Dietary GSPs inhibit <i>in vivo</i> tumor xenograft growth in athymic nude mice.

<p>(<b>A</b>) Dietary administration of GSPs (0.5%, w/w) inhibits the growth of Miapaca-2 cells grown as xenografts in athymic nude mice. Average tumor volume ± SD/mouse (mm3) in each treatment group was measured on weekly basis. Tumor xenograft tissues were harvested at the termination of the experiment at 11 weeks and the wet weight of the tumor/mouse in each group is reported in grams as the mean ± SD. Statistical significance of difference between control and GSPs-treated groups was analyzed by one-way ANOVA followed by the Bonferroni <i>t</i> test, n = 8. Statistical significance <i>vs</i> tumors from non-GSPs-treated control mice, *<i>P</i><0.005. (<b>B</b>) The immunohistochemical detection of TUNEL-positive cells in tumor xenograft tissues from mice administered GSPs in the diet and mice not receiving GSPs are shown, and resultant data on TUNEL-positive cells are summarized. (<b>C</b>) Immunohistochemical detection of activated caspase-3-positive cells in tumor xenograft tissues from GSPs-treated and non-GSPs-treated mice. Immunohistochemical data in terms of percentage of positive cells are presented as the mean ± SD of 5–6 tumor samples from each group. TUNEL-positive or caspase-3-positive cells were counted in four to five different fields, and data are summarized in terms of percent positive cells from 5–6 tumor xenograft samples. Representative micrographs are shown. (<b>D</b>) Dietary GSPs inhibit the expression of anti-apoptotic proteins while increasing the expression of pro-apoptotic protein, Bax, and the levels of activated caspases-3 in tumor xenograft tissues. Tumor lysates were prepared from the tumor xenografts collected at the termination of the experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043064#pone-0043064-g005" target="_blank">Figure 5A</a> and subjected to western blot analysis, as described in Materials and methods. Representative blots are shown from independent experiments from at least five tumors from five different mice per group with identical observations.