Immediate action of imatinib mesylate on calcium signaling in gastrointestinal stromal tumors (GIST).

Abstract

e20503 Background: In the majority of gastrointestinal stromal tumors (GISTs) mutation in tyrosine kinase receptors, c-KIT or PDGFRA, is thought to be the primary event. Both of these receptors are targets of tyrosine kinase inhibitors (TKIs) like imatinib mesylate. Downstream of the receptors the cellular signaling machinery is still obscure. In many cell types, including interstitial cells of Cajal, intracellular calcium (Cai) is accepted as an essential part of cell signaling and cellular function, including apoptosis. In this study we have evaluated Cai handling using electrophysiology and cell imaging in primary gastric GISTs. Methods: GIST cell cultures were prepared from primary untreated GISTs taken directly from surgery. Samples were also taken for pathology examination, verifying GIST diagnosis, and analyzed for mutations, showing a c-KIT exon 11 mutation in all included material. Measurements of Cai by Fura-2/AM fluorescence, patch-clamp techniques and molecular biology techniques were used in this study. Results: Cai measurements traced a persistent increase in Cai after stimulation with thapsigargin and a decrease upon stimulation with high levels of K+. After a short 10 uM imatinib mesylate exposure the thapsigargin effect was completely abolished. The source of Cai is most likely an influx through the plasma membrane. Furthermore, RT-PCR and Western-blot experiments confirmed the existence of store operated calcium channels including transient receptor potential canonical type (TRPC) ion channels and its associated proteins. Conclusions: This study shows that Imatinib mesylate, the most widely used TKI, has immediate and potent action on Cai handling in primary untreated GIST cells. Cai handling in GIST is complex and is likely to involve release from intracellular stores and additional influx through TRPC channels, as monitored by patch-clamp and the expression of TRPC channels and its regulatory proteins. We propose that alterations in Cai handling can, at least partly, explain the apoptotic effect of TKIs in GISTs.