Oral administration of BBE decreases HE tumor growth in vivo and prolongs survival. (a) Female 129 P/3 mice (n = 6 per group) injected with EOMA cells were treated with a daily oral gavage feeding of vehicle control (ddH₂O) or BBE at the doses indicated starting immediately after EOMA cell injection. Tumor specimens were collected 7 days after EOMA cell injection, and volume determined using caliper measurements (length × width × height). Tumor volume was significantly decreased in all BBE treated mice compared to vehicle fed controls (mean ± SD; **p < 0.01 ANOVA); (b) Effects of BBE on survival was compared between mice treated with oral BBE at 20 mg/kg (n = 18) versus vehicle control (n = 6). Kaplan–Meier survival curve shows a significant increase in survival time for mice receiving BBE compared to those given vehicle control (p < 0.02 log-rank analysis).

Oral administration of BBE protects against oxidative events in lipid and aqueous compartments in the blood. Mouse blood was collected at time of necropsy performed 7 days after EOMA cell injection (n = 6 mice per treatment group). (a) Oxidative end products in the lipid compartment were detected using the TBARS assay. A dose-dependent statistically significant decrease in lipid peroxidation was seen for all doses of BBE treatment compared to vehicle-treated controls (samples run in triplicate ×2; **p < 0.01 ANOVA); (b) BBE improved serum glutathione redox state (GSH/GSSG). A dose-dependent increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was observed up to a dose of 20 mg/kg of BBE. There was a slight increase in GSH/GGSG ratio observed at the 200 mg/kg dose. The difference in GSH/GSSG ratio was statistically significant for all doses compared to the vehicle treated (ddH₂O) control (mean ± SD; *p < 0.03; **p < 0.01 ANOVA).

In vitro assays of BBE toxicity. LDH assay to test cell viability showed that the BBE treatment in vitro is nontoxic to EOMA cells compared to untreated controls with no difference in the amount of LDH secreted or retained by untreated vs. BBE treated cells. Results are normalized by reporting secreted LDH as a percentage of total LDH (LDH cell sups + LDH cell lysate) for each sample. Samples run in triplicate × 3 (mean ± SD, ANOVA).

BBE inhibited JNK signal transduction pathway in EOMA cells. (a) EOMA cells were pretreated with BBE (150 μg/ml) for 16 h, and then JNK phosphorylation was either stimulated with TNF-α (400 IU/ml) × 15 min, or inhibited using JNK inhibitor SP600125 (30 μM) × 15 min. JNK phosphorylation was detected using a primary antibody specific for the phosphorylated JNK isoforms and compared to the total amount of JNK available using a different primary antibody to detect all JNK isoforms on a duplicate sample in a separate well. Results for each well were normalized based on relative protein content using a protein stain provided in the kit and measuring absorbance at 595 nm. Results shown in the figure represent the ratio of phosphorylated JNK versus total JNK for each sample. Samples run in triplicate ×2 (mean ± SD; *p < 0.01 ANOVA); (b) BBE inhibited cJun phosphorylation. EOMA cells were pretreated with BBE (150 μg/ml) for 16 h and stimulated with TNF-α (400 IU/ml) × 1 h. Nuclear extract from EOMA cells (2 ug/well) were placed in a 96-well plate coated with a consensus oligonucleotide for cJun binding within an AP-1 promoter sequence. A primary antibody was used to detect only phosphorylated c-Jun bound to the consensus oligonucleotide and a secondary HRP-conjugated antibody used for colorimetric measurement. Pretreatment of EOMA cells with BBE significantly inhibited TNF-α induction of c-Jun phosphorylation. Samples were run in triplicate ×2 (mean ± SD; *p < 0.01 ANOVA); (c) BBE inhibited AP-1 activation. The pAP-1luc plasmid was used to detect binding of AP-1 to transcriptional activation sites contained within the promoter for the firefly luciferase reporter gene. AP-1 binding was detected using a luminometer to quantify luciferase levels Treatment of EOMA cells with BBE (150 μg/ml) for 16 h inhibited AP-1 promoter binding activity compared to both basal levels of expression in untreated cells and cells stimulated with TNF-α (400 IU/ml) × 1 h. Samples were run in quadruplicate ×2 (mean ± SD; *p < 0.01, ANOVA).

BBE inhibited MCP-1 protein expression and NF-κB transactivation. (a) Pretreatment of EOMA cells with BBE (150 μg/ml) for 16 h significantly inhibited both basal as well as TNF-α (400 IU/ml × 2 h) inducible MCP-1 protein expression detected by ELISA. Samples were run in triplicate ×3 (mean ± SD; *p < 0.01 ANOVA). (b) EOMA cells were pretreated for 16 h with BBE (150 μg/ml) and then stimulated with TNF-α (400 IU/ml) for 15 min. Pyrollidine dithiocarbamate (PDTC, 0.2 mM), a standard inhibitor of NF-κB, was used as control and added at the same time as the TNF-α. Nuclear extract from EOMA cells (10 μg/well) were placed in a 96-well plate coated with a consensus oligonucleotide for p65 binding. A primary antibody was used to detect p65 bound to the consensus oligonucleotide and a secondary HRP-conjugated antibody used for colorimetric measurement. Pretreatment of EOMA cells with BBE significantly inhibited TNF-α induction of p65/NF-κB nuclear translocation. Samples were run in triplicate × 2 (mean ± SD; * p < 0.01 ANOVA).

BBE inhibited the angiogenic activity of EOMA cells. (a) EOMA cells grown on Matrigel demonstrate angiogenic behavior/tube formation which is inhibited by treatment with BBE (150 μg/ml) or JNK inhibitor SP600125 (30 μM). Imaging of EOMA cells grown on Matrigel was done using calcein AM and DAPI was used to label nuclei. (a-1) untreated, (a-2) vehicle control, (a-3) BBE treatment (150 μg/ml), (a-4) SP600125 (30 μM) (a-d scale bar = 200 μm), (b) Tube formation on Matrigel was quantified using the AxioVision software. * p < 0.01; (c) BBE inhibited EOMA cell migration. EOMA cells cultured in LSM (0.5% FBS), treated with BBE (150 μg/ml) and placed in the upper chamber of a transwell system showed decreased migration toward the lower chamber compared to cells in the same conditions treated with vehicle control (0.3% DMSO). Cell migration was also inhibited by treatment with the JNK inhibitor SP600125 (30 μM). Lower chambers were filled with NGM, which contains a higher serum content (10% FBS), than the LSM. No cells were placed in the lower chambers. There were 6 samples per treatment group with 3 cell migration measurements per sample *p < 0.01 ANOVA. (d) BrdU assay showed a significant decrease in the rate of EOMA cell proliferation with BBE treatment (150 μg/ml) compared to vehicle treated cells. A significant difference was also seen when treating cells with JNK inhibitor SP600125 (30 μM) suggesting that the observed effects of BBE on cell proliferation may be mediated through JNK. Samples run in triplicate × 3 (mean ± SD *p < 0.01, ANOVA)