Nitric Oxide and Tumor Necrosis Factor-α Production by Oleanolic Acid via Nuclear Factor-κB Activation in Macrophages

Abstract

Oleanolic acid (OA), a pentacyclic triterpene acid, is reported to have antitumor activities; however, the mechanism underlying its antitumorigenic effects is poorly understood. To further determine the mechanism of OA, we investigated the effects of OA on the release of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) and on the level of inducible nitric oxide synthase (iNOS) and TNF-α gene expression in mouse macrophages. We found that OA elicited a dose-dependent increase in NO and TNF-α production. Reverse transcription–polymerase chain reaction showed that the increased NO and TNF-α secretion were due to an increase in iNOS mRNA and TNF-α mRNA, respectively. Since iNOS and TNF-α transcription have recently been shown to be under the control of the NF-κB transcription factor, the effects of OA on NF-κB activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-κB binding sites linked to the luciferase gene revealed that the increased levels of iNOS mRNA and TNF-α mRNA induced by OA were mediated by the NF-κB transcription factor complex. Using DNA fragments containing the NF-κB binding sequence, OA was shown to activate the protein/DNA binding of NF-κB to its cognate site as measured by EMSA. These results demonstrate that OA stimulates NO and TNF-α release and is able to upregulate iNOS and TNF-α expression through NF-κB transactivation, which may be the mechanism whereby OA elicits its biological effects.