Molecular structure of CAPE

MDA-231 and MCF-7 Cells (3 × 10⁵) were cultured and incubated with CAPE for 72 h then treated with Annexin V-FITC and propidium iodide (PI) according to the Materials and Methods. After 72h, 40 µM CAPE caused an increase in Annexin V-positive apoptotic cells (Upper Right and Lower Right quadrant (UR+LR)) as compared to 0 µM in MDA-231 cells. In MCF-7 cells, 20 µM CAPE treatment resulted in an increase in Annexin V-positive apoptotic cells (UR+LR) when compared to 0 µM

a) Bovine capillary endothelial (BCE) cells were grown for 24 h in 24-well plates coated with Matrigel under normoxic or hypoxic conditions without treatment (controls), with 1.5-µM CAPE or bFGF (basic fibroblast growth factor, positive control). 1.5-µM CAPE inhibited tube formation under both normoxic and hypoxic conditions. b) MDA-231 cells were cultured at treated with different concentrations of CAPE (0–40 µM) and VEGF quantified in the media according to the Materials and Methods. CAPE inhibits VEGF secretion by MDA-231 cells in a dose dependent fashion from 0µM to 40 µM CAPE. This inhibition was statistically significant from 10µM CAPE (*p = 0.005), 20 µM CAPE (**p < 0.0001) up to 30µM CAPE (***p < 0.0004); n= 3. The experimental differences were determined by two-tailed Student’s t-test and p ≤0.05 was taken as a significant difference in all cases.

MDA-231 or MCF-7 cells/in 0.2 ml/flank (1:1 Matrigel and medium) were subcutaneously injected into each of the two flanks of female nude mice and fed with a diet containing cape or control diet. CAPE decreased the tumor volume of mouse xenografts by about 60% for MCF-7 (CAPE 50 nmol/mouse/day) xenografts with tumor volume greater than the means and 40% for MDA 231 xenografts (CAPE 10 nmol/mouse/day). The most consistent effects were evident using 50 nmol CAPE/ mouse/day diet, which caused statistically significant decline in growth of large tumors in MCF-7-inoculated mice 19, 26, 32, and 53 days after cell implantation, with 30% 38% 42% and 61% decreases in tumor volume, respectively, with p < 0.005 at CAPE 50 and 250 nmol/mouse/day at the end of the experiment. In MDA-231 cells, CAPE diet caused decreases in tumor volume at all the doses tested but, notably, there were more significant decreases in tumor volume (~40%; **p = 0.009 ) at the lowest dose of 10 nmol CAPE/mouse/day as compared to 50 and 250 nmol CAPE diets that yielded about 27% decrease in tumor volume (*p = 0.02). The experimental differences were determined by two-tailed Student’s t-test and p ≤0.05 was taken as a significant difference in all cases

The cell lines were cultured and then incubated with different concentrations of CAPE and analyzed using the MTT assay as described in the Materials and Methods section. a) The viability of MDA 231 (ER−) and MCF-7 (ER+) breast cancer cells was decreased by CAPE in a dose-dependent manner. b) In contrast, exposure to CAPE had no significant cytotoxic effect on the control cells, the immortal but non-tumorigenic MCF-10A (ER−) cells and MCF-12A (ER+) cells. All the determinations were carried out on 3–7 replicates. Growth of MDA-231 cells (first left bar) was dose-dependently (1–5 µM CAPE) and significantly inhibited (30.6%±6.9; **p<0.0001 at 72 h) when compared to untreated controls, while growth of control MCF-10A cells (second bar) was not affected at all. MCF-7 cell growth (third bar) was also dose- and time-dependently inhibited by 1–5 µM CAPE when incubated for 72 h with 5 µM CAPE suppressing cell growth down to 25.8%±9.3; *p<0.0001 of the untreated cells, while the growth of control MCF-12A cells (right bar) was decreased to 74.3%±1.9, with the overall difference being about 50% between the two ER (+) cell lines (Figure 2a). As shown in Figure 2b, the viability of MDA-231 and MCF-7 breast cancer cells was further decreased by higher doses of CAPE (up to 40 µM), with an IC₅₀ of approximately 15 µM. The experimental differences were determined by two-tailed Student’s t-test and p ≤0.05 was taken as a significant difference in all cases

MDA-231 and MCF-7 cells were treated with varying doses of CAPE for 48 h and cell cycle progression was determined using flow cytometry. CAPE exposure resulted in cell cycle arrest in the S-phase in a dose dependent manner for both MDA-231 (a) and MCF-7 (b) cells with an increase in the S-phase compartment and subsequent decrease in G0/G1 and G2/M phases. This figure is a representative of 3 independent experiments

a) MDA-231 cells were cultured and treated with CAPE for 48 h and protein lysates made as described in Materials and Methods. In MDA-231 cells, immunoblotting show CAPE-induced dose-dependent decreases in protein expression:IκB-β, p52 (3-fold) and p-IκB-α (20-fold), and the NF κB subunits p50, Rel B (1.5-fold), p-p65, p105 (2-fold), and p100 (5-fold). There were no changes observed in IκB-α and C-rel protein levels (blots not shown). Autoradiographs were scanned and bands quantified by a densitometry scanner. Densitometry results were expressed relative to a normalized protein control (β actin). b) CAPE inhibits both the total and phosphorylated forms of EGFR protein expression in a dose dependent manner. Representative blots are shown