Cell cycle.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell cycle analysis was performed after permeabilization using 7-AAD. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Chemical structures.

<p>Structures of compounds <b>1</b> (1,2-bis(4-chlorophenyl)disulfide), <b>2</b> (4-chloro-<i>N</i>-(4-chlorophenyl)benzamide), <b>3</b> ((<i>E</i>)-1,2-bis(4-chlorophenyl)diazene), <b>4</b> (1,2-bis(4-chlorophenyl)hydrazine), <b>5</b> (1,2-bis(4-methoxyphenyl)disulfide), and <b>6</b> (1,2-bis(4-nitrophenyl)disulfide).</p

Conformational analysis of compounds 1, 2 and 4.

<p>(A) A systematic approach was chosen to explore the conformational space around the three rotable bonds. (B-D) Global minimum energy conformations (<i>left panel</i>) and energy plots (<i>right panel</i>) of compounds <b>1</b> (B), <b>2</b> (C), and <b>4</b> (D).</p

Pdcd4 stabilizing activity.

<p>HEK293 cells stably expressing the Pdcd4 stability reporter Pdcd4_(39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Pdcd4 stabilizing activity was determined relative to Δ(RLU_DMSO–RLU_TPA). Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Erioflorin stabilizes Pdcd4_(39–91)luc from TPA-induced degradation.

<p>(A) Structure of erioflorin (Mwt = 348.4). (B) Stably Pdcd4_(39–91)luc expressing HEK293 cells were treated for 8 h with TPA (10 nM) with increasing concentrations of erioflorin (0.0625–10 µM). Pdcd4 stabilizing activity was determined relative to Δ(RLU_control–RLU_TPA-only). (C) Stably Pdcd4_(mut39–91)luc expressing HEK293 cells were treated as in (B). Luciferase activity is given relative to TPA-treated controls. All data are presented as means ± SEM (n≥3, *p<0.05, **p<0.01).</p

Nonspecific activity.

<p>HEK293 cells stably expressing Pdcd4_(mut39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Luciferase activity is given relative to TPA-treated controls. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Erioflorin inhibits cell proliferation and alters cell cycle progression.

<p>(A, C, E) MCF7, HeLa and RKO cells were seeded at 10–20% confluency one day prior to the experiment and treated with DMSO (black diamonds) or erioflorin (2.5 (white triangles) and 5 µM (gray squares)). Cell confluency was followed for six days. (B, D, F) MCF7, HeLa and RKO cells were serum deprived for 48 h and treated with erioflorin (5 µM) for 16 h. After propidium iodide staining, distribution of the cells to the different phases of the cell cycle (subG1 (white), G1 (light gray), S (dark gray), G2/M (black)) was determined. (G) RKO colon carcinoma cells were subjected to a scratch wound assay. After administration of the scratch, medium was changed to control or erioflorin (5 µM) containing medium. Wound closure was measured after 24 h and relative wound closure is given as the ratio of the width of the scratch at 24 h and to 0 h. All data are given as means ± SEM (n≥3, *p<0.05, **p<0.01, ***p<0.001).</p

Proliferation.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 5 days with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (1 μM (green), 3 μM (violet), 10 μM (yellow), and 30 μM (red)). Confluency was assessed every 4 h in an IncuCyte instrument. Proliferation kinetics shown are representative for at least 3 independent experiments and represent means of 3 wells treated in parallel (* p<0.05, ** p<0.01, *** p<0.01).</p

Erioflorin stabilizes Pdcd4 without affecting phosphorylation events.

<p>(A + B) HEK293 cells were transiently transfected with Pdcd4_(39–91)luc (A) or Pdcd4_(mut39–91)luc (B) <i>firefly</i> reporter vectors, in combination with expression vectors for either wildtype (S6K_wt = white bars) or constitutively active p70^S6K (S6K_ca = black bars) and a <i>renilla</i> luciferase vector one day prior to the experiment. Transfected cells were treated for 8 h with rapamycin (100 nM) or erioflorin (5 µM). <i>Firefly</i> normalized to <i>renilla</i> luciferase activity is presented relative to DMSO-treated controls. (C) HEK293 cells were treated for 8 h with TPA (10 nM) with or without erioflorin (0.625–5 µM). Whole-cell extracts were subjected to western analysis and probed with the indicated antibodies. Blots are representative of at least three independent experiments. Densitometric analysis and quantification of nucleolin-normalized Pdcd4 and phospho-S6 protein levels is shown relative to the DMSO control. (D) HEK293 cells were treated with DMSO (black diamonds), TPA (10 nM) with (white triangles) or without (gray squares) erioflorin (5 µM) for 8 h and cycloheximide (10 µM) was added for 1, 2 or 4 h. Pdcd4 protein levels were analyzed densitometrically, normalized to nucleolin and the half-life was calculated. All data are presented as means ± SEM (n≥3, *p<0.05, **p<0.01, ***p<0.001).</p

Erioflorin inhibits AP-1- and NF-κB-trancriptional activities.

<p>(A) HEK293 cells were co-transfected with an AP-1 <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TPA (10 nM) with or without erioflorin (2.5 and 5 µM). Relative AP-1 activity was normalized to <i>renilla</i> luciferase and presented relative to TPA-only treated cells. (B) HEK293 cells were co-transfected with a NF-κB <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TNFα (20 mg mL^–1) with or without erioflorin (2.5 and 5 µM). Relative NF-κB activity was normalized to <i>renilla</i> luciferase and presented relative to TNFα-only treated cells. All data are given as means ± SEM (n≥3, **p<0.01).</p