Sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables such as broccoli, possesses anti-inflammatory and chemopreventive activities. Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins mediating inflammatory processes, is inappropriately over-expressed in various cancers. In the present study, we investigated the effect of SFN on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in human breast epithelial (MCF-10A) cells. Treatment of MCF-10A cells with SFN (12.5 and 25 μM) significantly reduced COX-2 expression induced by TPA (10 nM). NF-κB has been known as a key transcription factor responsible for regulating the COX-2 gene expression. SFN suppressed the TPA-induced DNA-binding and transcriptional activity of NF-κB in MCF10A cells. SFN also inhibited nuclear translocation of p65, a functionally active subunit of NF-κB. Moreover, the phosphorylation and subsequent degradation of IκB-α were suppressed by SFN in TPA-stimulated MCF-10A cells. SFN inhibited TPA-induced IκB kinase (IKKα and IKKβ) activity in a concentration-dependent manner. MCF-10A cells transfected with a dominant-negative construct of IKKα or IKKβ were less responsive to TPA in terms of NF-κB-regulated COX-2 expression. The IκB kinase inhibitor Bay11-7082 suppressed the TPA-induced COX-2 expression as well as NF-κB activation. IKK-related kinase NAK (NF-κB-activating kinase) has been known as a direct upstream kinase of IKKβ. TPA treatment induced NAK activity which was suppressed by SFN. TPA-induced COX-2 expression was inhibited in MCF-10A cells transiently transfected with NAK siRNA. In addition, SFN inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and the activities of ERK and p38 mitogen-activated protein kinase (MAPK) in TPA-stimulated MCF-10A cells. MCF-10A cells treated with U0126 and SB203580, specific inhibitors of ERK and p38 MAPK, respectively exhibited reduced COX-2 expression after TPA-stimulation. To investigate the association of IKK and ERK, MCF-10A cells were treated with U0126, and then the IKK kinase assay was performed. Interestingly, U0126 suppressed the catalytic activity of IKKα, but not that of IKKβ. In conclusion, SFN inhibited TPA-induced NF-κB activation in MCF-10A cells by targeting IKK and MAPKs, thereby down-regulating COX-2 expression.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA