Stereo-specific Induction of NF- κ B Activation by Isochamaejasmin *

The root of Stellera chamaejasme L. is a traditional Chinese herb termed Rui Xiang Lang Du and has been used to treat solid tumors, tuberculosis and psoriasis. Exactly how Stellera chamaejasme L. regulates cellular responses remains unclear. We examined four biflavonoids isolated from Stellera chamaejasme L. , including isochamaejasmin, two of its stereo-isomers and a methyl derivative, in functional assays originally designed to screen ligands for the G protein-coupled formyl peptide receptor-like 1 (FPRL1). Isochamaejasmin was found to induce the expression of a NF- κ B-directed reporter gene in transfected HeLa cells with an EC 50 of 3.23 µ M, independently of FPRL1. The isochamaejasmin-stimulated NF- κ B reporter activity was accompanied by nuclear translocation of NF- κ B proteins and was blocked by a dominant negative construct of I κ B α . Isochamaejasmin also induced time-dependent phosphorylation of the mitogen-activated protein kinases ERK1/2 and p38, and a novel protein kinase C, PKC δ . Likewise, inhibition of these kinases with the respective pharmacological inhibitors significantly reduced the isochamaejasmin-stimulated NF- κ B activation. Interestingly, the two stereoisomers and the methyl derivative did not induce detectable activation of NF- κ B and were more cytotoxic than isochamaejasmin, which could partially rescue cycloheximide-induced apoptosis. Inhibition of NF- κ B activation reversed the anti-apoptotic effect of isochamaejasmin. These results provide the first evidence for a potential mechanism of action by Stellera chamaejasme L. , and indicate that structurally similar compounds derived from Stellera chamaejasme L. may have different pharmacological properties. Western blot analysis. Cells grown in six-well plates were stimulated with selected compounds for indicated times. The reaction was stopped by addition of 1 ml ice-cold PBS, and the cells were collected. After centrifugation, the cell pellet was resuspended in 1 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Nonidet P-40, 150 mM NaCl, 1 mM Na 3 VO 4 , 5 mM NaF, and protease inhibitors). The contents were incubated on ice for 15 min, centrifuged, and the supernatant was transferred to fresh Eppendorf tubes. Samples were analyzed by SDS-PAGE and Western blotting with antibodies against either phospho-extracellular signal-related kinase (ERK) 1/2 or p38 (Cell Signaling Technologies, Beverly, MA) at 1:1000 dilution for 18 h. A similar experiment was performed with an anti-phospho-PKC δ antibody (Thr505), from the same vendor. The membrane was washed and incubated with 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody for 1 h. Excess antibody was removed by washing, and immunocomplexes were visualized using enhanced chemiluminescence detection (Pierce, Rockford, IL) according to the manufacturer’s instructions. apoptosis reversed by isochamaejasmin in the We further cell-permeable NF-κ Our results showed that the NF- κ B-inhibitory peptide (SN50), but not the control peptide ablated the reversal effect of isochamaejasmin for 24 hours at 37 ° C A peptide inhibitor of NF- κ B, SN50, or the negative control peptide (SN50M) were added 30 minutes prior to the above reagents (B). MTT assay was performed thereafter. The cell viability is percentage change in cell numbers (means triplicate based on three separate experiments.