Figure 6

Restoration of Sod3 in 66 cl4-VEGF-C KD cells partially rescues resistance to oxidative stress and tumor progression. (A) Expression of Sod3 in 66 cl4-VEGF-C KD cells (KD2 in Figure 5). Empty vector was also introduced into 66 cl4-scram and 66 cl4-VEGF-C KD2 cells as a control. Sod3 expression was assessed in each cell line using a real-time PCR SYBR Green assay (top). Expression of secreted Sod3 in the media of 66 cl4-scram, 66 cl4-VEGF-C KD and 66 cl4-VEGF-C KD + Sod3 cells was measured by Western blot analysis (bottom). (B) Flow cytometry was performed to measure cell death induced by H₂O₂ under each condition shown. Three independent experiments were performed, and the data were combined for quantitation. (C) Cells from the 66 cl4-scram, VEGF-C KD and VEGF-C KD + Sod3 lines were injected into the fourth mammary fat pad of female BALB/c mice. Tumor growth in the mice was measured using calipers and calculated using the formula V = 1/2(W)(W)(L) (top). A representative picture of tumors from each group shows that restoration of Sod3 in VEGF-C KD cells partially rescues the size of tumors compared to scramble control tumors (bottom). (D) Representative in vivo image of 66 cl4-scram, VEGF-C KD and VEGF-C KD + Sod3 groups at day 60 after injection (top). Quantitation of bioluminescence imaging (in photons per second) emanating from the region surrounding the lungs (bottom). Mice that did not develop primary tumors were excluded from the quantitation. (E) Incidence of tumor formation and metastasis in the groups of mice injected with 66 cl4-scram, VEGF-C KD or VEGF-C KD + Sod3 cells. Restoration of Sod3 expression in the VEGF-C KD cells increased the number of mice that developed primary tumors and metastases, although not to the levels observed in the scramble control group. Fisher's exact test (two-sided) results indicated a significant increase in the number of mice that developed metastases when Sod3 expression was restored in the VEGF-C KD tumors.