4-Isopropyl-2,6-bis(1-phenylethyl)aniline 1, an Analogue of KTH-13 Isolated from Cordyceps bassiana, Inhibits the NF-κB-Mediated Inflammatory Response

Abstract

The Cordyceps species has been a good source of compounds with anticancer and anti-inflammatory activities. Recently, we reported a novel compound (4-isopropyl-2,6-bis(1-phenylethyl)phenol, KTH-13) with anticancer activity isolated from Cordyceps bassiana and created several derivatives to increase its pharmacological activity. In this study, we tested one of the KTH-013 derivatives, 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 (KTH-13-AD1), with regard to anti-inflammatory activity under macrophage-mediated inflammatory conditions. KTH-13-AD1 clearly suppressed the production of nitric oxide (NO) and reactive oxygen species (ROS) in lipopolysaccharide (LPS) and sodium nitroprusside- (SNP-) treated macrophage-like cells (RAW264.7 cells). Similarly, this compound also reduced mRNA expression of inducible NO synthase (iNOS) and tumor necrosis factor-α (TNF-α), as analyzed by RT-PCR and real-time PCR. Interestingly, KTH-13-AD1 strongly diminished NF-κB-mediated luciferase activities and nuclear translocation of NF-κB family proteins. In accordance, KTH-13-AD1 suppressed the upstream signaling pathway of NF-κB activation, including IκBα, IKKα/β, AKT, p85/PI3K, and Src in a time- and dose-dependent manner. The autophosphorylation of Src and NF-κB observed during the overexpression of Src was also suppressed by KTH-13-AD1. These results strongly suggest that KTH-13-AD1 has strong anti-inflammatory features mediated by suppression of the Src/NF-κB regulatory loop.

Figure 1

Effects of KTH-13-AD1 on macrophage-mediated inflammatory responses. (a) Chemical structure of KTH-13-AD1. (b) NO production in LPS-treated RAW264.7 cells or induced by sodium nitroprusside (SNP), as determined by Griess assay. ((c), left panel) The ROS scavenging effect of KTH-13-AD1 was measured with SNP (0.25 mM)-treated RAW264.7 cells, using DHR123 (20 μM). The level of ROS was determined through flow cytometric analysis. ((c), right panel) The radical scavenging activity of KTH-13-AD1 was determined using DPPH (250 μM) assay. (d) The effect of KTH-13-AD1 on the viability of RAW264.7 cells was evaluated using MTT assay. ^∗ P < 0.05 and ^∗∗ P < 0.01 compared to the vehicle control or normal group.

Figure 2

Effects of KTH-13-AD1 on transcriptional activation of LPS-mediated inflammatory responses. (a, b) mRNA levels of proinflammatory genes (iNOS and TNF-α) in LPS-treated RAW264.7 cells pretreated with KTH-13-AD1 (150 μM), as analyzed by RT-PCR (a) and real-time PCR (b). (c, d) Transcriptional activation of inflammatory responses was examined using a luciferase reporter gene assay with HEK293 cells transfected with NF-κB-Luc or AP-1-Luc, as well as with β-gal (as a transfection control) under either treatment with PMA or cotransfection with FLAG-MyD88 or CFP-TRIF (1 μg/mL each). Luciferase activity was measured using a luminometer. (e) The nuclear translocation levels of AP-1 (c-Jun and c-Fos) and the NF-κB (p65 and p50) family in LPS-treated RAW264.7 cells (5 × 10⁶ cells/mL) pretreated with KTH-13-AD1, as examined using nuclear fractionation and immunoblotting analysis. ^∗∗ P < 0.01 compared to the vehicle control.

Figure 3

Effect of KTH-13-AD1 on the activation of upstream signaling cascades of NF-κB. (a, b, c) Phospho- and total protein levels of IκBα, IKKα/β, AKT, p85, Src, Syk, and β-actin from cell lysates were determined through immunoblotting analysis. (d) The inhibitory activity of KTH-13-AD1 on the phosphorylation of p85 and Src, triggered by HA-Src overexpression in HEK293 cells, was evaluated using immunoblotting analysis. (e) NF-κB-mediated luciferase activity triggered by HA-Src overexpression in HEK293 cells, either in the presence or absence of KTH-13-AD1, was evaluated using luminometric analysis. ^∗ P < 0.05 compared to the vehicle control.

Figure 4

Effect of KTH-13-AD1 on the phagocytic uptake of RAW264.7 cells. RAW264.7 cells preincubated with KTH-13-AD1 were treated with FITC-dextran (1 mg/mL) for 20 min. The level of dextran uptake was determined through flow cytometric analysis. ^∗ P < 0.05 compared to the vehicle control.

Figure 5

The putative inhibitory pathway of macrophage-mediated inflammatory signaling responses, as suppressed by KTH-13-AD1.