Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

Abstract

BackgroundFor Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the need for combination therapies. In GBM, both the epidermal growth factor receptor (EGFR) and the notch signaling pathways are often deregulated and linked to cellular growth, invasion and angiogenesis. Several studies have confirmed cross-talk and co-dependence of these pathways. Therefore, this study aimed at testing a combination treatment strategy using inhibitors targeting the notch and EGFR pathways.MethodsFor evaluation of cell viability a standard MTT assay was used. Western blotting (WB) and Q-RT-PCR were employed in order to assess the protein- and mRNA expression levels, respectively. In order to determine angiogenic processes, we used an endothelial spheroid sprouting assay. For assessment of secreted VEGF from GBM cells we performed a VEGF-quantikine ELISA.ResultsGBM cells were confirmed to express EGFR and Notch and to have the capacity to induce endothelial cell sprouting. Inhibition of EGFR and Notch signaling was achieved using either Iressa (gefitinib) or the gamma-secretase inhibitor DAPT. Our data showed that DAPT combined with Iressa treatment displayed increased inhibitory effect on cell viability and abrogated expression and activation of major pro-survival pathways. Similarly, the combinational treatment significantly increased abrogation of GBM-induced endothelial cell sprouting suggesting reduced GBM angiogenesis.ConclusionThis study finds that simultaneous targeting of notch and EGFR signaling leads to enhanced inhibitory effects on GBM-induced angiogenesis and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0309-2) contains supplementary material, which is available to authorized users.

Reciprocal H3.3 gene editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling

“…Dose–response curves and IC50s were plotted and determined using the GraphPad Prism software. We first performed a drug treatment time course from 1 to 6 days using a saturating concentration of each drug based on the range of reported IC50s in the literature for various cell line treatments 17 , 29 , 47 – 50 . We confirmed that 3 days of treatment was sufficient to obtain significant differences in cell viability in a dose-dependent manner across our cell lines.…”

Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

“…Cell growth was measured using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Sigma, MO, USA) by the addition of 20 μl of MTT solution (5 mg/ml, dissolved in sterile water) to each well and incubation for 4 h before the addition of 100 μl of solubilization buffer (10 % sodium dodecyl sulfate, 0.01 M HCl). Absorbance at 570 nm was measured the next day using Synergy 2 microplate reader with Gen5 software [ 32 ].…”

Role of Notch Signaling Pathway in Glioblastoma Multiforme Pathogenesis

“…Unfortunately, this combined treatment is not sufficient to fully block endothelial cells from sprouting, probably due to other angiogenesis factors. Moreover, by blocking EGFR signaling, Hes1 levels decreased, suggesting that EGFR signaling stimulates Notch pathway activity [148]. Like DAPT, LLNle and L-685,458 are also able to kill GSCs, inhibiting NICD generation and inducing proteasome inhibition, proteolytic stress, and mitotic arrest [149].…”