Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death

Abstract

Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -β). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations.

AMPK activation protects astrocytes from hypoxia‑induced cell death

“…Glucose and lactate concentrations were measured in cell-free supernatants with a biochemistry analyzer Hitachy 917 (Roche). For the analysis of oxygen consumption, cultured cells were overlaid with paraffin oil and oxygen concentrations were measured every 15 min utilizing a fluorescence-based assay with a sensor at the bottom of each well (Oxodish, PreSens), as previously described (27).…”

“…Glucose and oxygen concentrations are two decisive determinants in cerebral ischemic lesions. Therefore, the present study first employed an established paradigm (26)(27)(28) of severe hypoxia with 0.1% oxygen, varied glucose concentrations and complete glutamine deprivation. cell death was measured by flow cytometry [fluorescence-activated cell sorting (FAcS)] as a percentage of PI-positive cells (for exemplary PI FAcS plots please see Fig.…”

Rescue of 2-Deoxyglucose Side Effects by Ketogenic Diet

“…LNT-229 cells were a kind gift of Dr. N de Tribolet (Lausanne, Switzerland) [37] and were cultured in high glucose Dulbecco’s modified eagle medium (DMEM) (4.5 g/L corresponding to 24.975 mM glucose) containing 10% foetal calf serum (FCS) (Biochrom KG, Berlin, Germany), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Life Technologies, Karlsruhe, Germany) at 37 °C in a cell culture incubator (Binder, Tuttlingen, Germany) under a CO 2 atmosphere (5%). The experiments were performed in serum free DMEM, with 2 mM of glucose and an incubation time of 6 h.…”

“…The cell-free supernatant was collected and the glucose and lactate concentrations were measured using the biochemistry analyzer Hitachi 917 [37]. …”

“…The secondary anti-rabbit and anti-goat antibodies were purchased from Santa Cruz Biotechnology. Enhanced chemiluminescence was used for detection [37]. …”