Figure 2. Overexpression of CuZnSOD or EcSOD decreases hydroethidine fluorescence, cell growth and clonogenic survival in cell lines expressing K-ras.

A. The H6c7 cell line and its derivatives, Kras+, and KrasT, were infected with either AdEmpty, AdCuZnSOD, or AdEcSOD at 100 MOI. AdCuZnSOD 100 MOI increased CuZnSOD protein in all of the cell lines compared to controls and cells infected with AdEmpty vector (100 MOI). AdEcSOD 100 MOI also increased EcSOD protein in all of the cell lines compared to controls and cells infected with the AdEmpty vector (100 MOI). There are two bands noted on the EcSOD Western blot. The top band (32 kDa) is the EcSOD protein with heparin binding domain and the bottom band (29.5 kDa) corresponds to the EcSOD protein without heparin binding domain.

B. Intracellular hydroethidine fluorescence decreased in H6c7, Kras+, and KrasT cells infected with AdCuZnSOD, or AdEcSOD. Intracellular superoxide levels as measured by DHE decreased significantly 24–48 hours after infection with AdCuZnSOD, or AdEcSOD 100 MOI compared to the same cells infected with the AdEmpty vector. *p < 0.05 vs AdEmpty, means ± SEM, n = 3.

C. Cell growth was decreased after infection with the AdCuZnSOD and AdEcSOD vectors. The H6c7 cell line and its derivatives, Kras+, and KrasT, were transduced with AdEmpty, AdCuZnSOD or AdEcSOD at 100 MOI. AdCuZnSOD and AdEcSOD demonstrated reductions in cell growth in cells expressing the K-ras oncogene. Mean in vitro cell growth on day 6 is shown. Each point represents the mean values, n = 3. * p < 0.05 vs 100 MOI AdEmpty.

D. Clonogenic survival. CuZnSOD and EcSOD overexpression decreased clonogenic survival only in the cell lines with expression of K-ras oncogene. Dark bars represent the surviving fraction of cells treated with the AdEmpty vector (100 MOI), while the open bars represent the surviving fraction treated with AdCuZnSOD or AdEcSOD. Each point represents the mean values, with p < 0.05 vs. AdEmpty, n = 3.