The Effect of Normoxic and Hypoxic U-87 Glioblastoma Paracrine Secretion on the Modulation of Brain Endothelial Cells

Abstract

Background: Glioblastoma multiforme (GBM) is a highly invasive brain tumour, characterized by its ability to secrete factors promoting its virulence. Brain endothelial cells (BECs) in the GBM environment are physiologically modulated. The present study investigated the modulatory effects of normoxically and hypoxically induced glioblastoma U-87 cell secretions on BECs. Methods: Conditioned media (CM) were derived by cultivating U-87 cells under hypoxic incubation (5% O2) and normoxic incubation (21% O2). Treated bEnd.3 cells were evaluated for mitochondrial dehydrogenase activity, mitochondrial membrane potential (ΔΨm), ATP production, transendothelial electrical resistance (TEER), and endothelial tight-junction (ETJ) gene expression over 96 h. Results: The coculture of bEnd.3 cells with U-87 cells, or exposure to either hypoxic or normoxic U-87CM, was associated with low cellular viability. The ΔΨm in bEnd.3 cells was hyperpolarized after hypoxic U-87CM treatment (p < 0.0001). However, normoxic U-87CM did not affect the state of ΔΨm. BEC ATP levels were reduced after being cocultured with U-87 cells, or with hypoxic and normoxic CM (p < 0.05). Suppressed mitochondrial activity in bEnd.3 cells was associated with increased transendothelial permeability, while bEnd.3 cells significantly increased the gene expression levels of ETJs (p < 0.05) when treated with U-87CM. Conclusions: Hypoxic and normoxic glioblastoma paracrine factors differentially suppressed mitochondrial activity in BECs, increasing the BECs’ barrier permeability.

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice

“…For the first assumption, we found controversial data. While a previous study showed that puromycin-purified rat BMECs culture exhibited improved barrier properties, including TEER values (Calabria et al, 2006 ), several works also demonstrated that BMECs co-cultured with pericytes and astrocytes might enhance the conditions of the primary culture system (Abbott, 2002 ; Siddharthan et al, 2007 ; Wisniewska-Kruk et al, 2012 ; Jamieson et al, 2019 ; Rado et al, 2022 ). On the other hand, cell culture seeding density impact the viability and suitability of the cell culture over time.…”

“…Nuclei are stained with DAPI (blue), N = , Scale bar µm. -Kruk et al, 2012;Jamieson et al, 2019;Rado et al, 2022). On the other hand, cell culture seeding density impact the viability and suitability of the cell culture over time.…”

Targeting mitochondrial metabolism by the mitotoxin bromoxib as a therapeutic approach for the treatment of leukemia and lymphoma

“…This chemical property justi es the term 'protonophore', as positively charged protons characterized by their hydrophilic nature, typically require channels, transporters, or passive diffusion facilitated by protonophores to traverse membranes [28,29]. Prominent examples of protonophores are compounds like CCCP, 2,4-dinitrophenol, or triphenylphosphonium, which are known for their cytotoxic potential in various cancer cell types, including breast cancer, lung cancer, neuroblastoma, and glioblastoma cells [30][31][32]. These agents function by disrupting the normal proton gradient across the inner mitochondrial membrane, thereby uncoupling oxidative phosphorylation (OXPHOS) and diminishing ATP production.…”

The Paracrine Effect of Hypoxic and Normoxic Cancer Secretion on the Proliferation of Brain Endothelial Cells (bEnd.3)

“…Mitochondria are an essential regulator of cell proliferation [ 22 ]. In our previous studies [ 23 , 24 ], we showed that mitochondrial activity of endothelial cells (bEnd.3) was suppressed after cultivation with paracrine factors secreted from breast and GBM cancer cells. Mitochondria regulate cell signalling pathways, cell death and gene expression, including cell division [ 25 ].…”