Inhibition of proteasome system by natural compounds: source of new anti-inflammatory and anti-cancer drugs

G COSTA1,2, V FRANCISCO2,3, J LIBERAL2,3, MT CRUZ2,3, MC LOPES2,3, MT BATISTA1,2. 1Center for Pharmaceutical Studies, University of Coimbra, 3000-548 Coimbra, Portugal, 2Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal, 3Center for Neurosciences and Cell Biology, University of Coimbra, 3000-548 Coimbra, Portugal

Objectives: The proteasome system plays a central role in the selective degradation of intracellular proteins involved in the control of inflammation, cell cycle regulation and gene expression. Therefore, proteasome inhibition is a potential biological target for the treatment of cancer and inflammatory conditions. In this work, we evaluated the effect of a lipid–free infusion (extract) obtained from Cymbopogon citratus (DC) Stapf. (Cy) on the proteasome activity, using an in vitro model of inflammation.

Methods: Cy dry leaves were extracted by infusion (5g/150ml H2O). Subsequently, lipids and essential oils were removed with n-hexane and the extract was freeze-dried. Phenolic acids fraction (PAF) was obtained by reverse phase liquid chromatography and, subsequently, a molecular exclusion Sephadex® gel column, using a water-methanol and water-ethanol gradient, respectively. Chemical composition of both infusion and PAF was attained by HPLC–PDA, according to our previous work [1]. Chlorogenic acid quantification was assessed by HPLC-PDA, using the external standard method. As an in vitro model of inflammation, mouse macrophage-like cell line RAW 264.7 was stimulated with lipopolysaccharide (LPS). The effect of Cy infusion, PAF and chlorogenic acid on the chymotrypsin-like activity of proteasome was assayed in cytosolic extracts using the fluorogenic peptide Suc-LLVY-AMC.

Results: Chlorogenic acid was found to be the most predominant phenolic acid in Cy, its percentage was 0.149% and 3.332%, in the infusion and PAF, respectively. The in vitro results showed, for the first time, that Cy infusion and PAF inhibited the proteasome activity. Moreover, we found that chlorogenic acid strongly inhibits the proteasome.

Conclusion: Our results revealed Cy phenolic compounds, namely the chlorogenic acid, as potential new anti-cancer and anti-inflammatory drugs through inhibition of proteasome system.

Acknowledgements: Research supported by the FCT project PTDC/SAU-FCF/105429/2008 and FEDER/COMPETE (FCOMP-01-0124-FEDER-011096) and a FCT PhD fellowship (SFRH/BD/46281/2008)

References: 1. Figueirinha, A. et al. (2008) Food Chem. 110: 718-728.