OBJECTIVES: Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) is a member of the HECT E3 ubiquitin ligases, and is elevated in prostate, bladder and colorectal cancers, and promotes colonic cell population expansion. Up to now, molecular mechanisms of how Nedd4 functions, have not been well understood. MATERIALS AND METHODS: In this study, shRNA was used to reduce expression of Nedd4 in the human prostate carcinoma cell line DU145. To analyse effects of Nedd4 on cell proliferation, MTT and colony formation assays were performed. DAPI staining and FACS analysis were used to investigate outcomes of Nedd4 activity, on apoptosis. Results of Nedd4 expression on lysosomal membrane permeabilization and autophagy were further investigated using acridine orange (AO) staining, immunofluorescence and western blot analysis. RESULTS: We found that in HeLa cells, expression of Nedd4 promoted cell proliferation, whereas its knockdown inhibited colony formation and induced apoptosis in DU145 cells. Furthermore, down-regulation of Nedd4 in DU145 cells promoted lysosomal membrane permeabilization. We also found that down-regulation of Nedd4 inhibited autophagy in both DU145 and A549 cells. Investigation into mechanisms involved revealed that knockdown of endogenous Nedd4 expression notably increased activated mTOR (p-mTOR) levels, which suggests that mTOR signalling was involved in the Nedd4-mediated autophagy. CONCLUSIONS: Our results indicate that expression of Nedd4 promoted cell proliferation and colony formation but prevented apoptosis. Moreover, Nedd4 promoted autophagy and was associated with the mTOR signalling pathway.
OBJECTIVES:Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) is a member of the HECT E3 ubiquitin ligases, and is elevated in prostate, bladder and colorectal cancers, and promotes colonic cell population expansion. Up to now, molecular mechanisms of how Nedd4 functions, have not been well understood. MATERIALS AND METHODS: In this study, shRNA was used to reduce expression of Nedd4 in the humanprostate carcinoma cell line DU145. To analyse effects of Nedd4 on cell proliferation, MTT and colony formation assays were performed. DAPI staining and FACS analysis were used to investigate outcomes of Nedd4 activity, on apoptosis. Results of Nedd4 expression on lysosomal membrane permeabilization and autophagy were further investigated using acridine orange (AO) staining, immunofluorescence and western blot analysis. RESULTS: We found that in HeLa cells, expression of Nedd4 promoted cell proliferation, whereas its knockdown inhibited colony formation and induced apoptosis in DU145 cells. Furthermore, down-regulation of Nedd4 in DU145 cells promoted lysosomal membrane permeabilization. We also found that down-regulation of Nedd4 inhibited autophagy in both DU145 and A549 cells. Investigation into mechanisms involved revealed that knockdown of endogenous Nedd4 expression notably increased activated mTOR (p-mTOR) levels, which suggests that mTOR signalling was involved in the Nedd4-mediated autophagy. CONCLUSIONS: Our results indicate that expression of Nedd4 promoted cell proliferation and colony formation but prevented apoptosis. Moreover, Nedd4 promoted autophagy and was associated with the mTOR signalling pathway.
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