BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 5. NF-kB is activated downstream of the BLT2-Nox1-ROS signaling pathway. (A–C) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 mM), LY255283 (10 mM), or DMSO vehicle for 6 h (A), were transfected with BLT2 or control siRNAs for 48 h (B), or were incubated with NAC (5 mM), DPI (0.5 mM), or DMSO for 6 h (C). Cell lysates were then subjected to immunoblot analysis with antibodies to phosphorylated IkB-a (p-IkB-a) and to a-tubulin (loading control). Numbers under lanes indicate relative density of p-IkB-a from three independent experiments. (D and E) MDA-MB-231 and MDA-MB-435 cells were transiently transfected with control or BLT2 siRNAs and the pNF-kB-Luc reporter plasmid for 24 h, deprived of serum for 12 h, and then harvested (D). Alternatively, the cells were transiently transfected with pNF-kB-Luc, incubated with NAC (5 mM), DPI (0.5 mM), or DMSO for 6 h, and harvested (E). All cells were then assayed for relative luciferase activity. Data are expressed relative to the corresponding control value and are means6SD from three independent experiments. *P,0.05, {P,0.01, {P,0.005. doi:10.1371/journal.pone.0049186.g005 At the moment, the exact mechanism by which IL-8 trigger invasiveness is poorly understood in breast cancer cells. Previously, however, it was shown that up-regulation of matrix metallopro- teinases (MMPs), especially MMP-2 and MMP-9, is involved in IL-8-induced invasiveness [31]. Moreover, we recently observed that MMPs are indeed downstream components of BLT2 signaling, potentially mediating the invasiveness and metastasis of ovarian and bladder cancer cells [14,15]. Thus, we speculate that MMPs may act downstream of BLT2-IL-8 cascade in MDA- MB-231 and MDA-MB-435 cells, thereby mediating the inva- siveness. A detailed mechanism for the potential link between IL-8 and MMPs remains to be further characterized. As summarized in Figure 8, we have shown that a BLT2–Nox1–ROS–NF-kB signaling cascade up-regulates the production of IL-8 in MDA- MB-231 and MDA-MB-435 cells and thereby contributes to the invasiveness and metastasis of these aggressive breast cancer cells. Our findings have thus revealed a previously unrecognized role for BLT2 in the invasiveness of aggressive breast cancer cells, and they should both contribute to a better understanding of the molecular mechanisms of breast cancer progression as well as provide potential targets for the development of new therapeutics for this condition. Materials and Methods Materials Fetal bovine serum (FBS), horse serum, Dulbecco’s modified Eagle’s medium–F12 mixture (DMEM/F12), RPMI 1640, and nonessential amino acids were obtained from Life Technologies (Gaithersburg, MD). LTB4, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid (12-HHT), MK886, baicalein, OKY-046, U75302 and LY255283 were from Cayman Chemical (Ann Arbor, MI). Bovine serum albumin, dimethyl sulfoxide (DMSO), cholera enterotoxin, hydrocortisone, recombinant human epidermal growth factor, bovine insulin, diphenylene iodonium, and N-acetylcysteine were from Sigma-Aldrich (St. Louis, MO). Bay11-7082 and Bay11- 7085 were from Calbiochem (La Jolla, CA). All other chemicals were obtained from standard sources and were of molecular biology grade or higher. Cell Culture The human breast cancer cell lines MDA-MB-231 and MDA- MB-435 were obtained from the Korean Cell Line Bank (Seoul, Korea) and Jackson Laboratory (Bar Harbor, ME), respectively. PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49186