Background: Reactive oxygen species (ROS) play a role in the development of a malignant phenotype through oxidative damage to DNA. Antioxidant enzymes (AE) such as superoxide dismutases (cytosolic: SOD-1; mitochondrial: SOD-2) convert O₂^- to H₂O₂ and H₂O, while catalase converts H₂O₂ to O₂ and H₂O. It appears that SOD-2 may have an important role in tumor progression such as invasion and migration. Previous studies utilizing immunohistochemistry have shown a correlation between increased expression of SOD-2 and matrix metalloproteinase (MMP) expression in advanced stage breast, esophageal and gastric cancers. The role of oxidative stress and SOD-2 in bladder cancer has not been evaluated previously. Our purpose was to evaluate the effect of SOD-2 expression on MMP-9 activity and to determine the ability of bladder cancer cells to invade and migrate.
Methods:We transfected the 253J cell line with a CMV vector containing SOD-2 construct. We determined intracellular levels of SOD-2 with SOD activity gel and the MMP-9 activity through gelatin zymography. Then the three cell lines (253J, 253J-CMV, and 253J-SOD2) plated (in triplicates) at a density of 1.0 x 10⁵ cells/well in a BD Matrigel-coated Invasion Chamber and incubated at 37^oC in 5% CO₂ for 22 hours. The cells that invaded through the matrigel and migrated through the 8 µm membrane were stained with a fluorescent stain (Hoechst 33258). The invading cells were quantified in 5 high power fields (100X) from each membrane.
Results: There was a five-fold increase in the SOD-2 activity in 253J-SOD2 cells when compared to the controls. There was a corresponding increase in the expression and activity of MMP-9 in the cells expressing increased levels of SOD-2. The 253J-SOD2 cells invaded through the matrix at a significantly higher rate (214.11 cells/membrane +/- 103.11 cells/membrane) than the 253J-CMV (62.44 cells/membrane +/- 28.23 cells/membrane) or the 253J (29.78 cells/membrane +/- 20.35 cells/membrane) (p < 0.001).
Conclusions: Increased expression of SOD-2 results in an increased MMP-9 activity, and the ability to invade and migrate through extracellular matrix. Thus SOD-2 may play an important role in tumor progression and metastasis.

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