Histology of MDA-MB-231 xenografts.

<p>Tumors dissected from animals were sectioned and stained with (A) H&E, and (B) mitotic figures (arrowheads), and (C) apoptotic bodies (arrows) evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>.</p

Expression of CD31 and VEGF in tumors.

<p>The expression of (A) CD31, and (B) VEGF were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are mean ± SD (n = 6–10), * significantly different from control (p<0.05) by ANOVA.</p

NAHA inhibits invasive behavior of breast cancer cells and capillary morphogenesis of endothelial cells.

<p>(A) Cell adhesion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and cell adhesion to vitronectin determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (B) Cell migration. Cell migration of MDA-MB-231 cells was determined after 5 hours of incubation in the presence of NAHA (0–50 µM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (C) Cell invasion. Invasion of MDA-MB-231 cells through Matrigel was determined after 24 hours of incubation in the presence of NAHA (0–50 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (D) uPA secretion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, and the expression of uPA detected in conditioned media from the same amount of cells with anti-uPA antibody by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The results are representative of three independent experiments. (E) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, media collected and secretion of VEGF determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (F) HAECs were treated with NAHA (0–50 µM) for 16 hours. Capillary morphogenesis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05.</p

NAHA inhibits growth of breast cancer cells.

<p>(A) Structure of NAHA, <i>2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide</i>. (B) MDA-MB-231, (C) MCF-7, (D) MCF-10A, (E) HMEC cells were treated with NAHA (0–50 µM). Cell proliferation was determined by the tetrazolium salt method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Cell viability was determined by trypan blue staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are the means ± SD. Similar results were obtained in at least two additional experiments. * p<0.05 for cell proliferation, # p<0.05 for cell viability. (F) Anchorage-independent growth (colony formation) of MDA-MB-231 cells was assessed on 1% agarose after incubation with NAHA (a – 0, b – 10 µM, c -25 µM, d – 50 µM) for 14 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. (G) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and whole cell extracts were subjected to Western blot analysis with anti-Cdk2 and anti-CDC20 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The equal protein loading was verified with anti β-actin antibodies. The rfesults are representative of three independent experiments.</p

NAHA inhibits growth of breast cancer cells <i>in vivo</i>.

<p>(A) Tumor volume. MDA-MB-231 cells were inoculated s.c. in the right flank of each mouse. Seven days after inoculation, mice were randomly divided into 3 groups (n = 10) and treated with NAHA (i.p., 10 mg or 50 mg/kg of body weight/3 times per week) or vehicle (n = 11) for 32 days. Tumor volume was measured three times a week. Data are mean ± SD, * significantly different from control (p<0.05) using a two-sample Student t-test. (B) Tumor weight, * p<0.05. (C) Body weight of mice.</p

Effect of GLT on the PhIP/DSS induced colon carcinogenesis and inflammation.

<p>Tumor incidence are summarized using percentage of animals with tumors and compared across groups using Fisher's exact test and the Bonferonni correction for multiple comparisons: ^a p<0.001 PhIP/DSS vs control, PhIP, DSS; ^b p<0.02 PhIP/DSS+500 GLT vs PhIP/DSS.</p><p>Tumor multiplicity are summarized using mean ± SD and compared across all group using Kruskal-Wallis one way analysis of variance on ranks and the Dunn's method for the multiple comparisons: ^a p<0.05 PhIP/DSS vs control PhIP, DSS; ^b p<0.05 PhIP/DSS+500 GLT vs PhIP/DSS.</p><p>Neoplastic index is summarized using median (min, max) and compared across groups using the Kruskal-Wallis test. Comparisons of each group to control performed using Mann-Whitney U tests with significance levels adjusted using the Bonferroni correction: ^a p<0.001 control vs PhIP; ^b p<0.001 control vs PhIP/DSS.</p><p>Data for colon length summarized using mean ± SD and compared across all group using ANOVA and Dunnett's post hoc test: ^ap<0.001 control vs DSS, control vs PhIP/DSS; ^bp<0.001 PhIP/DSS vs PhIP/DSS+100 GLT, PhIP/DSS vs PhIP/DSS+500 GLT.</p

GLT suppresses PhIP/DSS induced formation of colon tumors and inhibits focal hyperplasia and ACF formation.

<p>(A) Schematic of the animal treatment. The details of the treatment are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. (B) Body weight of control animals (black circle), animals treated with PhIP (white square), DSS (black square), PhIP/DSS (white circle), PhIP/DSS+GLT 100 mg/kg of body weight (black triangle), and PhIP/DSS+GLT 500 mg/kg of body weight (white traingle) during the experiment. (C) H&E staining of representative samples from animal experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#pone-0047873-g001" target="_blank">Figure 1A</a>. (D) Focal hyperplasia was evaluated by the histological analysis after H&E staining in colon tissue samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Results are means ± SD (n = 6–9 mice/per group). (E) ACF formation was evaluated after methylene blue staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Results are means ± SD (n = 10 foci/3 mice/per group), *p<0.05 by ANOVA.</p

GLT down-regulates expression of COX-2 in colon tissue.

<p>(A) Immunohistochemistry and (B) quantification of COX-2 positive cells were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047873#s4" target="_blank"><i>Materials and Methods</i></a>. Box plots represent 5^th/10^th percentiles, horizontal bars represent median values, and whiskers indicate minimum to maximum values. Significant differences (*p<0.05) were observed among PhIP/DSS vs. control, PhIP/DSS vs PhIP/DSS+GLT 100, and PhIP/DSS vs. PhIP/DSS+GLT 100.</p

Effect of GLT on the lipid metabolism.

<p>Values are Mean ± S.D. (n = 6), HDL, high-density lipoprotein. No significant difference from the control group.</p

Effect of GLT on the liver function and serum glucose.

<p>Values are Mean ± S.D. (n = 6), ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase. No significant difference from the control group.</p