Anti-inflammatory triterpenoid saponins from the stem bark of Kalopanax pictus.

Five new compounds, 16,23,29-trihydroxy-3-oxo-olean-12-en-28-oic acid (1), 4,23,29-trihydroxy-3,4-seco-olean-12-en-3-oate-28-oic acid (2), 3ß,6ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-glucopyranoside (3), 3-O-[2,3-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (4), and 3-O-[3,4-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (5), as well as 10 known compounds (6-15), were isolated from the stem bark of Kalopanax pictus. Compounds 1-5 and 7-14 inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values ranging from 0.6 to 16.4 µM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed on the basis of decreases in COX-2 and iNOS gene expression in HepG2 cells. The structure-activity relationship of the compounds with respect to anti-inflammatory activity is also discussed.

Up regulation of GW112 Gene by NF kappaB promotes an antiapoptotic property in gastric cancer cells.

To clarify the regulatory mechanism of GW112 gene expression, 5'-flanking region of the human GW112 gene was isolated and characterized in the present study. 5'-RACE analysis showed a single transcription start site, which is located 142 nucleotides upstream of the translation initiation site. Transient transfection studies with serial deletion constructs and close examination of the sequences identified a putative NF kappaB binding sequence between -442 and -430, which could be responsible for efficient expression of the GW112 gene. Indeed, GW112 gene was found to be regulated by NF kappaB signals including overexpressed p65 and I kappaB alpha, IKK inhibitor, and proteasome inhibitor. Binding of NF kappaB to its putative site was confirmed by EMSA and ChIP assays. These results suggest that NF kappaB is an essential regulatory factor for GW112 transcription. Based on this finding, we next confirmed that inhibition of GW112 expression could induce apoptosis in the presence of cytotoxic agent in gastric cancer cells. Furthermore, knocking-down or overexpression of GW112 gene in gastric cancer cells demonstrated that GW112 has an antiapoptotic property against the cytotoxic agents-induced apoptosis. Taken together, these results suggest that GW112 could be an important mediator in NF kappaB-dependent tumorigenesis of digestive tract tissues.

Activation of the thyroid-stimulating hormone beta-subunit gene by LIM homeodomain transcription factor Lhx2.

Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone alpha-subunit gene, the role of Lhx2 in regulating TSH beta-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH beta-subunit gene was examined. In the thyrotrope-derived TalphaT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH beta-subunit reporter gene. Deletion studies provided evidence that the -177 to -79 region of the TSH beta-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHbeta promoter, -118 to -108 and -86 to -68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the -86 to -68 region, substantially decreased Lhx2 responsiveness of the TSH beta-subunit reporter gene. In addition, a DNA fragment containing the -177 to -79 region of the TSHbeta promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH beta-subunit gene.