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


METHODS
For 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.


RESULTS
GBM 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.


CONCLUSIONS
This 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.
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Mikkel
0000-0001-9068-6939
M
Staberg
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Signe Regner
SR
Michaelsen
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Louise Stobbe
LS
Olsen
Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Copenhagen University Hospital and University of Copenhagen, Copenhagen, Denmark.
Mette Kjølhede
MK
Nedergaard
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Mette
M
Villingshøj
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Marie-Thérése
MT
Stockhausen
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.|Brain Tumor Biology Group, Danish Cancer Society Research Center, Copenhagen, Denmark.
Petra
P
Hamerlik
Department of Radiation Biology, The Finsen Center, Copenhagen University Hospital, Copenhagen, Denmark.
Hans Skovgaard
HS
Poulsen
Angiogenesis; DAPT; EGFR; Endothelial spheroid sprouting; Gamma-secretase inhibitor; Glioblastoma; Iressa; Notch; Tyrosine-kinase inhibitor