Structural requirements of sesquiterpene lactones to inhibit LPS-induced nitric oxide synthesis in RAW 264.7 macrophages
Introduction
Sesquiterpene lactones are biologically active natural products found almost exclusively in species of the Compositae (Asteraceae) family. Common structural features of this class of compounds are a terpenoid C-15 skeleton and a γ-lactone moiety. Other structural elements can vary considerably. Depending on the basic skeleton, different subgroups are distinguished, e.g., germacranolides, eudesmanolides, guaianolides and pseudoguaianolides. Pharmacological activities described for sesquiterpene lactones include antimicrobial,1 antiviral,2 cytotoxic or antitumor,1, 3 and anti-inflammatory properties.4, 5, 6, 7, 8 Recently, several sesquiterpene lactones, i.e., parthenolide, isohelenin, dehydrocostus lactone, and yomogin, were shown to inhibit the expression of inducible nitric oxide synthase (iNOS) in various cell systems.9, 10, 11 iNOS is an enzyme which is induced in different cell types by pro-inflammatory stimuli.12 Excessive production of nitric oxide (NO) by this enzyme is considered as a promoter of tissue injury in inflammation. iNOS is, therefore, an important target involved in inflammatory and also immunoregulatory processes.13 The ability of sesquiterpene lactones to modulate iNOS expression is suggested to contribute to the anti-inflammatory properties of these compounds.9
In this context, studies aimed at elucidating the structural requirements of sesquiterpene lactones regarding their potential to inhibit iNOS-dependent NO synthesis are important to find new potent inhibitors. The primary objective of this study was, therefore, to investigate sesquiterpene lactones from different structural subgroups for their potential to inhibit iNOS-dependent NO synthesis in order to find structural requirements for this biological activity.
So far, the best characterized biological activities of sesquiterpene lactones are their cytotoxicity1, 3, 14, 15 and the ability of sesquiterpene lactones to inhibit the activation of the transcription factor nuclear factor kappaB (NF-κB).5, 6, 7, 8, 9 Both activities are of interest in the context of the present study: in vitro cytotoxicity may simulate inhibition of NO synthesis and is undesirable from a pharmacological point of view. Activation of NF-κB was shown to be necessary for iNOS expression.16 Moreover, Wong et al. provided evidence that sesquiterpene lactones inhibit iNOS expression via inhibition of NF-κB.9 Therefore, our second point of interest was to learn whether the structural requirements of sesquiterpene lactones needed for the inhibition of nitric oxide synthesis correlate with those necessary for their cytotoxic potential and their ability to inhibit activation of NF-κB.
For these studies RAW 264.7 macrophages activated by bacterial endotoxic lipopolysaccharide (LPS) were chosen since induction of iNOS was shown to be dependent on activation of NF-κB in this cell system.16 Sesquiterpene lactones 1–11 (Chart 1) were selected from different structural subgroups. First, the influence of 1–11 on iNOS-dependent NO synthesis was determined by measuring nitrite accumulation in cell culture supernatants. Second, using the determined effective concentrations of 1–11 the influence on cell viability and NF-κB activation was measured.
Section snippets
Inhibition of LPS-induced nitrite accumulation in RAW 264.7 cells by sesquiterpene lactones 1–11. Structural requirements
In order to investigate whether sesquiterpene lactones 1–11 are able to inhibit inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) synthesis, we determined nitrite accumulation (20 h) in cell culture supernatants of lipopolysaccharide (LPS)-activated (1 μg/mL) and sesquiterpene lactone-treated RAW 264.7 macrophages seeded in 96-well plates. Maximum concentration of sesquiterpene lactones 1–11 employed was 100 μM. Table 1 shows that indeed all non-glycosylated sesquiterpene
Discussion
The present study demonstrates that (i) all sesquiterpene lactones tested, except the glycosylated 8, are able to inhibit iNOS-dependent nitrite accumulation dose-dependently with IC50 values ranging from 0.5 μM to 36.8 μM; (ii) a high inhibitory activity correlated with the existence of an α-methylene-γ-lactone moiety but not necessarily with a bi-functionality of the respective compound; (iii) the inhibitory activities were not due to cytotoxicity. The cytotoxic potential observed at higher
Conclusion
We conclude that the most important structural requirement for inhibition of iNOS-dependent NO synthesis seems to be the α-methylene-γ-lactone moiety. Additional other functional elements like, e.g., an α,β-unsubstituted cyclopentenone seem to be less important. Data from the literature as well as our experiments support the idea that different biological activities of sesquiterpene lactones seem to have different structural requirements. However, the α-methylene-γ-lactone moiety seems to play
Materials
Helenalin and psilotropin were isolated from Psilostrophe cooperi (A. Gray) Green, ambrosin, hymenin, and bipinnatin from Hymenoclea salsola Torr. & A. Gray, and taraxinic acid-β-d-glucopyranosylester from Taraxacaum linearisquameum Soest; alantolactone, gaillardin, glaucolide A, psilostachyin, and xanthatin were a kind gift from Dr. E. Rodriguez (Cornell University, Ithaca, NY).
Hydrogenation of alantolactone
Hydrogenation was performed in a Parr hydrogenation apparatus at room temperature: 10 mg of alantolactone were
Acknowledgements
We would like to thank U. Rüberg and Ch. Gerhäuser for their excellent technical assistance.
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