Bcl-2 and Mn-SOD antisense oligodeoxynucleotides and a glutamine-enriched diet facilitate elimination of highly resistant B16 melanoma cells by tumor necrosis factor-α and chemotherapy

(1)Mitochondrial glutathione ( mtGSH) depletion increases sensitivity of Bcl- 2- overexpressing B16 melanoma ( B16M)- F10 cells ( high metastatic potential) to tumor necrosis factor- alpha( TNF- alpha)- induced oxidative stress and death in vitro. In vivo, mtGSH depletion in B16M- F10 cells was achieved by feeding mice ( where the B16M-F10 grew as a solid tumor in the footpad) with an L- glutamine ( L- Gln)- enriched diet, which promoted in the tumor cells an increase in glutaminase activity, accumulation of cytosolic L- glutamate, and competitive inhibition of GSH transport into mitochondria. L- Gln- adapted B16M- F10 cells, isolated using anti- Met- 72 monoclonal antibodies and flow cytometry- coupled cell sorting, were injected into the portal vein to produce hepatic metastases. In L- Gln- adapted invasive ( iB16M- Gln(+)) cells, isolated from the liver by the same methodology and treated with TNF- alpha and an antisense Bcl- 2 oligodeoxynucleotide, viability decreased to similar to 12%. iB16M-Gln (+) cell death associated with increased generation of O-2(center dot). and H2O2, opening of the mitochondrial permeability transition pore complex, and release of proapoptotic molecular signals. Activation of cell death mechanisms was prevented by GSH ester- induced mtGSH replenishment. The oxidative stress- resistant survivors showed an adaptive response that includes overexpression of manganese-containing superoxide dismutase ( Mn- SOD) and catalase activities. By treating iB16M- Gln (+) cells with a double anti- antisense therapy ( Bcl- 2 and SOD2 antisense oligodeoxynucleotides) and TNF- alpha, metastatic cell survival decreased to similar to 1%. Chemotherapy ( taxol plus daunorubicin) easily removed this minimum percentage of survivors. This contribution identifies critical molecules that can be sequentially targeted to facilitate elimination of highly resistant metastatic cells.