BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 1. Up-regulation of BLT2 in highly invasive breast cancer cells. (A and C) Semiquantitative RT-PCR (left panel) and quantitative real- time PCR (center and right panels) analysis of BLT1 and BLT2 mRNAs in MCF-10A (control) as well as MDA-MB-231 (A) and MDA-MB-435 (C) cells. Data are representative of three independent experiments. (B and D) Flow cytometric analysis of surface BLT2 expression. All quantitative data are expressed relative to the value for MCF-10A cells and are means6SD from three independent experiments. *P,0.05. doi:10.1371/journal.pone.0049186.g001 IkB-a phosphorylation in MDA-MB-231 and MDA-MB-435 cells (Figure 5C). Consistent with these results, the activity of an NF- kB–dependent luciferase reporter gene was also significantly inhibited by BLT2 depletion (Figure 5D) as well as by NAC or DPI (Figure 5E). Together, these results suggested that NF-kB activation occurs downstream of BLT2-ROS signaling in MDA- MB-231 and MDA-MB-435 cells. BLT2–ROS–NF-kB Signaling Induces IL-8 Expression in MDA-MB-231 and MDA-MB-435 Cells IL-8 expression is associated with the invasiveness and metastatic potential of aggressive breast cancer cells [23,25]. We therefore examined the possible role of IL-8 in regulation of the invasiveness of MDA-MB-231 and MDA-MB-435 cells by the BLT2 signaling cascade. The amounts of IL-8 mRNA and protein were markedly increased in MDA-MB-231 and MDA-MB-435 cells compared with control MCF-10A cells (Figure 6A). Further- more, the abundance of IL-8 mRNA in MDA-MB-231 and MDA- MB-435 cells was markedly reduced by exposure to LY255283, whereas it was little affected by treatment with U75302 (Figure S3A). In addition, depletion of BLT2 by RNAi resulted in a similar marked reduction in the amounts of IL-8 mRNA (Figure S3A) and protein (Figure 6B) in these cells, indicating that BLT2 signaling up-regulates IL-8 expression. Among the BLT2 ligands tested, we observed that only LTB4 could enhance the levels of IL-8 (Figure S4A) and invasiveness (Figure S4C) in MDA-MB-231 and MDA- MB-435 cells. In addition, LTB4 synthesis inhibitor MK886 suppressed the levels of IL-8 (Figure S4B) and invasiveness (Figure S4D), while 12-lipoxygenase inhibitor (baicalein) and thrombox- ane synthesis inhibitor (OKY-046) had no effect (data not shown). PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49186