Cancer Res Treat.  2016 Apr;48(2):687-697. 10.4143/crt.2014.320.

The Effect of Chemoradiotherapy with SRC Tyrosine Kinase Inhibitor, PP2 and Temozolomide on Malignant Glioma Cells In Vitro and In Vivo

• Eom KY
• Cho BJ
• Choi EJ
• Kim JH
• Chie EK
• Wu HG
• Kim IH
• Paek SH
• Kim JS
• Kim IA

• KMID: 2454347
• DOI: http://doi.org/10.4143/crt.2014.320

Abstract

PURPOSE
We investigated the effect of chemoradiotherapy with PP2 and temozolomide (TMZ) on malignant glioma cells using clonogenic assays and in vivo brain tumor model.
MATERIALS AND METHODS
The effect of PP2 on radiosensitivity of U251 and T98G cells was investigated using clonogenic assays. The expression of E-cadherin, matrix metalloproteinases 2 (MMP2), Ephrin type-A receptor 2 (EphA2), and vascular endothelial growth factor (VEGF) was measured by Western blotting and an accumulation of γH2AX foci 6 hours after radiotherapy was measured after PP2 treatment. The effect of PP2 on migration, invasion, and vasculogenic mimicry formation (VMF) of U251 cells was evaluated. In an orthotopical brain tumor model with U251 cells, PP2 was injected intraperitoneally with or without oral TMZ before, during and after whole brain radiotherapy. Bioluminescence images were taken to visualize in vivo tumors and immunohistochemical staining of VEGF, CD31, EphA2, and hypoxia-inducible factor 1a was performed.
RESULTS
PP2 increased radiosensitivity of U251 and T98G cells without decreasing survival of normal human astrocytes. Chemoradiotherapy with PP2 and TMZ resulted in increased accumulation of γH2AX foci. PP2 induced overexpression of E-cadherin and suppression of MMP2, VEGF, and EphA2. PP2 also compromised invasion, migration, and VMF of U251 cells. In brain tumors, chemoradiotherapy with PP2 and TMZ decreased tumor volume best, but not statistically significantly compared with chemoradiotherapy with TMZ. The expression of VEGF and CD31 was suppressed in PP2-treated tumors.
CONCLUSION
PP2 enhances radiosensitivity of malignant glioma cells and suppresses invasion and migration of U251 cells. Chemoradiotherapy with PP2 and TMZ resulted in non-significant tumor volume decrease.

Keyword

SRC tyrosine kinase inhibitor; PP2; Temozolomide; Glioblastoma; Radiotherapy

MeSH Terms

Astrocytes
Blotting, Western
Brain
Brain Neoplasms
Cadherins
Chemoradiotherapy*
Glioblastoma
Glioma*
Humans
In Vitro Techniques*
Matrix Metalloproteinases
Protein-Tyrosine Kinases*
Radiation Tolerance
Radiotherapy
Tumor Burden
Tyrosine*
Vascular Endothelial Growth Factor A
Cadherins
Matrix Metalloproteinases
Protein-Tyrosine Kinases
Tyrosine
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1. Clonogenic assays of U251 and T98G cells after treating PP2, temozolomide (TMZ), or both. Cell surviving curves after treating PP2 (10 μM), TMZ (25 μM), or both were generated. The points indicate the mean surviving fractions at each dose level (±standard error). The lines are fitted to linear-quadratic equations. An addition of PP2 to TMZ showed an additive cytotoxic effect both in U251 and T98G cells. Sensitizer enhancement ratios (SER) were defined as isoeffective dose at surviving fraction 0.5 or 0.05. PP2 induced radiosensitization in U251 and T98G cells irrespective of MGMT promoter methylation. EGFR, epidermal growth factor receptor; RT, radiotherapy.

