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J. Zhang
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-063 - XIAP Inhibits Mature Smac Induced Apoptosis by Degrading It through Ubiquitination in NSCLC (ID 5287)
14:30 - 14:30 | Author(s): J. Zhang
- Abstract
Background:
X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancers. However, their relationship is still unknown in lung cancer.
Methods:
RT-PCR and Western blot were performed to explore the correlation between Smac and XIAP at the level of mRNA and protein in NSCLC patients. Full-length XIAP, Smac and mature Smac were generated by PCR and cloned into pcDNA3.0-Flag/Myc. The location of mature Smac and full-length Smac was detected by immunofluorescence. MTT assay and Flow cytometry were detected the cell viability and apoptosis of transfected cells. Caspase-3 activity was masured by Caspase-3 activity assay. Co-immunoprecipitation assay was done to reveal the direct relation between XIAP and Smac. Nude mouse xenograft experiment further proved the relation and the function of Smac and XIAP in vivo.
Results:
In this study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspases-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it.
Conclusion:
In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC.
