gms | German Medical Science

64th Annual Meeting of the German Society of Neurosurgery (DGNC)

German Society of Neurosurgery (DGNC)

26 - 29 May 2013, Düsseldorf

CAIX regulates extracellular pH and invasion in glioblastoma

Meeting Abstract

  • Martin Andreas Proescholdt - Department of Neurosurgery, University Regensburg Medical Center, Regensburg, Germany
  • Marsha Merrill - Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA
  • Eva-Maria Stoerr - Department of Neurosurgery, University Regensburg Medical Center, Regensburg, Germany
  • Annette Lohmeier - Department of Neurosurgery, University Regensburg Medical Center, Regensburg, Germany
  • Alexander Brawanski - Department of Neurosurgery, University Regensburg Medical Center, Regensburg, Germany

Deutsche Gesellschaft für Neurochirurgie. 64. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC). Düsseldorf, 26.-29.05.2013. Düsseldorf: German Medical Science GMS Publishing House; 2013. DocMI.13.07

doi: 10.3205/13dgnc389, urn:nbn:de:0183-13dgnc3891

Published: May 21, 2013

© 2013 Proescholdt et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.



Objective: Malignant gliomas are highly invasive tumors. Metabolically these tumors are characterized by glycolysis leading to increased levels of lactic acid. The hypoxia-inducible carbonic anhydrase (CA) IX has been shown to moderate the extrusion of hydrogen ions into the extracellular space. Since the acidification of the extracellular environment contributes to host tissue invasion due to activation of lysosomal proteolytic enzymes such as cathepsin B we hypothesized that CA IX plays an important role in this context. We therefore induced glycolysis in glioblastoma cells and investigated the cathepsin B expression, its subcellular distribution and secretion parallel to the invasive behavior of the cells upon CAIX knockdown.

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.