High-grade glioma motility reduced by genetic knockdown of KCC3

Cell motility is dependent on a coordinated reorganization of the cytoskeleton, membrane recycling, and focal adhesion to the extracellular matrix. Each of these cellular processes involves re-distribution of cell water, which is facilitated by the transport of inorganic ions (with obligatory water movement). Scratch-wound healing assays of Wistar C6 glioblastoma cells demonstrated cell motility in advance of cell proliferation. Although bumetanide inhibition of Na-K-2Cl cotransport activity did not affect cell motility, treatment of glioma cells with furosemide to inhibit K-Cl cotransport activity prevented ~75% of wound closure in a reversible reaction. Genetic silencing of KCC3 with short hairpin interfering RNA reduced protein expression by 40 - 60%, K(+) influx by ~50%, and cell motility by ~50%. Appearance of KCC1 mRNA and KCC3 mRNA at 25 PCR cycles versus KCC4 mRNA at 35 PCR cycles, suggests more KCC1/KCC3 expression in both primary rat astrocytes and C6 glioma cells. Altogether, these experiments suggest that the presence/function of multiple isoforms of the Na(+-)independent K-Cl cotransporter may have a role in glioma cell motility.