Table 1.
Clinical characteristics in archival GBM patients.
Fig 1.
Expression of NCL in glioma cells and NHA and its binding affinity to AS1411.
(a) Relative mRNA level of NCL in 40 clinical GBM tissues analyzed by Real-time qPCR. (b) Real-time qPCR detection of mRNA level of NCL in clinical GBM tissues compared to normal brain tissues (c) Immunohistochemical staining of NCL in GBM and normal brain tissues (Astrocytes). Scale bar equals 50 μm. (d) Real-time qPCR detection of NCL mRNA in human glioma cells: U87, U251, SHG44 and normal NHA cell. (e) Immunoblotting detection of NCL protein in U87, U251 and SHG44 cells compared to NHA, β-actin was used as loading control. (f) Immunofluorescence detection of the expression and location of NCL in U87 and NHA cells. (g) Immunofluorescence detection of FITC-AS1411 (green) and NCL (red) co-localizations in glioma cells. Error bars indicate ± s.d. *P< 0.05, **P< 0.01, two-tailed student’s t-test. Scale bar equals 20 μm.
Fig 2.
Short term AS1411 exposure induced growth suppression in glioma cells.
(a) MTT analysis of cell viability after exposure to AS1411 from 0 to 80 μM for 48, 72 and 96h in U87, U251, SHG44 and NHA cells, respectively. (b) After 48 h treatment. Cell viability inhibition induced by AS1411 in glioma cells was compared to that in NHA cells. (c) 10×Light microscope images indicated the growth inhibition and cell structure destroy in U87 and U251 cells induced by AS1411 of 0 to 5μM and CRO of 5μM for 48 h. Error bars indicate ± s.d. *P<0.05, two-tailed student’s t-test. Scale bar equals 10 μm.
Fig 3.
Expression of Bcl-2, p53, Akt1 after AS1411 treatment and NCL silencing in U87 and U251 cells.
(a) Real-time qPCR detection of Bcl-2, p53 and Akt1 mRNA in U251 and U87 cells after treatment with AS1411 (5 μM) for 48 h, 72 h and 96 h. (b) Immunoblotting analysis of Bcl-2, p53 and Akt1 in U251 and U87 cells treated with AS1411 (5 μM) for 48 h, β-actin used as loading control. (c) Real-time qPCR detection of Bcl-2, p53 and Akt1 mRNA after siRNA NCL silencing in U87 cells and U251 cells for 48 h. Immunoblotting detection of p53, Bcl-2 and Akt1 after siRNA NCL silencing in U87 and U251 cells for 48 h. (d) Error bars indicate ± s.d. *P<0.05, **P< 0.01, two-tailed student’s t-test.
Fig 4.
G2/M cell cycle arrest evoked by AS1411.
(a) Cell cycle arrest induced by AS1411 in U87, U251, SHG44 and NHA cells. 48 h after treatment with AS1411 (5μM), cells were collected and stained with propidium iodide (PI); DNA content was determined by flow cytometry. This assay was performed in triplicate. CRO (5μM) was used as negative control. (b) Histograms showing the percentage of glioma cells and NHA in G0-G1, S, and G2-M phases in four independent experiments. (c) The G2/M cell cycle related protein cyclin A1, cyclin B1 and cyclin D1 was detected by immunoblotting after treatment with AS1411 (5μM) for 48 h, CRO (5μM) was used as negative control, β-actin was used as loading control. (d) Histograms showing the percentage of glioma cells in G0-G1, S, and G2-M phases after the NCL overexpression. Error bars indicate ± s.d. **P<0.01, two-tailed student’s t-test.
Fig 5.
Apoptosis induced by AS1411 in glioma cells.
(a) Apoptotic cell death induced by AS1411 in U87, U251, SHG44 and NHA cells. 48h after treatment, cells were collected and stained with PI and Annexin V–FITC, Annexin V-positive/PI-negative cells were measured by flow cytometry. This experiment was repeated three times. (b) Histograms showing the percentage of cells in apoptosis. U87, U251, SHG44 and NHA cells in four independent experiments. Annexin V-positive cells were considered as apoptotic cells. (c) Histograms showing the percentage of glioma cells apoptosis after the Bcl-2 overexpression and p53 siRNA silencing. Error bars indicate ± s.d. **P<0.01, two-tailed student’s t-test.
Fig 6.
Migration and invasion inhibition induced by AS1411.
(a) AS1411 inhibited the migration of glioma cells in vitro. The migration capabilities of U87, U251, SHG44 and NHA cells were assessed after pretreatment with AS1411 of 0 to 10 μM and CRO of 10μM. Akt1 overexpression antagonized AS1411 induced migration inhibition. (b) AS1411 inhibited the invasion of glioma cells in vitro. The invasive capabilities of U87, U251, SHG44 and NHA were assessed after pretreatment with AS1411 of 0 to 10μM and CRO of 10μM. Akt1 overexpression antagonized AS1411 induced invasion inhibition. Error bars indicate s.d. *P<0.05, **P<0.01, two-tailed Student’s t-test.
Fig 7.
Tumor growth inhibition and life expansion induced by AS1411 in vivo.
Subcutaneous tumors generated from U87 cells were allowed to reach a volume of 200 mm3 and were treated with AS1411 and CRO. (a) Survival of brain tumor–bearing mice was recorded and represented in a Kaplan–Meier plot. (b) Tumor volume during the course of treatment. (c) AS1411 treatment (20 days) induced down-regulation of Bcl-2 and up-regulation of p53 protein in tumor tissue. *P<0.05, **P<0.01, two-tailed Student’s t-test.