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J. Biol. Chem., Vol. 278, Issue 41, 39591-39599, October 10, 2003

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Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium^*

Angel Ortega , Paula Ferrer , Julian Carretero, Elena Obrador, Miguel Asensi, José A. Pellicer and José M. Estrela 

From the Department of Physiology, University of Valencia, Av. Blasco Ibañez 17, Valencia 46010, Spain

B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from 19% (controls) to 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). L-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of L-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.

Received for publication, April 10, 2003 , and in revised form, June 25, 2003.

^* This work was supported by Ministerio de Ciencia y Tecnologáa Grants SAF99–112 and VIN01–013 and Generalitat Valenciana Grant GV01-140 (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.

These two authors held fellowships from the MCYT.

To whom correspondence should be addressed. Tel.: 34-963864646; Fax: 34-963864642; E-mail: jose.m.estrela{at}uv.es.

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