title: MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies.
abstract: 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.
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Kuan Lin
authors.initials: KL
authors.lastname: Kuo
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: I Lin
authors.initials: IL
authors.lastname: Ho
authors.affiliation: Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Chung Sheng
authors.initials: CS
authors.lastname: Shi
authors.affiliation: Graduate Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: June Tai
authors.initials: JT
authors.lastname: Wu
authors.affiliation: Department of Pathology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Wei Chou
authors.initials: WC
authors.lastname: Lin
authors.affiliation: Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Yu Chieh
authors.initials: YC
authors.lastname: Tsai
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Hong Chiang
authors.initials: HC
authors.lastname: Chang
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Chien Tso
authors.initials: CT
authors.lastname: Chou
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Chen Hsun
authors.initials: CH
authors.lastname: Hsu
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Ju Ton
authors.initials: JT
authors.lastname: Hsieh
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Shih Chen
authors.initials: SC
authors.lastname: Chang
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Yeong Shiau
authors.initials: YS
authors.lastname: Pu
authors.affiliation: Department of Urology, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan. Electronic address: khhuang123@ntu.edu.tw.
authors.forename: Kuo How
authors.initials: KH
authors.lastname: Huang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002277:Carcinoma / Q000188:drug therapy* / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D003517:Cyclopentanes / Q000008:administration & dosage*; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D000075687:NEDD8 Protein; D011743:Pyrimidines / Q000008:administration & dosage*; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D014452:Ubiquitins / Q000037:antagonists & inhibitors / Q000235:genetics; D014571:Urologic Neoplasms / Q000188:drug therapy*; D023041:Xenograft Model Antitumor Assays
chemical_list: D003517:Cyclopentanes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011743:Pyrimidines; D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human; C539933:pevonedistat
keywords: Apoptosis; MLN4924; Neddylation; Urothelial carcinoma