Abstract 4510: Similarities in trans-Golgi localization of chlorotoxin in tumor and in normal vascular endothelial cells

Abstract

Abstract Introduction: Chlorotoxin (TM601), a scorpion derived 36-amino acid peptide is a new experimental drug currently in Phase II clinical trials for adult recurrent glioma. Despite its known specificity for tumor cells, its exact mode of action is not known but anti-angiogenic effects have been observed. Here we provide evidence that TM601 localizes near the Golgi complex in human glioma (U373, U87), lung carcinoma (A549), and in normal human vascular endothelial cells (HUVEC) but not in normal human dermal fibroblasts (HDF) and astrocytes (NHA). TM601 enters glioma cells via clathrin-mediated transport and affects level of several Golgi associated peptides. Methods: Cells were treated with or without 10 µM TM601 or fluorescent TM601 for 1 or 24 hours. We evaluated: 1) TM601 cellular localization in relation to the Golgi (antibodies to TM601 and Golgin97 marker) and lysosomes (fluorescent trackers) using immunocytochemistry or direct immunofluorescence visually and quantitatively by confocal microscopy; 2) TM601 cellular entry in U373 and HDF cells using an inhibitor of clathrin-dependent endocytosis (chlorpromazine 10 µg/ml) or inhibitor of macropinocytosis (amiloride 300 µM). Cell lysates were prepared and proteins evaluated by Western blot; 3) TM601 effects on the U373 proteome in the sucrose fractionated Golgi-enriched fraction using liquid chromatography coupled to tandem mass spectrometry. Results: 1) In all tumor cells and HUVEC cells, TM601 was observed as an intense granular staining at the trans-Golgi. In normal fibroblasts and astrocytes it was found at a low level in the cytoplasm. In U373 glioma, but not in the normal HDF and NHA cells, TM601 showed a statistically significant colocalization with the LysoTracker (p=3.9E-05) that stained Golgi associated lysosomal vesicles. 2) A statistically significant effect on TM601 cellular level in U373 was observed after treatment with chlorpromazine and in HDF (but not U373) with amiloride. 3) Preliminary data (n=2) of Golgi-enriched fractions from TM601 treated U373 cells showed that TM601 treatment significantly affected levels of several peptides. Further evaluation by Western blots is planned. Summary: Our data show similarity between glioma, lung carcinoma and the normal vascular endothelial cells in the cellular uptake/transport of TM601 toward Golgi complexes. These cells differ drastically from other normal cells such as HDF and NHA which present low cytoplasmic level of TM601. It is expected that endothelial cell transport of TM601 may occur via receptor mediated clathrin dependent endocytosis pathway as in the glioma cells. In addition, our preliminary data showed TM601 effect on the level of several Golgi associated proteins in glioma cells. A similar study with endothelial cells is planned. The information obtained from these studies will assist in understanding how TM601 affects angiogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4510.