gms | German Medical Science

Objective: The Warburg effect describes the metabolic switch of tumor cells away from mitochondrial ATP generation to aerobic glycolysis, which is even enhanced by hypoxia, one characteristic of glioblastomas and a well-known factor in maintaining stemness. However, O₂ levels fluctuate within the tumor, requiring metabolic flexibility for progression. We therefore compared the effects of acute and chronic hypoxia versus oxygenation on glioma cells.

Method: Stem-like glioblastoma cell lines (GS lines) were established from 4 glioblastomas either under normoxia (21% O₂, GS^N lines) or hypoxia (1% O₂, GS^H lines). To mimick fluctuating O₂ conditions, GSN lines were exposed to short-term (48 hrs) hypoxia, and GSH lines to short-term normoxia. Gene expression analysis (microarray, qPCR, western blot, immunohistochemistry), metabolic flux analysis using mass spectrometry and functional analysis for all conditions were performed.

Results: Bioinformatic analyses revealed that short-term hypoxia predominantly induced adaptive metabolic pathways and cell cycle arrest, whereas long-term hypoxia activated neurodevelopmental processes. Detailed analysis showed that expression of glycolytic enzymes was upregulated by short-term hypoxia, whereas pentose phosphate pathway (PPP) enzymes were downregulated. The opposite pattern was found for oxygenation. Querying the Rembrandt database revealed that despite downregulation by hypoxia, expression of PPP enzymes is increased in glioblastomas, whereas expression of the hypoxia-inducible enzymes of the parallel preparatory phase of the glycoytic pathway is decreased. Immunohistochemistry showed strong staining for PPP enzymes in the main population glioblastoma cells, except for pseudopalisading cells. Glycolytic enzymes displayed an inverse pattern. Furthermore, a decreased PPP but increased glycolytic activity under hypoxia could be shown by mass spectrometric analysis of lactate labeling patterns using [1,2-¹³C₂]-D-glucose. In addition, acute and chronic hypoxia increased cell migration, but decreased proliferation, whereas acute oxygenation had the opposite effect.

Conclusions: Our findings extend Warburg’s observation by showing that in most tumor cells the PPP is favored over the parallel preparatory phase of glycolysis, supplying metabolites for biomass production. However, under acute severe hypoxic stress the PPP is suppressed, causing a switch to direct glycolysis for increased migration and decreased proliferation as protection against hypoxic stress.