Chlorotoxin Inhibits Glioma Cell Invasion via Matrix Metalloproteinase-2

Abstract

Primary brain tumors (gliomas) have the unusual ability to diffusely infiltrate the normal brain thereby evading surgical treatment. Chlorotoxin is a scorpion toxin that specifically binds to the surface of glioma cells and impairs their ability to invade. Using a recombinant His-Cltx we isolated and identified the principal Cltx receptor on the surface of glioma cells as matrix metalloproteinase-2 (MMP-2). MMP-2 is specifically upregulated in gliomas and related cancers, but is not normally expressed in brain. We demonstrate that Cltx specifically and selectively interacts with MMP-2 isoforms, but not with MMP-1, -3, and -9, which are also expressed in malignant glioma cells. Importantly, we show that the anti-invasive effect of Cltx on glioma cells can be explained by its interactions with MMP-2. Cltx exerts a dual effect on MMP-2: it inhibits the enzymatic activity of MMP-2 and causes a reduction in the surface expression of MMP-2. These findings suggest that Cltx is a specific MMP-2 inhibitor with significant therapeutic potential for gliomas and other diseases that invoke the activity of MMP-2.

A role for ion channels in glioma cell invasion

“…ClC-3 channels were reduced by exposure to Cltx, which indicates internalization of ClC-3 channels. Similar experiments with antibodies to MMP-2 show that MMP-2 also decreases on the cell surface (Deshane et al, 2003). The internalization of ClC-3 channels induced by Cltx is prevented by simultaneously treating the cells with the sterol-binding drug filipin (5 μg ml −1 ) (Fig.…”

“…Based on the above findings, which indicate that Cltx causes internalization of ClC-3 channels into caveolar rafts, we did not expect Cltx to block Transwell migration in the presence of filipin because Cl − channels can no longer be internalized. This is the case (Deshane et al, 2003). Thus, the mechanisms by which NPPB and Cltx inhibit Transwell migration differ because NPPB inhibits the function of ClC-3 channes, whereas Cltx depletes functional ClC-3 channels from the membrane.…”

“…These results were surprising because they indicate that Cltx, unlike the related scorpion peptides, does not bind directly to a Cl − channel but that it binds to a cell-surface protein complex that contains MMP-2 and MT1-MMP (Deshane et al, 2003). Binding of Cltx causes internalization of this complex, along with ClC-3 channels, into caveolar rafts, which depletes the available membrane-associated Cl − channels.…”

“…The purified proteins were separated on SDSPage and identified by mass spectrometry. Unexpectedly, these studies isolated a protein complex consisting of MT1-MMP, MMP-2 and TIMP-2 (Deshane et al, 2003), which are all involved in the enzymatic degradation of the extracellular matrix (Chen and Wang, 1999;Forsyth et al, 1999). This protein complex is instrumental in facilitating the invasion of glioma cells (Belien et al, 1999;Nakada et al, 2003).…”

“…Through interaction with MMP-2/MT1-MMP it induces the internalization into caveolar rafts and, within 10-30 minutes depletes Cl − channels from the cell surface. Internalization occurs in conjunction with the Cltx peptide (Deshane et al, 2003) and this feature has been exploited recently to manufacture a Cltx-based multi-functional nanoprobe that either targets glioma cells or detecting them by magnetic resonance imaging and fluorescence microscopy (Veiseh et al, 2005). Moreover, the finding that the molecule is captured intracellularly by tumor cells and, hence, cannot be cleared explains why [131]I-labeled Cltx is still visualized in tumors 8 days after administration in clinical trials (Hockaday et al, 2005).…”