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J. Biol. Chem., Vol. 280, Issue 8, 6950-6959, February 25, 2005

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Acceleration of Glutathione Efflux and Inhibition of -Glutamyltranspeptidase Sensitize Metastatic B16 Melanoma Cells to Endothelium-induced Cytotoxicity^*

María Benlloch, Angel Ortega¶, Paula Ferrer, Ramón Segarra, Elena Obrador, Miguel Asensi, Julián Carretero||, and José M. Estrela**

From the Departamento de Fisiología, Universidad de Valencia, 46010 Valencia, Spain and the ^||Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain

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 -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 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.

Received for publication, July 28, 2004 , and in revised form, November 19, 2004.

^* This work was supported by Grants SAF2003-01886 and VIN01-013 from the Ministerio de Educación y Ciencia and Grant GRUPOS03/185GVA from the Generalitat Valenciana (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a fellowship from the Ministerio de Educación y Ciencia.

^¶ Recipient of a fellowship from the Generalitat Valenciana.

^** To whom correspondence should be addressed: Dept. of Physiology, Faculty of Medicine and Odontology, 17 Ave. Blasco Ibañez, 46010 Valencia, Spain. Tel.: 34-963864646; Fax: 34-963864642; E-mail: jose.m.estrela{at}uv.es.

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