Figure 6.

I3C effects on the growth as well as PTEN, NEDD4-1 and phosphorylated Akt levels in melanoma cell-derived tumor xenografts in athymic mice. (A) One million G-361 melanoma cells were implanted in each lateral flank of NIH III athymic mice, and after palpable tumors were detected (mean starting volume of 146 ± 10 mm³), the mice were injected subcutaneously with either I3C (200 mg/kg body weight) or with the DMSO vehicle control over an 18-day time course. The resulting tumor volumes were measured and calculated as described in the Methods section. The data represents the means ± S.E.M. (**P< 0.01) for 5 mice / group, each with two tumors, one in each flank. The micrograph insert shows tumors harvested at day 18 from I3C treated or vehicle control treated animals. (B) At terminal sacrifice, the G-361 cell derived tumor xenografts were harvested, and a portion of each tumor was fixed in 4% paraformaldehyde as described in the methods section. 10 μm cryostat sections from tumors excised from I3C treated and untreated animals were analyzed for PTEN expression by immunofluorescence studies using primary antibodies to PTEN. The staining patterns were compared to DAPI staining of the cell nuclei and merged images are shown in the bottom set of panels. (C) The protein densities of PTEN, NEDD4-1 and phosphorylated Akt protein in tumor xenografts from I3C treated and untreated animals were determined by immunofluorescence staining of tumor sections. The results from three independent experiments were quantified.