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

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

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 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

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

Avicin D induced cell killing is GR-independent.

<p>(A) Western blot comparing the GR expression in wild type HEK293T cells transfected with the mock DNA and plasmid containing the wild type GRα. (B) HEK293T cells transfected with mock DNA or wild type GRα were treated with different concentrations of avicin D for 72 hrs. Cell viability was measured using the MTT assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (C) Jurkat cells were treated with different concentrations of avicin D or Dex for 72 hrs. At the end of 72 hrs, cell viability was evaluated using the MTT assay, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>.</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