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Some recent evidence has suggested a protective role of zinc against cancer. The mechanism by which zinc exerts this action has not been defined and, in particular, it has not been clarified whether zinc may directly act on cancer cells and the molecular mechanisms involved in this effect. In this study, we examined the in vitro effect of zinc on the apoptosis of mouse TS/A mammary adenocarcinoma cells, studying the zinc-dependent modulation of the intracellular levels of reactive oxygen species (ROS) and of p53 and Fas/Fas ligand pathways. We showed that zinc concentrations ranging from 33.7 to 75 muM Zn(2+) induced apoptosis in mammary cancer cells. The apoptosis was associated with an increased production of intracellular ROS, and of p53 and Fas/Fas ligand mRNA and protein. Zn(2+) induced a faint metallothionein response in TS/A cells in comparison with mouse lymphocytes. The treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and Fas ligand protein induced by zinc. The data demonstrate that zinc exerts a direct action on mammary cancer cells inducing ROS-mediated apoptosis and that the effect may be mediated by the ROS-dependent induction of p53 and Fas/Fas ligand.

Numerous studies have demonstrated the efficacy of cytokine gene-engineered tumor cells to induce tumor rejection and specific memory acquisition into syngeneic immunocompetent mice by activation of host-dependent antitumor responses. A progressive immune dysfunction, mainly involving thymus-dependent specific immunity, occurs during aging. In this study we evaluated whether the injection of IL-2 gene-transfected tumor cells in old mice causes an immune activation which results in tumor rejection and induction of specific immune memory as occurs in young animals. Young and old mice were inoculated with syngeneic parental mammary adenocarcinoma cells (TS/A p.c.) or with TS/A cells engineered to release IL-2 (TS/A-IL2). Three clones of TS/A-IL-2 cells were used producing low (30 U, B1.30), intermediate (3600 U, B6.3600), or high (6000 U, B4.6000) IL-2. While the B1.30 clone grew in 100% of mice, the B6.3600 and B4.6000 clones were promptly rejected in both young and old animals. In young mice, rejection was associated with a large neutrophil and macrophage infiltration, with a minor number of CD4+ and CD8+ lymphocytes. In old mice, neutrophils and macrophages were the main cells involved in tumor rejection whereas both CD4+ and CD8+ lymphocytes were scarcely present in tumoral infiltrate. A lower number of apoptotic tumor cells was found in TS/A-IL2-challenged old mice in comparison with young animals. To test whether the injection of TS/A-IL2 cells induced a specific immune memory, mice with no tumors after the challenge with B6.3600 and B4.6000 clones received a lethal challenge of TS/A p.c. 90% and 30% of young mice previously injected with B4.6000 or B6.3600 clones, respectively, rejected TS/A p.c. In old mice, B4.6000 cells did not confer protection, whereas only 10% of mice which received B6.3600 cells were able to reject TS/A p.c. Neither the graft of a young thymus or the adoptive transfer of young T lymphocytes to old mice induced specific immune memory for TS/A p.c. in old animals. These data suggest the necessity to refine antitumor vaccination procedures in aging.