Diallyl trisulfide inhibits phorbol ester-induced COX-2 expression in human mammary epithelial cells: IKKβ as a prime molecular target

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Multiple lines of genetic and pharmacologic evidence suggest that aberrant overexpression of cyclooxygenase-2 (COX-2) is implicated in carcinogenesis. Therefore, targeted inhibition of COX-2 is regarded as an effective and promising strategy for cancer prevention or treatment. Data from the population-based and laboratory studies support that consumption of garlic is associated with the reduced risk of several types of malignancies. Chemopreventive effects of garlic have been attributed to its oil-soluble sulfur ingredients, such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), but their underlying molecular mechanisms remain largely unresolved. In the present study, we have investigated the effects of DAS, DADS, and DATS on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in human mammary epithelial (MCF10A) cells. Treatment of MCF10 cells with each of the aforementioned allylsulfur compounds before TPA (10 nM) treatment, DATS (10 nM) strongly inhibited the TPA-induced COX-2 expression compared with other allyl sulfides. DATS also strongly inhibited the NF-κB DNA binding activity and degradation of IκBα compared with other allyl sulfides tested. DATS inhibited the degradation of IκBα, DNA binding and transcriptional activity of NF-κB, and catalytic activity of IκB Kinase β (IKKβ) in a concentration dependent manner. It has been reported that the DNA binding and the transcriptional activities of nuclear-translocated NF-κB areinfluenced by the cellular redox status, and cysteine thiols of NF-κB or its regulator IKKβ play a crucial role in activation of NF-κB signaling. We observed that the thiol reducing agent dithiothreitol (DTT) and the antioxidant N-acetylcysteine (NAC) attenuated DATS-induced inhibition of IKKβ activity, degradation of IκBα, and NF-κB DNA binding in TPA-stimulated MCF10A cells. Moreover, DTT attenuated the inhibitory effect of COX-2 expression by DATS in TPA-stimulated MCF10A cells. To determine whether thiol modification of IKKβ is important role in DATS-induced down regulation of NF-κB DNA binding activity and COX-2 expression, MCF10A cells were translently transfected with wild type IKKβ or mutant IKKβ (Cys179A) in which Cys179 was replaced by alanine. Inhibitory effects of DATS on IKKβ activity and NF-κB DNA binding activity were blunted in the MCF10A cells transfected with mutant IKKβ (Cys179A). Moreover, DATS failed to suppress COX-2 expression in the MCF10A cells transfected with mutant IKKβ (Cys179A). In another experiment, DATS also inhibited the catalytic activity of IKKβ in the human mammary cancer cells, which is associated with induction of apoptosis. In conclusion, one of the plausible mechanisms by which DATS exerts anti-inflammatory as well as antiproliferative effects involves suppression of IKKβ catalytic activity through thiol modification.