Korean J Physiol Pharmacol.  2018 Nov;22(6):617-625. 10.4196/kjpp.2018.22.6.617.

The Nedd8-activating enzyme inhibitor MLN4924 suppresses colon cancer cell growth via triggering autophagy

• Lv Y
• Li B
• Han K
• Xiao Y
• Yu X
• Ma Y
• Jiao Z
• Gao J

• KMID: 2430084
• DOI: http://doi.org/10.4196/kjpp.2018.22.6.617

Abstract

Neddylation is a post-translational protein modification process. MLN4924 is a newly discovered pharmaceutical neddylation inhibitor that suppresses cancer growth with several cancer types. In our study, we first investigated the effect of MLN4924 on colon cancer cells (HCT116 and HT29). MLN4924 significantly inhibited the neddylation of cullin-1 and colon cancer cell growth in a time and dose-dependent manner. MLN4924 induced G2/M cell cycle arrest and apoptosis in HCT116 and HT29 cells. Moreover, MLN4924 also triggered autophagy in HCT116 and HT29 cells via suppressing the PI3K/AKT/mTOR pathway. Inhibiting autophagy by autophagy inhibitor 3-MA or ATG5 knockdown reversed the function of MLN4924 in suppressing colon cancer cell growth and cell death. Interestingly, MLN4924 suppresses colon cell growth in a xenograft model. Together, our finding revealed that blocking neddylation is an attractive colon cancer therapy strategy, and autophagy might act as a novel anti-cancer mechanism for the treatment of colon cancer by MLN4924.

Keyword

Autophagy; Cell growth; Colon cancer; MLN4924; Neddylation

MeSH Terms

Apoptosis
Autophagy*
Cell Cycle Checkpoints
Cell Death
Colon*
Colonic Neoplasms*
Heterografts
HT29 Cells
Humans
Protein Processing, Post-Translational

Figure

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  Fig. 1 MLN4924 inhibits the neddylation of cullins-1 and the cell proliferation in colon cancer cells (A) MLN4924 inhibits cullins-1 neddylation. HCT116 and HT29 cells were treated with vehicle or 0.3 µM MLN4924 for 12, 24 or 48 h. The expression of cullins-1 and cullins-1-nedd8 was determined by western blot. GAPDH was used for loading control. (B) HCT116 and HT29 cells were treated with vehicle or different concentration of MLN4924 for 24, 48 or 72 h. The cell proliferation was measured by BrdU assays. (C) MLN4924 inhibited cell clonogenic ability. HCT116 and HT29 cells were seeded in 6-well culture plate at 1000 cells per well and treated with vehicle or MLN4924 for 14 days, followed by crystal violet staining and colony counting. The statistical analysis results were shown in the right panels. All values were represented as means±SD calculated from three independent experiments. ***p<0.001.

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  Fig. 2 MLN4924 induced G2 cell cycle arrest and apoptosis in colon cancer cells HCT116 and HT29 cells were treated with vehicle or 0.3 µM MLN4924 for 12, 24 or 48 h. The expression of p-H2A, T-H2A, p-CHK2, T-CHK2, p21 and p27 was determined by western blot. (B) HCT116 and HT29 cells were treated the same as in (A) and were analyzed by FACS after staining with propidium iodide for cell cycle analysis at 24 h. (C) HCT116 and HT29 cells were treated the same as in (A) and analysis of apoptosis by FACS using AnnexinV/PI double-staining at 12, 24 or 48 h. (D) HCT116 and HT29 cells were treated the same as in (A). The expression of cleaved caspase-3 and cleaved PARP was determined by western blot. All values were represented as means±SD calculated from three independent experiments. **p<0.01, ***p<0.001.

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  Fig. 3 MLN4924 induced autophagy in colon cancer cells (A) HCT116 and HT29 cells were treated with vehicle or 0.1 µM MLN4924 for 48 h, then they were analyzed by transmission electron micrograph (TEM), the black arrows indicated the autophagosomes. (B) HCT116 and HT29 cells were treated with vehicle or 0.3 µM MLN4924 for 12, 24 or 48 h. The expression of LC3 and p62 was determined by western blot. GAPDH was used for loading control. (C) HCT116 and HT29 cells were transfected with GFP-mRFP-LC3 vector then they were treated with vehicle or 0.3 µM MLN4924 for 24 h. Images were took with a confocal microscope and the merged region enclosed within the white square has been enlarged in the right panel.

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  Fig. 4 The PI3K/Akt/mTOR pathway contributes to MLN4924 triggered autophagy HCT116 and HT29 cells were treated with vehicle or 0.3 µM MLN4924 for 12, 24 or 48 h. The expression of p-AKT, p-mTOR and p-70S6K were determined by western blot. GAPDH was used for loading control.

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  Fig. 5 Autophagy is involved in MLN4924-induced cell growth inhibition in colon cancer cells. (A) HCT116 and HT29 cells were treated with 3-MA (3 mM) in the absence or presence of 0.3 µM MLN4924 for 24, 48 or 72 h, the cell proliferation was measured by BrdU assays. (B) HCT116 and HT29 cells were treated the same as in (A) for 48 h, The expression of LC3, p62, cleaved caspase-3 and cleaved PARP was determined by western blot. GAPDH was used for loading control (C) HCT116 and HT29 cells were treated the same as in (A) for 48 h, double stained with Annexin V/PI and analyzed by FACS. (D-F) HCT116 and HT29 cell lines with stable depletion of ATG5 were treated with vehicle or 0.3 µM MLN4924 for 48 h. (D) The cell proliferations was measured by BrdU assays. (E) The expression of ATG5, cleaved caspase-3 and cleaved PARP was determined by western blot. GAPDH was used for loading control. (F) Double stained with Annexin V/PI and analyzed by FACS. All values were represented as means ± SD calculated from three independent experiments. ***p<0.001.

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  Fig. 6 MLN4924 suppresses colon cancer cell growth in mouse tumor xenograft. Six weeks old female nude mice bearing HCT116 and HT29 tumors were randomly divided into two groups (ten mice per group): (A) vehicle control, (B) intraperitoneal administration with MLN4924 (60 mg/kg) three times per week. (A) Tumor volumes were measured at 5-day intervals for 40 days and expressed as the mean±SD (n=10) in tumor volume-time curves. (B) six weeks after treatment, tumor tissue samples from each treatments group was subjected to either hematoxylin-eosin (H&E) staining or TUNEL assay.

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