Article Figures & Data
Figures
- Figure 1.
A, Aurora A expression in human neuroblastoma cell lines determined by quantitative RT-PCR (i) and Western blot analysis (ii). Western blots were probed with anti-Aurora A antibody and reprobed with anti-β-actin antibody to show equal protein loading. Breast tumor cell line MCF-7 and normal breast epithelial cell line MCF-10A were used as positive and negative controls for Aurora A expression, respectively. B, analysis of Aurora A expression in neuroblastoma and ganglioneuroma tumor tissue samples by Western blot. Representative neuroblastoma tumor samples are shown, grouped by INSS stage.
- Figure 2.
Aurora A mRNA expression level is significantly associated with decreased progression-free survival in patients with neuroblastoma. A, effect of Aurora A mRNA expression on progression-free survival in all patients in this study (P < 0.0001). B, effect of Aurora A mRNA expression on progression-free survival in INSS stage III and IV patients (P < 0.021).
- Figure 3.
Inhibition of Aurora A expression causes a proliferation defect and enhances chemosensitivity in both S-type and N-type human neuroblastoma cell lines. Aurora A expression in S-type SK-N-AS and N-type SH-SY5Y-Luc cells stably transduced with pSR-Sh-scramble control and pSR-Sh-Aurora A (#1 and #2 sequence). A, cells were analyzed by Western blot after 10 d of puromycin (2 μg/mL) selection. B, inhibition of Aurora A expression causes a proliferation defect. The SK-N-AS and SH-SY5Y-Luc Sh-Control and Sh-Aurora A #2 cell lines were plated in 96-well plates at 1 × 103 cells per well. The Cell Counting Kit-8 tetrazolium salt–based proliferation assay was used to quantify cellular proliferation relative to day 1 absorbance measured at 450 nm. These experiments were done in triplicate and reported as mean ± SD. C, SK-N-AS and SH-SY5Y-Luc Sh-Control and Sh-Aurora A #2 stably transduced cell lines were plated in 0.3% agarose/DMEM on top of a 0.5% agarose/DMEM layer. After 14 d of growth, colonies were stained with MTT (bottom) and colonies >30 cells were counted (top). A similar result was observed using Sh-Aurora A #1 cells (data not shown). D, inhibition of Aurora A expression increases chemosensitivity. SK-N-AS and SH-SY5Y-Luc Sh-Control and Sh-Aurora A #2 cell lines were plated in 96-well plates at 1 × 104 cells per well. After 24 h of growth, all of the cell lines were treated with the indicated micromolar concentration of doxorubicin for the indicated amount of time. Cell viability was determined with the Cell Counting Kit-8 cell viability assay relative to the 0 μmol/L group. All experiments were done in triplicate and statistical significance was determined by Student's t test where P < 0.05 was statistically significant. *, P < 0.05; **, P < 0.001 compared with the Sh-Control group.
- Figure 4.
A, growth-inhibitory effect of Aurora A inhibitor MLN8054 on cultured NB cell lines in vitro. N-type and S-type NB cells were plated in 96-well flat-bottomed plates at a concentration of 5 × 103 or 1 × 104 per well and NB cells were treated with increasing concentrations of MLN8054 for 96 h. Cytotoxic activity was determined by the Cell Counting Kit-8 assay. B, effect of the Aurora A inhibitor MLN8054 on apoptosis and cell cycle distribution in two representative NB cell lines, SK-N-AS and SH-SY5Y-Luc. Cells were preincubated in the absence or presence of different concentrations of MLN8054 for 48 h and stained for Annexin V and propidium iodide [PI; (i)], stained with propidium iodide and analyzed for relative DNA content (ii), and harvested and submitted to immunoblotting of full-length (116 kDa) and cleaved (89 kDa) PARP (iii).
Tables
- Table 1.
Clinicopathologic characteristics of the patients with neuroblastoma and Aurora A mRNA and protein expression
Patient characteristics mRNA Protein n X ± SD P n X ± SD P Age at diagnosis (y) ≤1 28 3.10 ± 2.64 0.877 25 1.37 ± 1.67 0.29 >1 39 2.98 ± 3.30 28 1.82 ± 1.37 INSS stage I + II + IVS 31 1.98 ± 1.34 0.007 26 1.28 ± 1.24 0.0028 III + IV 36 3.94 ± 3.72 27 2.85 ± 2.23 MYCN status Nonamplified 49 2.64 ± 2.66 0.017 36 1.30 ± 1.04 0.0029 Amplified (>10 copies) 14 4.84 ± 3.90 11 2.50 ± 1.31 Histology Favorable 28 2.42 ± 2.00 0.007 23 1.03 ± 0.87 0.0006 Unfavorable 19 4.89 ± 3.94 14 2.26 ± 1.11 Disease risk Low + intermediate 42 2.31 ± 1.80 0.019 35 1.22 ± 0.94 0.011 High 22 4.74 ± 4.18 16 2.11 ± 1.47 Relapse Yes 19 4.59 ± 4.41 0.019 14 2.42 ± 2.07 0.044 No 43 2.63 ± 2.00 35 1.45 ± 1.17 NOTE: n, number of neuroblastoma tumor tissues tested; X, mean mRNA and protein expression levels between selected groups and determined by RT-PCR and Western blotting assay.
- Table 2.
Biological characteristics and sensitivity to MLN8054 in human neuroblastoma cell lines
Cell line Origin MYCN p53 status Cell type IC50 of MLN8054 (μmol/L) SK-N-SH NB tumor SC WT N + S 0.46 ± 0.01 (2) SH-SY5Y-Luc NB tumor SC WT N 0.24 ± 0.02 (4) IMR-32 NB tumor A WT N 0.30 ± 0.08 (2) LAN-1 NB tumor A Null N 0.67 ± 0.04 (4) JF NB tumor A N/D N 0.26 ± 0.06 (3) SH-EP NB tumor SC WT S 0.92 (1) NB19 NB tumor A WT S 0.50 (1) SMS-KCN NB tumor A N/D S 2.77 ± 0.18 (2) SK-N-AS NB tumor SC mt S 4.11 ± 1.03 (2) MCF-10A Breast normal epithelial WT 12.92 (1) MCF-7 Breast tumor WT 0.44 (1) NOTE: Numbers represent average ± SD IC50 derived from a Cell Counting Kit-8 proliferation assay. Values in parentheses represent the number of experiments completed for each cell line.
Abbreviations: NB, neuroblastoma; SC, single copy; A, amplified; WT, wild-type; mt, mutated; N/D, not described; N, neuroblastic; S, substrate adherent.
Supplementary Data, Shang, et al.
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Volume 8, Issue 8
