When activated, NF-B, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-B family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-B activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-B, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding factor was unaffected.Besides TNF, curcumin also blocked phorbol esterand hydrogen peroxide-mediated activation of NF-B. The TNF-dependent phosphorylation and degradation of IB␣ was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-B activation pathway at a step before IB␣ phosphorylation but after the convergence of various stimuli.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-cB in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-KcB by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose-and time-dependent manner. Besides TNF, CAPE also inhibited NF-cB activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-KcB activation. CAPE prevented the translocation of the p65 subunit of NF-cB to the nucleus and had no significant effect on TNF-induced IKcBa degradation, but did delay IucBa resynthesis. The effect of CAPE on inhibition of NF-cB binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-i, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-KcB activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.