Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC 50 ‫؍‬ 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has antimigratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.Traditionally, genetic mutagenesis has proven to be a useful tool in solving the function of a wide range of genes in biological process. Recently, a chemical genetic approach has been developed to elucidate the principles of a wide range of biological processes (for review, see Refs. 1-3). In chemical genetics, instead of site-specific mutation as in traditional genetics, the function of a protein is inhibited or activated using small chemicals. Therefore, chemical genetics seeks to identify novel small molecules that afford functional dissection of cell biological pathways. Such chemicals are useful as bioactive molecular probes and allow further analysis of the relationship between target processes or proteins within cells and their cellular function.Metastasis plays a major role in morbidity and mortality from breast cancer (4). The metastatic potential of cancer cells is related to the ability to digest the extracellular matrix, migrate, cross blood vessel walls, and reach the blood circulation (for review, see Refs. 5-8). Cell movement is a complex process involving a number of steps, including the disruption of cell-cell junctions, cytoskeletal rearrangements, and the constant remodeling of adhesive contacts with the extracellular matrix (for review, see Refs. 9 -11). Cell migration contributes to several other important pathological pro...

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of normal metabolism or as the result of defense and development. ROS readily oxidize methionine (Met) residues in proteins/peptides to form Met-Rsulfoxide or Met-S-sulfoxide, causing inactivation or malfunction of the proteins. A pepper (Capsicum annuum) methionine sulfoxide reductase B2 gene (CaMsrB2) was isolated, and its roles in plant defense were studied. CaMsrB2 was down-regulated upon inoculation with either incompatible or compatible pathogens. The down-regulation, however, was restored to the original expression levels only in a compatible interaction. Gain-of-function studies using tomato (Solanum lycopersicum) plants transformed with CaMsrB2 resulted in enhanced resistance to Phytophthora capsici and Phytophthora infestans. Inversely, loss-offunction studies of CaMsrB2 using virus-induced gene silencing in pepper plants (cv Early Calwonder-30R) resulted in accelerated cell death from an incompatible bacterial pathogen, Xanthomonas axonopodis pv vesicatoria (Xav) race 1, and enhanced susceptibility to a compatible bacterial pathogen, virulent X. axonopodis pv vesicatoria race 3. Measurement of ROS levels in CaMsrB2-silenced pepper plants revealed that suppression of CaMsrB2 increased the production of ROS, which in turn resulted in the acceleration of cell death via accumulation of ROS. In contrast, the CaMsrB2-transgenic tomato plants showed reduced production of hydrogen peroxide. Taken together, our results suggest that the plant MsrBs have novel functions in active defense against pathogens via the regulation of cell redox status.

Nuclear factor-B (NF-B) and the signaling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts. NF-B regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and antiapoptotic responses. In our search for NF-B inhibitors from natural resources, we identified cardamomin, 2Ј,4Ј-dihydroxy-6Ј-methoxychalcone, as an inhibitor of NF-B activation from Alpinia conchigera Griff (Zingiberaceae). In present study, we demonstrated the effect of cardamomin on NF-B activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and LPS-induced mortality. This compound significantly inhibited the induced expression of NF-B reporter gene by LPS or tumor necrosis factor (TNF)-␣ in a dose-dependent manner. LPS-induced production of TNF-␣ and NO as well as expression of inducible nitric-oxide synthase and cyclooxygenase-2 was significantly suppressed by the treatment of cardamomin in RAW264.7 cells. Also, cardamomin inhibited not only LPS-induced degradation and phosphorylation of inhibitor B␣ (IB␣) but also activation of inhibitor B (IB) kinases and nuclear translocation of NF-B. Further analyses revealed that cardamomin did not directly inhibit IB kinases, but it significantly suppressed LPS-induced activation of Akt. Moreover, cardamomin suppressed transcriptional activity and phosphorylation of Ser536 of RelA/p65 subunit of NF-B. However, this compound did not inhibit LPS-induced activation of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun NH 2 -terminal kinase, but significantly impaired activation of p38 mitogen-activated protein kinase. We also demonstrated that pretreatment of cardamomin rescued C57BL/6 mice from LPS-induced mortality in conjunction with decreased serum level of TNF-␣. Together, cardamomin could be valuable candidate for the intervention of NF-B-dependent pathological condition such as inflammation.

G-protein coupled receptor 43 (GPR43) serves as a receptor for short-chain fatty acids (SCFAs), implicated in neutrophil migration and inflammatory cytokine production. However, the intracellular signaling pathway mediating GPR43 signaling remains unclear. Here, we show that β-arrestin 2 mediates the internalization of GPR43 by agonist. Agonism of GPR43 reduced the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB), which was relieved by short interfering RNA (siRNA) of β-arrestin 2. Subsequently, mRNA expression of proinflammatory cytokines, interleukin (IL)-6 and IL-1β, was downregulated by activation of GPR43 and knockdown of β-arrestin 2 recovered the expression of the cytokines. Taken together, these results suggest that GPR43 may be a plausible target for a variety of inflammatory diseases.