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. 2017 Jul 17;7(1):5599.
doi: 10.1038/s41598-017-06098-y.

MLN4924 (Pevonedistat), a protein neddylation inhibitor, suppresses proliferation and migration of human clear cell renal cell carcinoma

Shuai Tong  1   2 , Yang Si  1   2 , Hefen Yu  1   2 , Lingqiang Zhang  3 , Ping Xie  4   5 , Wenguo Jiang  6   7   8
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VSports最新版本 - MLN4924 (Pevonedistat), a protein neddylation inhibitor, suppresses proliferation and migration of human clear cell renal cell carcinoma

"V体育官网" Shuai Tong et al. Sci Rep. .

Abstract

Neddylation is a post-translational protein modification associated with cancer development. MLN4924 is a neddylation inhibitor currently under investigation in multiple phase I studies on various malignancies, and its clincal name is Pevonedistat. It has been documented that MLN4924 blocks Cullins neddylation and inactivates CRLs and, in turn, triggers cell-cycle arrest, apoptosis, senescence and autophagy in many cancer cells. In this study, we investigated the anti-tumor effect of MLN4924 in human clear cell renal carcinoma (ccRCC). Levels of both Nedd8 activating enzyme E1 and Nedd8-conjugating enzyme E2 were higher in ccRCC tissues and RCC cancer cells than in normal. Moreover, MLN4924 treatment led to rapid inhibition of Cullin1 neddylation and notably suppressed growth and survival as well as migration in a dose-and time-dependent manner. Mechanistic studies revealed that MLN4924 induced the accumulation of a number of CRL substrates, including p21, p27 and Wee1 to trigger DNA damage and induce growth arrest at the G2/M phase. MLN4924 also induced anti-migration and anti-invasion by activating E-cadherin and repressing Vimentin. Taken together, this study provides the first evidence that neddylation pathway is overactive in ccRCC and that MLN4924 induces dose-dependent anti-proliferation, anti-migration, anti-invasion in ccRCC cells. The study thus indicates that MLN4924 has potential therapeutic value for the clinical treatment of renal cancer VSports手机版. .

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VSports手机版 - Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Expression of neddylation pathway proteins and MLN4924 effectively inhibited Cullin1 neddylation in human ccRCC cells. (a) Neddylation pathway proteins APP-BP1 (E1) and Ubc12 (E2) were analyzed by western blot using antibodies against indicated proteins. (b) The mRNA level of APP-BP1, Ubc12 and Cullin1 were analyzed in TCGA-KIRC dataset. Significance between the 2 populations was determined with a two tailed t-test. (c,d) Cells were treated with MLN4924 at indicated doses and for 12 hrs before being subjected to Western blot analysis. (e) Cells were treated with MLN4924 at 1 μM and for indicated dose-time before being subjected to Western blot analysis.
Figure 2
Figure 2
MLN4924 reduces human ccRCC cell viability. (a) Cells were treated with serial dilutions of MLN4924 for 72 hrs and cell viability was determined using CCK8 assays. Representative inhibitory curves from three independent experiments are shown for each cell lines. (b) Cells were seeded into 6-well plates petri-dishes at 500 cells per dish in triplicate and treated with MLN4924 for 12 days, followed by 0.01% (w/v) crystal violet staining and colony counting. Representative images of three independent experiments are shown for colony formation. (c) Graph of the relative number of colonies formed (the results of three independent experiments, expressed as mean ± SEM). (p < 0.05).
Figure 3
Figure 3
MLN4924 induces accumulation of CRL substrates in ccRCC cells. (a,c) Cells were treated with increasing concentrations of MLN4924 as indicated. Levels of p21, p27, Wee1 and γ-H2AX were examined by western blotting analysis in the whole cell lysates. (b) MLN4924 arrests cell cycle in the G2 phase in ccRCC cells. Cells were treated with DMSO control or MLN4924 at indicated concentrations for 12 hrs before subjected to flow cytometry assays. (d) MLN4924 led to DNA damage in ccRCC cells. ACHN cells were treated with MLN4924 at 1 μM and for 12 hrs, γ-H2AX staining was detected by immunofluorescence. (e) ACHN cells were treated with 1 μM MLN4924 for 12 hrs. The DNA tail moment for each experimental condition was quantified by alkaline comet assay. Representative images are shown. (the results of three independent experiments, expressed as mean ± SEM). (p < 0.05).
Figure 4
Figure 4
MLN4924 induces cell apoptosis in ccRCC cells. (a) Cells were treated with MLN4924 for 12 hrs, stained with Annexin-V-FITC and PI, and examined with flow cytometry assays. The percentages of ACHN and A498 cells in apoptosis are shown in (b). (c) Cells were treated with MLN4924 for 12 hrs, and levels of actived-caspase-3, Bax and Noxa were analyzed by western blotting in the whole cell lysates.
Figure 5
Figure 5
MLN4924 suppressed migration by up-regulating E-cadherin and down-regulating Vimentin. (a) Cells were treated with indicated concentrations of MLN4924 for 12 hrs before being subjected to Transwell migration analysis. Shown are representative images (b) or mean ± SD from 200 cells per well in triplicates. Similar results were obtained in three independent experiments. (c) Cells were treated with MLN4924 at indicated concentrations and subjected to Western blot analysis. (d) Cells were treated with MLN4924 at indicated concentrations, followed by total RNA isolation and qRT-PCR analysis for E-cadherin. Shown is mean ± SD. Similar results were obtained in three independent experiments.
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