Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize metastatic B16 melanoma cells to endothelium-induced cytotoxicity.

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of gamma-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to approximately 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.