Sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables, possesses 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 abnormally 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 (2.5, 12.5 or 25 μM) reduced COX-2 expression induced by TPA in a concentration-dependent fashion. NF-κB has been known as a key transcription factor responsible for regulating the COX-2 gene expression. To identify the critical transcription factor involved in regulating the TPA-induced COX-2 gene expression, MCF-10A cells were transiently transfected with site-specific mutant COX-2 promoter constructs. Destruction of consensus sequences responsible for the NF-κB binding site significantly decreased the COX-2 promoter activity, whereas destruction of the NF-IL6 site and the CRE site did not influence the promoter activity. SFN suppressed the TPA-induced DNA-binding and transcriptional activity of NF-κB as well as nuclear translocaion 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. The IκB kinase inhibitor Bay11-7082 and the IKKβ-specific inhibitor SC-514 abrogated the TPA-induced COX-2 expression as well as NF-κB activation and the catalytic activity of IKK. NF-κB-activating kinase (NAK) has been known as a direct upstream kinase of IKKα. TPA treatment induced NAK activity which was suppressed by SFN. Likewise, TPA-induced COX-2 expression was abrogated in MCF-10A cells transiently transfected with NAK siRNA. The catalytic activity of IKK was also reduced by siRNA knock down of NAK gene. In addition, SFN inhibited the phosphorylation and the activity of extracellular signal-regulated kinase (ERK) in TPA-stimulated MCF-10A cells. MCF-10A cells treated with U0126, a pharmacologic inhibitor of ERK, exhibited reduced COX-2 expression after TPA stimulation. Similarly, the dominant-negative mutant ERK abolished COX-2 expression. To investigate the association between ERK and IKK signaling, MCF-10A cells were treated with U0126, and then the IKK kinase assay was performed. Interestingly, U0126 suppressed the catalytic activity of IKK. In conclusion, SFN inhibited TPA-induced NF-κB activation in MCF-10A cells by targeting NAK and ERK, thereby suppressing IKK activity and down-regulating COX-2 expression.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA