic protein family that play central role in the carcinogenesis and resistance to cancer therapy. (—|X)-linked inhibitor of apoptosis protein (XIAP) is a member of IAPs family of caspase inhibitors that selectively binds and inhibits caspases-3, -7 and -9. Its anti-apoptotic activity can be negatively regulated by an interacting protein, XAF1 (XIAP-associated Factor 1). XAF1 has been implicated as a tumour suppressor gene and is expressed by all normal adult and fetal tissues However, its expression is absent or only at low level in cancer cells. XAF1 can directly inhibite the anti-caspase activity of XIAP. However, the direct effect of XAF1 on cell growth and the underlying mechanism have not been studied. In this study, we established stable transfectants expressing XAF1 to detect the effect of XAF1 on AGS cell growth and the putative mechanism.MethodsTransfectants of AGS cell stably expressing XAF1 and vector control were established, named as AGS/XAF1 and AGS/vector respectively, and identified by detection of XAF1 expression with western-blot. AGS cells were synchronized with double thymidine treatment. Cell cycle distribute was evaluated by flow cytometry while XAF1 expression was decected by western-blot. MTT assay was performed to assess the effect of XAF1 on cell proliferation. Cell cycle distribution of AGS/XAF1 and AGS/vector were also analyzed by flow cytometry. Cells transiently or stably transfected with XAF1 were observed under fluorescence microscopy after staining with MPM-2, a specific antibody that reconized mitosis specific phosphoy lated proteins. The stable transfectants were stained with both Hoechst 22358 and anti-α-tubulin to discriminate nucleus and cytoplasm, while anti-γ-tubulin was used to stain the centrosome. Cell apoptosis was assessed by flurorescence microscopy and flow cytometry. The activation of caspase-3 was dectected by western-blot.ResultsThe stable transfectants were established, the expression of XAF1 was increased in AGS/XAF1 and decreased in AGS/XAF1-AS. XAF1 protein was specifically expressed in G₂/M stage as determined in synchronized cells. MTT assay showed that over-expression of XAF1 suppressed serum-dependent AGS cell growth. Over-expression of XAF1 induced about 75% of cells arrested in G₂/M. Typical morphological features of apoptosis was not found. In contrast about 50% of cells with over-expression of XAF1 displayed multiple nuclei as judged by staining with Hoechst 22358. About 25% of AGS/XAF1 transfectant cells displayed more than three centrosomes per cell including a significant proportion with more than 10 centrosomes. When cells was treated with serum-deprivation, the AGS/XAF1 cells displayed a higher percentage of apoptotic cells and increased cleaved caspase-3 expression in comparison with vector control.ConclusionsXAF1 was specifically expressed in G₂/M stage. Over-expression of XAF1 suppressed serum-dependent cell growth, induced cell accumulation in mitosis, G₂/M cell cycle arrest and mitotic catastrophe. Over-expression of XAF1 increased the susceptibility of AGS cell to serum-deprivation-induced apoptosis. XAF1 promoting apoptosis may be secondary to mitotic catastrophe, implicating a novel XIAP-independent function pathway of XAF1. Our findings suggested that XAF1 may be a potential target for the therapy of gastrointestinal cancers.