The effects of cardamonin on LPS-induced inflammatory protein production in monocytes/macrophages

Hatziieremia, S., Gray, A. I., & †Plevin, R., Department of Pharmaceutical Sciences, †Department of Physiology & Pharmacology, University of Strathclyde, Glasgow G4 0NR, UK.

Chalcones are a group of naturally occurring polyphenolic compounds which have served as structural leads for drugs against diseases such as cancer and sepsis (Won et al., 2005). Cardamonin, a known chalcone isolated from A.absinthium was previously shown to exhibit potent anticancer properties. In this study, we have examined the inhibitory effect of cardamonin, on lipopolysaccharide (LPS)-induced responses in human monocytes THP-1 cells and murine macrophages RAW264.7 cells. The LPS-signalling cascade leading to TNFα production in THP-1 cells and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells is dependent upon activation of nuclear factor-kappa B (NFκB) and activation of members of mitogen-activated protein kinase (MAPK) family (Chan et al., 2001). We sought to correlate the cellular effects of cardamonin to inhibition of a specific signalling pathway.

TNFα production was measured in THP-1 monocytes by a double-antibody ELISA assay. Induction of iNOS and COX-2 in RAW264.7 macrophages and activation of MAP kinases and NF k B was assessed by Western blotting. Cell viability was determined in both THP-1 and RAW264.7 cells by Alamar BlueTM.

In THP-1 cells LPS (1µg/ml) stimulated a 14.9 ± 2.9 fold (p <0.05, n=3) increase in TNFα release that was significantly inhibited by pre-treatment with cardamonin (IC50=9.12±0.414 µM, n=3). Cell viability was not affected. Cardamonin also significantly inhibited LPS-induced iNOS expression (IC50=13.29±1.878 µM, n=3) in RAW264.7 macrophages but slightly enhanced COX-2 expression. In accordance with previous reports, in THP-1 and RAW264.7 cells, exposure to LPS (1µg/ml) stimulated the phosphorylation of the MAPKs ERK1/2, p38 and JNK in a time-dependent manner (Brugger et al., 1999; Swantek et al., 1997). Cardamonin had no significant effect on the activation of these MAPKs in either cell types. Furthermore, LPS-stimulation resulted in a substantial loss of IκBα and increase in levels of phosphorylated-NFκB in THP-1 and RAW264.7 cells. However, cardamonin did not affect either of these parameters. Another class of cytokine, IFNγ also stimulated iNOS induction in RAW264.7 cells; this effect was significantly inhibited by cardamonin.

These results demonstrate that cardamonin inhibits the expression of pro-inflammatory molecules induced by LPS such as TNFα and iNOS. However, cardamonin does not interfere with LPS-mediated signalling pathways and is likely to inhibit a site downstream, also activated by other classes of cytokine.

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SH is a recipient of a scholarship from the Greek Government.