Figure 6.
Suppression of lung metastasis of MDA-MB231.luc cells by EcSOD. To induce lung metastasis, athymic nude mice were injected with 2 × 106 of MDA-MB231.luc cells in the lateral tail vein. Overexpression of EcSOD was accomplished with AdEcSOD or AdΔHBD (1 × 1010 PFU) give intramuscularly 3 days before the breast cancer cell injection. AdEmpty was used as a vector control. (a, Top panel) Representative bioluminescent images of mice taken immediately post MDA-MB231.luc cell injection. (a, Bottom panel) Mice were imaged both dorsally and ventrally every week to monitor the metastasis of breast cancer cells. EcSOD-treated groups showed reduced development of lung metastasis compared with the AdEmpty-infected group. Time course images taken from the same animal in each group are shown here. (b) Luminescent signal (photon flux) of the metastasized MDA-MB231.luc cells was plotted over time showing a significant inhibition of lung metastasis by AdEcSOD and AdΔHBD versus AdEmpty (P<0.005). Data shown are mean values and s.d. from two independent experiments with ten mice per group. P-values were calculated using two-sided Student’s t-test. (c) Survival rate of mock (Control), AdEmpty, AdEcSOD and AdΔHBD-transduced athymic nude mice were plotted using Prism stats program (Kaplan–Meier survival) with the days elapsed following tail vein injection of MDA-MB231 luc cells. Survival curve for AdEcSOD and AdΔHBD were significantly different (P<0.0005) from both Control and AdEmpty-infected groups. (d) Plasma EcSOD activity levels in mice were evaluate using the WST SOD activity kit. Mice infected with AdEcSOD and AdΔHBD showed significantly higher levels of plasma EcSOD activities compared with AdEmpty-treated animals (P<0.0005). P-values were calculated using two-sided Student’s t-test. (e) Western blot analysis showing overexpression of both the full-length and truncated EcSOD in AdEcSOD- and AdΔHBD-treated animals. Plasma samples were analyzed using either an anti-human EcSOD antibody (top) or an anti-mouse EcSOD antibody (bottom). Low molecular weight heparin (LMWH) was administered intravenously at 1000 IU/kg of body weight to release full-length (FL) EcSOD into circulation and blood was collected from the animals after 30–60 min of heparin treatment. Protein concentration loaded per lane: 2 μg for the detection of human EcSOD (huEcSOD) and 8 μg for the detection of mouse EcSOD (moEcSOD).