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Figure 6. In vivo validation of withaferin A-mediated anti-cancer effects. (a) Representative bioluminescence images of control and withaferin A-treatment mice subcutaneously inoculated with floating (F) cells. Withaferin A treatment was initiated 2 weeks post-tumor implantation to allow tumor establishment. Note, inoculated F cells were found to start disseminating anteriorly during week 3-4 as compared to localized signal found in withaferin A-treated mice (left panels). The bioluminescent intensity was significantly lower in withaferin A-treated group than in the control group. The data were quantitatively represented by the fold change in bioluminescence intensity over time (right panel); (b) systematic injection of F cells mimicking metastatic model. Representative images of F cell-inoculated mice (via tail vein injection) demonstrated that withaferin A treatment given 2 weeks after intervenous tumor injection not only suppressed tumor growth but also controlled the spread of F cells; (c) immunohistochemical analysis of tumor samples harvested from both control and withaferin A-treated animals (from subcutaneous tumor samples). Representative sections of the tumor samples were stained with CXCR4, caspase-3, and poly ADP-ribose polymerase antibodies. Samples treated with withaferin A demonstrated decreased CXCR4 immunostaining, while increased caspase-3 and poly ADP-ribose polymerase immunostaining as compared with the control samples (×200)