1069. Increased Antioxidant Levels Following MnSOD-PL Transfection Results in Increased Radioprotection of Normal Tissue but Not Tumor Cells
Authors: Michael W. Epperly, Suhua Nie, Xichen Zhang, Joel S. Greenberger
Journal: Molecular Therapy (2005)

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Administration of Manganese Superoxide Dismutase Plasmid/Liposome complexes (MnSOD-PL) to the lung, esophagus, oral cavity, intestine and bladder has resulted in increased radioprotection of normal tissue. However, transfection of orthotopic tumors with MnSOD-PL has resulted in sensitization of the tumors to irradiation. One hypothesis is that the increased MnSOD expression produces increased hydrogen peroxide. Tumor cells may have a decreased ability to reduce hydrogen peroxide due to decreased antioxidant levels resulting in increased concentrations of hydrogen peroxide, leading to cell toxicity. Normal cells with higher levels of antioxidants may be able to reduce hydrogen peroxide and prevent cell death. To test this hypothesis, we measured antioxidant levels in a normal embryonic fibroblast cell and three tumor cell lines (3LL murine lung tumor, SSC-VII murine oral cavity cancer, or OC-19 human oral cavity cancer). Cells from each of the cell lines were transfected with a plasmid containing the human MnSOD transgene. Twenty-four hours later, the transfected cells as well as non-transfected cells were irradiated to 10 Gy. Irradiated cells as well as non-irradiated cells were harvested after 30 mins., and glutathione (GSH) or glutathione peroxidase (GPX) activities measured. All four cell lines following transfection with the MnSOD plasmid had significant increases in GSH levels. However, after irradiation, OC-19 and SCC-VII cells had significantly decreased GSH compared to GSH levels (65 |[plusmn]| 0.6 decreased to 43.6 |[plusmn]| 0.7 |[mu]|M, and 29.8 |[plusmn]| 1.9 decreased to 20.2 |[plusmn]| 1.4 |[mu]|M, respectively). In contrast the normal fibroblast cell line and the 3LL cell line showed no irradiation induced change in the level of GSH. GPX activity decreased following irradiation in all four nontransfected cell lines. Following transfection with the MnSOD plasmid there was significantly decreased GPX activity following irradiation in the 3LL, SCC-VII, and OC-19 lines (56.3 |[plusmn]| 2.5 to 49.0 |[plusmn]| 1.6, 66.1 |[plusmn]| 1.3 to 37.5 |[plusmn]| 1.1, 45.0 |[plusmn]| 1.6 to 37.1 |[plusmn]| 0.9 mU/mg protein, respectively). However, in the normal fibroblast cell line, there was no change in GPX activity following irradiation in transfection or the non-transfected cells (22.1 |[plusmn]| 3.1 and 21.5 |[plusmn]| 1.9, respectively). In vivo analysis of orthotopic SSC-VII tumors in the oral cavity of C3H/HeNHsd mice transfected by intravenous injections of MnSOD plasmid/liposome complexes showed decreases in total thiols following irradiation compared to nonirradiated orthotopic tumors (56.8 |[plusmn]| 1.1, and 33.9 |[plusmn]| 0.9 nmol/mg protein, respectively). Irradiation did not cause a decrease in protein SH groups in nontransfected SSC-VII tumors (56.8 |[plusmn]| 1.1 and 60.7 |[plusmn]| 6.4 nmol/mg protein, respectively). These data demonstrate that transfection with MnSOD-PL carries an irradiation induced decrease in antioxidants in tumor cells. Reduction in the antioxidant pools may increase the radiosensitivity of tumors.