The Anticancer Plant Triterpenoid, Avicin D, Regulates Glucocorticoid Receptor Signaling: Implications for Cellular Metabolism

Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from “ancient hopanoids,” avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use

Effect of avicin D on GRE-dependent gene expression.

<p>(A) A549 cells transfected with p (GRE)₂-50huIL6P-luc+ were treated with avicin D (1 µM) or Dex (1 µM) for 2–16 hrs. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (B) Effect of avicin D and RU486 on Dex-induced luciferase activity. A549 cells transfected with p (GRE)₂-50huIL6P-luc+ were pre-treated with avicin D (1 µM) or RU486 (1 µM) for 2 hrs, prior to being exposed to Dex (1 µM) or avicin D (1 µM) for 16 hrs. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU). (C) A549 cells were treated with Avicin D (1 µM) for 0–120 min or with Dex (1 µM) for 60 min. Western blot analysis of the nuclear extracts was performed using anti-phospho GR (Ser211) antibodies. Actin levels have been shown as a loading control.</p

Effect of avicin D on PEPCK and FASN expression.

<p>Hep G2 cells were either untreated (lane 1), treated with Dex for 24 hrs (lane 2), or treated with avicin D for 48 (lane 3) and 72 (lane 4) hrs. Cells were also either co-treated with avicin D and Dex for 48 h (lane 5), or pretreated with avicin D (24 hrs), followed by a 24 hr treatment with Dex (lane 6). Avicin D and Dex were used at 1 µM each in all cases. Western blot analysis of total cell lysates was performed using anti-PEPCK (A) and anti-FASN (B) antibodies.</p

Chemical structures of steroids and avicin D.

<p>(A) The basic ring structure of a steroid molecule. (B) Chemical structure of dexamethasone, a prototypical steroid. (C) Chemical structure of avicin D. Part 1 of the molecule has the core 5-ring structure which resembles the core structure of a steroid molecule, and part 2 has a side chain containing two units of acyclic monoterpenes, connected by a quinovose sugar.</p

A model of avicin D-GR interaction.

<p>The Avicin-D warhead was docked into the crystal structure of RU-486 bound to the antagonist form of glucocorticoid (pdb code: 1NHZ) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (A) Distances between the Sg of three cystein residues and the olefin groups of Avicin-D warhead (green carbon atoms). (B) Ribbon structure of 1NHZ with RU-486 (brown carbon atoms) and the model of the Avicin-D warhead (green carbon atoms). (C) Same as (B) except focused on the binding pocket of RU-486.</p

Avicin D binds to GR and translocates it into the nucleus.

<p>(A) A549 cells were incubated with 25 nM [³H] Dex in the presence or absence of 0–500 fold excess of cold Dex or cold avicin D. Following one hour incubation at 37°C, cells were lysed and radioactivity measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (B & C) A549 cells were treated with avicin D (1 µM) for 0–60 min, or with Dex (1 µM) for 60 min at 37°C. (B) At the end of the incubation, cells were fixed and immunostained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (C) Western blot analysis of the nuclear extracts of treated cells was performed using anti- GR antibody as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Actin expression was used as a loading control.</p

Role of different GR domains in avicin D-mediated inhibition of NF-κB activity.

<p>HEK 293T cells were transiently transfected with mock DNA, or plasmids carrying either wild type GRα or deletion variants of GR. After transfection, cells were pre-treated with Avicin D (1 µM) for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Luciferase activity was measured in the cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as % change over the untreated control, which in turn is taken as 100%.</p

Avicin D inhibits activation of NF-κB.

<p>(A) A549 cells were pre-treated with 1 µM each of either avicin D or Dex for 24 hrs. Following the pre-treatment, cells were exposed to TNF (1 nM) for another 24 hrs. At the end of the TNF treatment, cell supernatants were collected. IL-6 levels were measured using an ELISA kit. (B) A549 cells transfected with p (IL6κB)3-50huIL6P-luc+ were either untreated or pre-treated with Avicin D/Dex (1 µM each) for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Cell lysates were assayed for luciferase activity, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU). (C) Normal and GR over expressing HEK 293T cells were transfected with 100 ng of p (IL6κB)3-50huIL6P-luc+ as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Cells were next pre-treated treated with Avicin D/Dex (1 µM each) and treated with TNF (1 nM) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#pone-0028037-g006" target="_blank">Fig. 6B</a>. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as % change over the untreated control, which in turn is taken as 100%. (D) A549 cells transfected with p(IL6κB)3-50huIL6P-luc+ were pre-treated with avicin D/Dex (1 µM each) either as single agents or in combinations for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Cell lysates were assayed for luciferase activity and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU).</p