Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity

Abstract

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p ). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

High glycolytic activity of tumor cells leads to underestimation of electron transport system capacity when mitochondrial ATP synthase is inhibited

“…Figures 7 and 8 show that the glycolytic inhibitor 2-DG completely prevented the underestimation of max OCR (Figs 7A and 8A ) and SRC (Figs 7B and 8B ) due to oligomycin in T98G and U-87MG cells. These results suggest that underestimation of max OCR and SRC in the presence of oligomycin is associated with high cellular glycolytic activity, a predominant pathway for ATP regeneration in these tumor cells 19 . Next, cellular ATP content was measured under the same conditions used in the respirometry experiments, i.e., both cell lines were incubated in the presence or absence of oligomycin in sDMEM or sDMEM plus 40 mM 2-DG.…”

“…Incubating the cells in sDMEM containing 2-DG resulted in a significant drop in ATP levels of 75.6 ± 4.0% in T98G cells and 83.5 ± 1.9% in U-87MG cells (Figs 7C and 8C ), further suggesting that most cellular ATP in glioma cell lines is produced by glycolysis 19 . Induction of the Crabtree effect by 2-DG 22 may also contribute to ATP depletion under this condition.…”

“…Interestingly, a recent study with astrocytes revealed that ADP-stimulated GDH plays an important role under conditions of increased mitochondrial oxidative metabolism demand 35 . This pathway may play an important role in the supply of mitochondrial NADH to glutamine-addicted highly glycolytic tumor cells 19 , 36 . In addition, a high intramitochondrial ATP/ADP ratio may also limit max OCR by promoting partial inhibition of NADH oxidation.…”

“…The OCR in intact and permeabilized suspended cells was determined using a respirometer (OROBOROS Oxygraph-2k, Innsbruck, Austria), as previously described 14 , 19 . In intact tumor cells this was carried out by incubating an aliquot of the cell suspension (2–4 × 10 6 cells) at 37 °C in a 2 mL chamber containing the reaction medium, as described in the figure legends.…”

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma

“…Moreover, an altered redox homeostasis is observed in GBMs, and this triggers the activation of various survival pathways leading to disease progression and chemo resistance [24,[30][31][32][33]. It is necessary to consider, however, that changes in the ROS threshold by antioxidant compounds may differently affect the viability of tumor cells; for example, if we consider the literature on the biological effects of heme-oxygenase-1 (HO-1), we can observe that its upregulation can promote cell cycle arrest and cellular death in some tumors, whereas it has also been associated with tumor survival and progression in other neoplasms [34][35][36].…”