Targeting nrf2-mediated gene transcription by triterpenoids and their derivatives

Abstract

Chemoprevention represents a strategy designed to protect cells or tissues against various carcinogens and carcinogenic metabolites derived from exogenous or endogenous sources. Recent studies indicate that plant-derived triterpenoids, like oleanolic acid, may exert cytoprotective functions via regulation of the activity of different transcription factors. The chemopreventive effects may be mediated through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Activation of Nrf2 by triterpenoids induces the expression of phase 2 detoxifying and antioxidant enzymes such as NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) - proteins which can protect cells or tissues against various toxic metabolites. On the other hand, inhibition of other transcription factors, like NF-κB leads to the decrease in the pro-inflammatory gene expression. Moreover, the modulation of microRNAs activity may constitute a new mechanism responsible for valuable effects of triterpenoids. Recently, based on the structure of naturally occurring triterpenoids and with involvement of bioinformatics and computational chemistry, many synthetic analogs with improved biological properties have been obtained. Data from in vitro and in vivo experiments strongly suggest synthetic derivatives as promising candidates in the chemopreventive and chemotherapeutic strategies.

Keywords: Anti-oxidant response; Betulin; Chemoprevention; Oleanolic acid; Triterpenoids.

Fig. 1.. Schematic representation of a broad action of triterpenoids.

Fig. 2.. Nrf2 transcriptional regulation. Under normal conditions, Nrf2 is sequestered in cytoplasm by Keap1. Triterpenoids via oxidative modification of –SH groups in Keap1 or via phosphorylation of Nrf2 by various protein kinase signaling pathways including MAPK, PI3K, PKC and CK-2 facilitate the liberation process of Nrf2 from Keap1 as well as translocation of Nrf2 into the nucleus. After binding Maf proteins and antioxidant response element (ARE) activation, the increased transcription of Nrf2-regulated genes (e.g. HO-1, GST, NQO-1) can be achieved.

Fig. 3.. Schematic representation of the procedure of Nrf2 activity assessment. 24h after transfection with a reporter construct containing the antioxidant response element (ARE) sequence driving the expression of reporter gene, luciferase (ARE-luc plasmid), cells are stimulated by Nrf2 inducers (e.g. triterpenoids). Nrf2 translocates to nucleus and binds to ARE sequence what leads to increased chemiluminescence.

Fig. 4.. OA derivatives increase Nrf2 activity and its target gene expression. 24h after transfection with a reporter construct regulated by the antioxidant response element (ARE-luc), NIH3T3 cells were stimulated by PGJ2 (positive control), OA and its derivatives (A-Lac-O-Me, O-diol, Sc-O-ScIm). After next 24h luciferase was measured showing the increased activity of Nrf2 after stimulation by all compounds (A). Moreover, the effect of selected compound (A-Lac-O-Me) on Nrf2 protein level was checked using immunostaining analysis (B). 6h treatment with the OA derivatives led to the increased expression of HO-1 (gene regulated by Nrf2) as shown by real-time PCR (C). Nrf2 activation by triterpenoids might be cell-type dependent. After transfection of human endothelial cells (HMEC-1) and human keratinocytes (HaCaT) with ARE-luc plasmid and stimulation with three different concentrations of betulin, the increase in luciferase activity was observed only in HaCaT cells (D). (A, C, D) - mean of 3 independent experiments, *p<0.05, B - representative immunostaining for Nrf2 (green). DAPI staining was used to visualize nuclei (blue).