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. 2022 Jan-Dec:31:9636897221077921.
doi: 10.1177/09636897221077921.

Celecoxib Synergistically Enhances MLN4924-Induced Cytotoxicity and EMT Inhibition Via AKT and ERK Pathways in Human Urothelial Carcinoma

Shida Xiong  1 , Wei Huang  1 , Xiaoqiang Liu  1 , Qian Chen  1 , Yi Ding  1 , Haoxuan Huang  1 , Ru Zhang  2 , Ju Guo  1
Affiliations

Celecoxib Synergistically Enhances MLN4924-Induced Cytotoxicity and EMT Inhibition Via AKT and ERK Pathways in Human Urothelial Carcinoma

Shida Xiong et al. Cell Transplant. 2022 Jan-Dec.

Abstract

MLN4924 is a specific small-molecule inhibitor of NEDD8-activating enzyme (NAE) that blocks the neddylation modification cascade. Several I/II/III clinical trials suggested that MLN4924 exerts an antitumor effect against various malignancies. However, recent studies have also found that MLN4924 activates the PI3K/AKT and MAPK/ERK signal pathways, important regulators of tumorigenesis, and drug resistance in human urothelial carcinoma (UC). This study examined the synergistic effect of celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, on MLN4924-induced cytotoxicity and epithelial-mesenchymal transition (EMT) inhibition via AKT and ERK pathways in human UC. We performed both in vitro and in vivo experiments. Briefly, a combination of MLN4924 and celecoxib reduced the protein expression of p-AKT(S473) and p-ERK in UC cell lines. Moreover, celecoxib shifted the half-maximal inhibitory concentration (IC50) curve of MLN4924 to the left, and the combinational effect of MLN4924 and celecoxib showed significant synergism in T24 and 5637 cells. Also, celecoxib enhanced the MLN4924 antitumor effects of inhibiting UC cell growth, colony formation, migration, invasion, and inducing apoptosis. In addition, celecoxib potentiated the MLN4924-induced EMT, decreased the expression of N-cadherin and vimentin, and activated the expression of E-cadherin. Celecoxib also increased the expression of pro-apoptosis proteins PARP and BAX and reduced the expression of antiapoptosis protein Bcl2. In vivo study indicated that the combination of MLN4924 and celecoxib synergistically suppressed the tumor growth in a UC xenograft nude-mice model, which was further supported by immunohistochemistry of tumor tissues. To sum up, our study revealed that celecoxib synergistically enhanced MLN4924-induced cytotoxicity and EMT inhibition in UC. It also inhibited the activation of AKT and ERK pathways, which were activated by MLN4924. These discoveries provide a new drug combination strategy for UC treatment.

