MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been 
reported to elicit an anti-tumor effect on various malignancies. In this study, 
we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma 
(UC) in vitro and in vivo by using three human UC cell lines of various grading 
(T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by 
measuring viability (MTT), proliferation (BrdU incorporation), cell cycle 
progression (flow cytometry with propidium iodide staining) and apoptosis (flow 
cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, 
apoptosis-related molecules, and cell stress-related proteins were examined by 
Western blotting. The influence of tumor cell migration and invasion was 
analyzed by Transwell and wound healing assays. We also evaluated the effects of 
MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that 
MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, 
anti-invasion and apoptosis in human UC cells, accompanied by activations of 
Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of 
phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, 
MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, 
phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun 
activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC 
xenograft mouse model with minimal general toxicity. We concluded that MLN4924 
induces apoptosis and cell cycle arrest, as well as activation of cell stress 
responses in human UC. These findings imply MLN4924 provides a novel strategy 
for the treatment of UC.