• 

  Fig. 2. Representative images of γH2AX foci formation in U251 cells. γH2AX foci formation was measured in U251 cells after treating PP2 (10 μM), temozolomide (TMZ, 25 μM), or both. The nuclei stained by DAPI are shown in blue, while the γH2AX foci stained by FITC are shown in green. Increased accumulation of γH2AX foci after treating PP2 and TMZ was confirmed by indirect immunofluorescence 6 hours after radiotherapy (6 Gy), indicating delayed DNA double strand breakage repair. All differences between the groups (6 Gy vs. 6 Gy+TMZ, p=0.014; 6 Gy+TMZ vs. 6 Gy+PP2, p=0.020; 6 Gy+PP2 vs. 6 Gy+TMZ+PP2, p=0.016) were statistically significant.

• 

  Fig. 3. Representative images of modified Boyden chamber assay (A), wound healing assay (B), and vasculogenic mimicry (VM) formation assay (C). (A) U251 cells were treated with PP2 (10 μM), temozolomide (TMZ, 25 μM), or both for 24 hours, and cell invasion was measured using a Transwell system with 8-μm pores in polycarbonate membranes. Results are expressed as a ratio of radiotherapy alone (control). (B) U251 cells were grown to confluence in 6-well plates and then starved for 24 hours. A linear scratch was made in each hemisphere of the well with 1-mL pipette tip. Images were taken of the intersections of linear cell wound and each grid line. (C) Two hundred microliters ECM Matrigel was dropped in 48-well tissue culture plates and then incubated at 37°C for 2 hours. Column, bar, and asterisk represent the mean of three independent experiments, standard error, and statistical significance of p < 0.05 when compared with control, respectively.

• 

  Fig. 4. Western blotting of U251 cells after treating inhibitors. After PP2 (10 μM) treatment, E-cadherin was overexpressed, while expression of matrix metalloproteinase 2 (MMP2), Ephrin type-A receptor 2 (EphA2), and vascular endothelial growth factor (VEGF) were suppressed. TMZ, temozolomide.

• 

  Fig. 5. The surviving fraction at 2 Gy (SF2) of X-ray in normal human astrocytes. SF2 was measured after treating PP2 (10 μM), temozolomide (TMZ, 25 μM), or both. The points indicate the mean surviving fractions at each dose level. Column and bar represent mean of three independent experiments and standard error, respectively.

• 

  Fig. 6. (A) Representative bioluminescence images of human brain tumor xenograft in nude mice. U251 cells were injected orthotopically into thalamic area using a 26G needle with syringe. Bioluminescence images were obtained 7 days after injection, and then randomly assigned to control or experimental groups as follows: control, radiotherapy (XRT), XRT+PP2, XRT+temozolomide (TMZ), and XRT+TMZ+PP2, respectively. Chemoradiotherapy with TMZ exhibited marked tumor shrinkage and chemoradiotherapy with PP2 and TMZ led to non-visualization of tumors, indicating an additional radiosensitization by PP2. (B) In semi-quantitative analyses of relative luminescence area, combination of PP2 and TMZ resulted in a smaller area of luminescence than TMZ or PP2 alone, but it was not statistically significant. Bar represents standard error. (C) TMZ-treated groups had smaller mean intensity than control or radiotherapy alone. However, the difference between TMZ and PP2 plus TMZ group was not significant.

• 

  Fig. 7. Immunohistochemical stains of in vivo human brain tumors in nude mice. Immunohistochemical staining with antibodies against vascular endothelial growth factor (VEGF), CD31, Ephrin type-A receptor 2 (EphA2), and hypoxia-inducible factor 1α (HIF1α) was performed. The expression of VEGF and CD31 were down-regulated in PP2-treated tumors relative to those with XRT alone or XRT plus temozolomide (TMZ) tumors, suggesting that PP2 may suppress angiogenesis in in vivo tumor as well as in in vitro cells. Expression of EphA2 and HIF1α were not influenced by PP2 treatment in in vivo tumors. XRT, radiotherapy.

Reference

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