Epidermal Growth Factor (EGF) Augments the Invasive Potential of Human Glioblastoma Multiforme Cells via the Activation of Collaborative EGFR/ROS-Dependent Signaling

Abstract

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear–front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

Bioinspired Bola-Type Peptide Dendrimers Inhibit Proliferation and Invasiveness of Glioblastoma Cells in a Manner Dependent on Their Structure and Amphipathic Properties

“…Cells (2 × 10 4 /cm 2 ) were cultured in 12-well plates on sterile coverslips and treated with indicated concentrations of dendrons or bola dendrimers for 48 h. Immunolocalization of chosen cellular structures was performed according to previously described protocol [ 44 ]. Briefly, cells were fixed with 3.7% formaldehyde (20 min.…”

Human glioblastoma cell motility depends on the activity of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase

“…An example is the trafficking of energetically active mitochondria to membrane protrusions accompanying enhanced invasive properties of the LN229 GB cell line (Caino et al 2015). Another is the inhibitory effect of ROS scavenging on EGF-stimulation of T98G cell migration (Pudelek et al 2020). More broadly, ROS are known to promote activity of pro-migratory signaling pathways by direct redox modification of their protein components (Weng et al 2018) (Dustin et al 2020).…”

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner

“…Human GBM T98G (ATCC, CRL-1690) and U87 cells (ATCC, CRL-1690) were routinely cultivated in high glucose (4500 g/L) DMEM medium (Sigma, St. Louis, MO, USA), which was supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% Antibiotic-Antimycotic Solution (Sigma) [ 59 , 60 ]. For endpoint experiments, the cells were harvested with 0.25% trypsin in Ca 2+ /Mg 2+ -free PBS (Corning, Corning, NY, USA) supplemented with 0.5 mM EDTA (UltraPure TM ; Invitrogen, Carlsbad, CA, USA), counted with Z2 particle counter (Beckman-Coulter, Brea, CA, USA) and then seeded into multi-well tissue culture plates (Falcon).…”

“…For each condition, at least 5000 singlets were collected. The data were processed using IDEAS 6.2 software (Merck; [ 59 ]). The biability of cells was analyzed using Trypan blue assay (Sigma; No.…”