Figure 3. NBP-enhanced cancer-associated properties in breast cancer MCF7 cells.

(A) MCF10A cells (10A) were treated with NBP, and MCF7 cells were treated with NBP, NB, or PhIP (P) for 24 h. DNA damage was measured by a comet assay and normalized by the value of average tail moment determined in untreated counterpart cells, set as 1 (X, arbitrary unit). (B-1 to B-6) MCF7 cells were exposed to NBP, NB, or PhIP for five cycles (NBP5, NB5, and P5). The NBP20 and MCF10A-Ras (Ras) cell lines were used as comparisons. (B-1) To determine cellular acquisition of RDGF, cells were maintained in LM medium for 10 days. Cell colonies ≥0.5 mm diameter were counted. (B-2) To determine cellular acquisition of AIG, cells were seeded in soft agar for 14 days. Cell colonies ≥0.1 mm diameter were counted. Cellular migratory (B-3) and invasive (B-4) activities were determined by counting the numbers of cells translocated through a polycarbonate filter without or with coated Matrigel, respectively, in 10 arbitrary visual fields. (B-5) Relative level of ROS as fold induction (X, arbitrary unit) was normalized by the level determined in untreated cells, set as 1. (B-6) Cell lysates were analyzed by immunoblotting using specific antibodies to detect levels of H-Ras, p-Erk1/2, Erk1/2, and Nox-1, with β-actin as a control, and these levels were quantified by densitometry. Levels of H-Ras (Ras/actin) and Nox-1 (Nox/actin) were calculated by normalizing with the level of β-actin and the level set in untreated control cells as 1 (X, arbitrary unit). Levels of specific phosphorylation of Erk1/2 (p/Erk) were calculated by normalizing the levels of p-Erk1/2 with the levels of Erk1/2, then the level set in control cells as 1 (X, arbitrary unit). Columns, mean of triplicates; bars, SD. All results are representative of three independent experiments. Statistical significance is indicated by * P<0.05, ** P<0.01, and *** P<0.001.