Keywords: EMT; MLN4924; celecoxib; neddylation; urothelial carcinoma.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Celecoxib suppresses the activation of AKT and ERK signal pathways which activated by MLN4924, and enhances the suppression of growth in UC cells by MLN4924 (A–B) T24 and 5637 cells were incubated with various concentration of MLN4924 (0, 0.1, 0.3, 1 μM) with or without celecoxib (60 μM) for 24 h, followed by Western blotting using antibodies against p-AKT (S473), AKT, ERK, and p-ERK. β-actin was used as a loading control. (C–D) A total of 4000 T24 cells and 6000 5637 cells were seeded in triplicate in 96-well plates and treated with various concentrations of celecoxib, MLN4924, or the combination of MLN4924 and celecoxib (50 μM) for 48 h, followed by the CCK-8 assay (mean ± SEM, n = 3). (E) CI-fraction affected plot of the MLN4924/celecoxib combination in T24 and 5637 cells assessed with Compusyn Software (Fa, corresponding to the fraction of cell viability, the synergistic effect was considered if CI < 1, and additive if CI = 1, antagonistic if CI > 1). UC: urothelial carcinoma; CCK-8: cell counting kit-8; SEM: standard error of the mean; CI: combination index. **0.005 < P < 0.01, ***P < 0.005.
Figure 2.
Figure 2.
Celecoxib enhances the suppression of survival in UC cells by MLN4924. (A–B) A total of 300 T24 cells and 600 5637 cells were seeded in six-well plates and treated with various concentration of MLN4924 alone or in combination with celecoxib (40 μM). T24 and 5637 cell colonies were stained and counted after 7 and 12 days, respectively (>50 cells in a colony). Rate of colony formation (%) = colony number/(300 or 500) × 100% (mean ± SEM, n = 3, *0.01 < P < 0.05, **0.005 < P < 0.01, ***P < 0.005). UC: urothelial carcinoma; SEM: standard error of the mean.
Figure 3.
Figure 3.
Celecoxib enhances the induction of apoptosis induced by MLN4924. (A–B) Cells were treated with MLN4924 alone or in combination with celecoxib. After 48 h, the cells were harvested and stained using the FITC-Annexin V/PI apoptosis detection kit. Cells with Annexin V+ and PI + staining located in the right upper and lower quadrants were considered as apoptotic cells (mean ± SEM, n = 3, *0.01 < P < 0.05, **0.005 < P < 0.01, ***P < 0.005). (C) Cells were incubated with various concentrations of MLN4924 (0, 0.1, 0.3, 1 μM), with or without celecoxib (60 μM) for 24 h. Then cells were harvested for Western blotting using indicated antibodies. SEM: standard error of the mean; DMSO: dimethyl sulfoxide.
Figure 4.
Figure 4.
Celecoxib enhances the suppression of migration and invasion in UC cells by MLN4924. (A) T24 and 5637 cells were seeded in six-well plates and treated with MLN4924 (0.5 μM) with or without celecoxib treatment (50 μM) for 24 h. After serum starvation for 12–18 h, used a pipette tip to scratch across the well. The T24 and 5637 cells were photographed at 24 and 48 h, respectively. Data were shown as the relative wound area normalized to the control. (B) A total 3 × 105 T24 cells and 6 × 105 5637 cells were treated with the indicated agents and then seeded into the upper chamber containing serum-free medium. Then, T24 and 5637 cells were fixed and stained after 24 and 48 h, respectively, followed by photography and counting. (C) Cells were treated with the drugs for 24 h, followed by Western blotting using antibodies against N-cadherin, E-cadherin, and vimentin (mean ± SEM, n = 3, *0.01 < P < 0.05, **0.005 < P < 0.01, ***P < 0.005). UC: urothelial carcinoma; SEM: standard error of the mean; DMSO: dimethyl sulfoxide.
Figure 5.
Figure 5.
The xenograft model demonstrates the efficacy of the combination of MLN4924 and celecoxib in vivo. (A–B) Nude mice bearing T24 xenograft tumors were treated with DMSO (as control), MLN4924, celecoxib, or the MLN4924/celecoxib combination for 4 weeks (n = 6 for each group, one tumor has disappeared after given the combinational drugs for 1 week). Tumor volume = longest tumor diameter × (shortest tumor diameter)/2. (C–D) Tumors were fixed in 10% formalin and embedded in paraffin. Each sample was stained for Ki67 and cleaved caspase-3 with representative images shown. Positive cells were counted from three independent tumors in each group (mean ± SEM, n = 3, *0.01 < P < 0.05, **0.005 < P < 0.01, ***P < 0.005). SEM: standard error of the mean; DMSO: dimethyl sulfoxide.
Figure 6.
Figure 6.
Mechanism of action. MLN4924 treatment blocks neddylation by inactivation of NAE; yet, it also activates AKT and ERK pathways. Celecoxib downregulates the AKT and ERK, synergistically enhancing MLN4924-induced suppression of growth, survival, and EMT, and inducing apoptosis via Bcl2 signal pathways of UC cells. The combination of MLN4924 with celecoxib in vivo significantly suppresses the growth of UC by increasing the expression of caspase 3 and decreasing the expression of Ki67. NAE: NEDD8-activating enzyme; EMT: epithelial–mesenchymal transition; UC: urothelial carcinoma.
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References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70(1):7–30. - PubMed
    1. Tan WS, Rodney S, Lamb B, Feneley M, Kelly J. Management of non-muscle invasive bladder cancer: a comprehensive analysis of guidelines from the United States, Europe and Asia. Cancer Treat Rev. 2016;47:22–31. - PubMed
    1. Rajalingam K, Dikic I. SnapShot: expanding the ubiquitin code. Cell. 2016;164(5):1074.e1071. - PubMed
    1. Morrow JK, Lin HK, Sun SC, Zhang S. Targeting ubiquitination for cancer therapies. Future Med Chem. 2015;7(17):2333–50. - PMC - PubMed
    1. Mansour MA. Ubiquitination: friend and foe in cancer. Int J Biochem Cell Biol. 2018;101:80–93. - PubMed

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