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. 2013 Apr 1;19(7):1717-28.
doi: 10.1158/1078-0432.CCR-12-2383. Epub 2013 Feb 12.

The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo

Ning Zhou  1 , Kamini Singh, Maria C Mir, Yvonne Parker, Daniel Lindner, Robert Dreicer, Jeffrey A Ecsedy, Zhongfa Zhang, Bin T Teh, Alexandru Almasan, Donna E Hansel
Affiliations

The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo

Ning Zhou et al. Clin Cancer Res. .

Abstract

Purpose: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target VSports手机版. .

Experimental design: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR V体育安卓版. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. .

Results: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells V体育ios版. .

Conclusions: Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer VSports最新版本. .

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Conflict of interest statement

Conflict of interest statement:

We acknowledge no author relationships that we believe could be construed as resulting in a conflict of interest with regard to the experimental results and interpretations presented in the following manuscript.

"V体育官网" Figures

Figure 1
Figure 1
Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, Human samples of normal urothelium (N=10) and urothelial carcinoma of the bladder (N=8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma compared to the normal urothelium. B, Upregulation of these genes was validated by two-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N=3) and normal urothelium (N=3). 10 of 13 genes (asterisked) demonstrated statistical significance (T-test P-value < 0.05) for differential expression in urothelial carcinoma compared to normal urothelium.
Figure 2
Figure 2
MLN8237 induces cell cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 demonstrated specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, Propidium iodide staining with flow cytometry analysis was performed to assess cell cycle changes. T24, UM-UC-3, and RT4 cells were treated with 10 nM to 1 μM MLN8237 for 48 h. All three cell lines demonstrated dramatic cell cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also demonstrated a considerable increase in aneuploidy, whereas RT4 cells did not.
Figure 3
Figure 3
Cellular phenotypes of T24 and RT4 cells differ after MLN8237 treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, Immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells demonstrate a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic aurora A expression, whereas RT4 cells lacked cytoplasmic aurora A expression. E, Real-time imaging of T24 and RT4 cells treated with MLN8237 was performed over 48 h. T24 cells exhibited dramatic increases in cell size as a result of repeated cell cycle progressions without separation of daughter cells. RT4 cells appeared to become arrested after one failed mitotic attempt, preventing repeated cell cycle progressions that could otherwise result in increased ploidy.
Figure 4
Figure 4
MLN8237 induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 h. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 nM and 45 nM, respectively) and lowest potency in RT4 cells (IC50 of 120 nM). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 h after initiation of treatment. C, Annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 h after initiation of MLN8237 treatment. D, Clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nM MLN8237.
Figure 5
Figure 5
MLN8237 arrests tumor growth in vivo in a mouse xenograft model. A, Mice were treated with the drug at 30 mg/kg doses orally once daily for 4 weeks, and tumor volumes over time were charted. Mice in the treatment group showed arrest of tumor growth throughout the 4 week treatment regimen. Vehicle-treated control mice, on the other hand, showed a dramatic increase in tumor size, and all but one control mice was sacrificed by day 15 due to tumor size. B, Tumors were harvested and stained for hematoxylin/eosin, Ki67 for proliferation, and TUNEL for apoptosis. Tumors treated with MLN8237 showed decreased cellularity and regions of cell death and fibrosis, as well as decreased Ki67 staining and increased TUNEL staining, compared to tumors from control mice. Percentage of cells staining positive for Ki67 and TUNEL were counted in 5 visual fields per group and depicted in the bar graph.
Figure 6
Figure 6
Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 h (left column), or sequentially, with one drug for 48 h, followed by washout and the other drug for 48 h (middle and right columns). MTS assay was utilized to quantify the effect on cell viability of these combination treatments. MLN8237 demonstrated synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right columns), and antagonistic effects when dosed simultaneously (left column).
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References

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