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.
Benlloch
María
M
Departamento de Fisiología, Universidad de Valencia, 46010 Valencia, Spain.
Ortega
Angel
A
Ferrer
Paula
P
Segarra
Ramón
R
Obrador
Elena
E
Asensi
Miguel
M
Carretero
Julián
J
Estrela
José M
JM
D000818:Animals; D017209:Apoptosis; D002448:Cell Adhesion; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004727:Endothelium / Q000166:cytology*; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009362:Neoplasm Metastasis; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D018384:Oxidative Stress; D019253:Proto-Oncogene Proteins c-bcl-2; D014700:Verapamil / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors*
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D014700:Verapamil; D005723:gamma-Glutamyltransferase; C421109:multidrug resistance-associated protein 1