LAB-ANGIOGENESIS AND INVASION

OBJECTIVE: Glioblastoma multiforme is the most frequent primary brain tumor in adults. The major obstacle for successful treatment is the invasive growth pattern. Metabolically, glioblastomas are highly glycolytic, leading to increased levels of lactic acid production. The carbonic anhydrase IX (CAIX) moderates the extrusion of hydrogen ions into the extracellular space, which may enhance tumor invasion by activating proteolytic enzymes. We therefore induced glycolysis in glioblastoma cells and investigated the extracellular pH, cathepsin B expression, and its subcellular distribution and secretion parallel to the invasive behavior of the cells upon CAIX knockdown. METHODS: U251 glioblastoma cells were transfected with a CAIX siRNA construct and cultured in a Biocoat Matrigel invasion chambers with an 8-mm pore size membrane. The chambers were incubated in a humified 5% CO2 modular with either 21% oxygen and 25 mM glucose (control) or 0% oxygen plus 125 mM glucose (glycolysis). Invasion was quantified by counting the cells on the lower membrane surface. Extracellular pH was measured using a pH-meter. Cathepsin B expression and localization was investigated by RT-PCR, Western blot, and immunofluorescence staining, respectively. The cathepsin B secretion into the supernatant was measured using a cathepsin B activity assay. RESULTS: In vitro glycolysis caused a significant increase of cathepsin B expression and secretion combined with massive invasion of glioblastoma cells. In addition, the subcellular distribution of the enzyme was shifted to the cell periphery. The extracellular pH dropped significantly under glycolytic conditions, antagonized by CAIX knockdown. In addition, CAIX knockdown did not influence cathepsin B expression but attenuated the intracellular change of cathepsin B distribution and reduced both the cathepsin B secretion and glioma cell invasion. CONCLUSION: Our data demonstrate that CAIX moderates invasion in glycolytic glioma cells via acidification of the extracellular milieu and enhanced secretion of cathepsin B.