gms | German Medical Science

Objective: Malignant gliomas are highly invasive tumors. Metabolically these tumors are characterized by glycolysis leading to increased levels of lactic acid. The acidification of the extracellular space, which is promoted by the activity of carbonic anhydrase (CA) IX leads to host tissue invasion due to activation of lysosomal proteolytic enzymes such as cathepsin B. We therefore attempted to investigate the interrelationship between glycolytic metabolism, extracellular pH, expression of (CA) IX as well as subcellular distribution and secretion of cathepsin B with regard to the invasive behavior of glioblastoma cells.

Method: U251 glioblastoma cells were transfected with a CAIX siRNA construct and a non specific sequence siRNA as control. Cathepsin B expression and localization as investigated by quantitative RTPCR, Western blot and immunofluorescence staining respectively, the cathepsin B secretion into the supernatant was measured using a cathepsin B activity assay. The pH-dependent lysosome trafficking was analyzed by horseradish peroxidase labeling in bicarbonate free buffer (5 mg/ml). For invasion assays, a Matrigel invasion chamber was used. The chambers were incubated in a 5% CO₂ modular with either 21% oxygen and 25 mM glucose in the culture medium (ctrl.) or 0% oxygen plus 125 mM glucose (glycolysis). Invasion was quantified by counting the cells which invaded into the lower culture compartment. Extracellular acidification was investigated by pH measurement of the supernatant.

Results: In vitro glycolysis caused a significant drop of extracellular pH (pHe) combined with massive invasion of glioblastoma cells, which was antagonized by CAIX knockdown. The cathepsin B expression was induced under glycolytic conditions, which was not influenced by CAIX interference. In contrast, CAIX knockdown reduced the subcellular distribution change of cathepsin B towards the cell periphery, which was induced by glycolysis. Also, the secretion of cathepsin B, which was strongly increased under glycolysis, was significantly reduced by CAIX knockdown. Lysosome labeling revealed the identical pattern of bidirectional, pH-dependent movement indicating the possible mechanism of subcellular cathepsin B transport.

Conclusions: Our data demonstrate that CAIX moderates invasion in glycolytic glioma cells via acidification of the extracellular milieu and enhanced secretion of cathepsin B.