Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

We have found that in human gliomas, lung carcinoma and normal vascular endothelial cells, TM601 localizes near trans-Golgi while in normal human dermal fibroblasts (NHDF) and astrocytes it is dispersed in the cytoplasm. The uptake of TM601 by U373 glioma cells is rapid, concentration and time dependent, not affected by inhibitors such as filipin (caveolae-dependent endocytosis) and amiloride (non-selective macropinocytosis), but significantly affected by chlorpromazine (clathrin-dependent intracellular transport of coated pits) resulting in intracellular build-up of the drug and clathrin near the Golgi. In contrast, TM601 uptake by NHDF cells was significantly affected by amiloride indicating that macropinocytosis is the dominant uptake route of TM601 in these cells.In conclusion, we found a distinct cellular localization pattern and uptake of TM601 by glioma cells differing from that found in normal cells. Further insight into the cellular processing of TM601 should assist in the development of effective anti-glioma therapeutic modalities.TM601 is a pure, chemically synthesized chlorotoxin of 36 amino acids which was first purified from the venom of the scorpion Leiurus quinquestriatus. For clinical development, chlorotoxin has been manufactured using solid phase chemical syntheses, and it is called TM601. It is known that TM601 has a similar homology and structure as other venom peptides and in that resembles members of the family of small disulfide-rich proteins characterized by a knotted topology [1]. It was first found to be an inhibitor of small conductance chloride channels [2]. Both inhibition of invasion and inhibition of metalloproteinase-2 (MMP-2) activity have been previously observed in glioma cells treated with chlorotoxin [3,4]. Recently, a similar in vitro finding was reported for human umbilical vein endothelial cells (HUVEC) treated with TM601 [5]. TM601 is not cytostatic or cytotoxic to tumor or vascular endothelial cells in vitro. However, poten