GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells Tsuyoshi Honda, 1 Simona Coppola, 2 Lina Ghibelli, 2 Song H Cho, 1 Shunsuke Kagawa, 3 Kevin B Spurgers, 1 Shawn M Brisbay, 1 Jack A Roth, 3 Raymond E Meyn, 4 Bingliang Fang, 3 and Timothy J McDonnell 1 1 Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; 2 Institut tor Vergata Rome, Italy; 3 Department of Thoracic and Cardiovascular Surgery; and 4 Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. Cancer Gene Therapy (2004) 11, 249–255. doi:10.1038/sj.cgt.7700684 Published online 5 March 2004 Keywords: apoptosis; bax; redox; glutathione A poptosis is a highly conserved process throughout evolution and has a major role in maintaining tissue homeostasis. 1 A number of genes that regulate apoptosis are also conserved including members of the bcl-2 gene family. Members of this family can be divided into two functional groups: antiapoptotic genes that inhibit apop- tosis and proapoptotic genes that facilitate apoptosis. Bcl- 2 is the prototypic member of the former group and Bax, bcl-2-associated X protein, is the prototypic member of the latter. 2 Bax was first identified as a 21 kDa protein that coimmunoprecipitated with bcl-2. 3 Relative levels of hetero and homodimers comprising these proteins, including bcl-2 homodimers, may function in the manner of a rheostat to regulate apoptosis. Expression of Bax may be sufficient to induce a common pathway of apoptosis. It has been demonstrated that bax directly induces cytochrome c release from mitochondria, 4 sug- gesting that it is necessary for Bax protein to anchor to a membrane of the mitochondria to activate the apoptotic pathway. Glutathione (GSH) is a tripeptide, g-glutamyl cysteinyl glycine, which is considered to be a key molecule in the antioxidant pathway of cells. A major role of GSH is to maintain the intracellular redox state by scavenging reactive oxygen species. GSH has also been implicated in protection against the induction of apoptosis and necrotic cell death in a variety of cell types. 5,6 However, the function of GSH in apoptosis regulation is not completely understood. In this study, we determined that the H322 NSCLC cell line was significantly resistant to apoptosis induction following adenovirus bax gene transfer compared to other NSCLC cell lines. The resistance of H322 cells correlated with a significantly higher level of intracellular GSH compared to the sensitive cell lines. To gain a better understanding of the effect of GSH depletion on Bax-mediated apoptosis and to confirm if GSH depletion is a useful strategy to enhance apoptotic induction by Bax cotransfection, we performed GSH depletion in H322 and H226 cells after Bax transduction using buthionine sulfoximide (BSO), a specific inhibitor of de novo GSH synthesis. Quantification of GSH after treatment confirmed that BSO leads to a decrease in intracellular reduced GSH during the first 24 hours and resulted in a significant augmentation in Ad-bax- mediated cell death. This enhanced sensitivity correlated with an increase in both mitochondrial membrane- Received August 1, 2003. Address correspondence and reprint requests to: Dr Timothy J McDonnell, MD, PhD, Department of Molecular Pathology, Box 89, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. E-mail: tmcdonne@mail.mdanderson.org Cancer Gene Therapy (2004) 11, 249–255 r 2004 Nature Publishing Group All rights reserved 0929-1903/04 $25.00 www.nature.com/cgt