In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas

Abstract

Treatment for glioblastoma consists of radiotherapy and temozolomide-based chemotherapy. However, virtually all patients recur, leading to a fatal outcome. Receptor tyrosine kinase (RTK)-targeted therapy has been the focus of attention in novel treatment options for these patients. Here, we compared the efficacy of imatinib, sunitinib, and cediranib in glioblastoma models. In the present work, the biologic effect of the drugs was screened by viability, cell cycle, apoptosis, migration, and invasion in vitro assays or in vivo by chick chorioallantoic membrane assay. Intracellular signaling was assessed by Western blot and the RTK targets were identified using phospho-RTK arrays. The amplified status of KIT, PDGFRA, and VEGFR2 genes was assessed by quantitative polymerase chain reaction. In a panel of 10 glioblastoma cell lines, we showed that cediranib was the most potent. In addition, cediranib and sunitinib synergistically sensitize the cells to temozolomide. Cediranib efficacy was shown to associate with higher cytostatic and unique cytotoxic effects in vitro and both antitumoral and antiangiogenic activity in vivo, which could associate with its great capacity to inhibit mitogen-activated protein kinase (MAPK) and AKT pathways. The molecular status of KIT, PDGFRA, and VEGFR2 did not predict glioblastoma cell responsiveness to any of the RTK inhibitors. Importantly, phospho-RTK arrays revealed novel targets for cediranib and sunitinib therapy. In conclusion, the novel targets found may be of value as future biomarkers for therapy response in glioblastoma and lead to the rational selection of patients for effective molecular targeted treatment.

Figure 1

Effect of cediranib, sunitinib, and imatinib on U251 cellular survival, cell cycle, apoptosis, migration, and invasion. (A) Cellular viability was measured at 24, 48, and 72 hours by MTS. (B) Cell cycle analysis was done at a 48-hour time point by flow cytometric analysis of PI-stained cells. (C) Apoptosis we assessed by Western blot for PARP cleavage.

Figure 2

Effect of cediranib, sunitinib, and imatinib on U251 cellular migration and invasion. (A) Migration was measured at 48 hours by wound healing migration assay. The relative migration distance was calculated as indicated in Materials and Methods section and the results are expressed as the means ± SD. (B) Invasion was assessed by Matrigel invasion assay. The results were expressed in relation to the DMSO control (considered as 100% of invasion) as the mean percentage of invasion ± SD. (C) Representative pictures of invasion assay (x40 magnification).

Figure 3

In vivo effect of cediranib on glioblastoma growth and angiogenesis. (A) Representative pictures (x16 magnification) of CAM assay in ovo and ex ovo at days 14 and 17. (B) Tumor growth was measured as described in Materials and Methods section. The results were expressed as mean percentage of tumor growth from day 14 (considered as 0%) to day 17 of development ± SD. (C) Counting of the blood vessels ex ovo at day 17. The results were expressed as the mean number of vessels for each group of treatments ± SD. Differences with P < .05 on the Student's t test were considered statistically significant (*). A total of 12 eggs were used for tumor formation (six were treated with cediranib and six with DMSO alone).

Figure 4

Cediranib, sunitinib, and imatinib targets on glioblastoma cells. (A) Representative pictures of phospho-RTK arrays for U251 cell line. Each RTK is represented in duplicate in the arrays (two spots side by side), and four pairs of phosphotyrosine positive controls are located in the corners of each array. (B) Other cell lines were treated with cediranib, sunitinib, and imatinib at the indicated concentrations, and the RTKs that were targeted after drug treatment are shown in the table.

Figure 5

Effect of cediranib, sunitinib, and imatinib on intracellular signaling pathway activation in U251 cells. The cells were incubated with increasing concentrations of the three drugs by 2 hours. By Western blot, we assess the activation levels of several intermediates of the MAPK (p-ERK1/2 and p-p38), AKT (p-AKT), JAK/STAT (p-STAT3), and SRC (non-p-SRC Tyr⁵²⁷ and p-SRC Tyr⁴¹⁶) pathways. The levels of phosphorylation were compared with the corresponding total proteins.