Hoechst 33342 and acridine orange-stained MCF-7, MDA-MB-231 and MCF-12A cells at 400× magnification after 48 h exposure.

<p>Vehicle-treated cells (A–C, G–I and M–O) in various stages of the cell cycle are observed. Formation of apoptotic bodies are observed in ESE-16-treated cells (G–I). An increase in the number of cells in metaphase is observed in ESE-16-treated MCF-12A (<i>P–</i>R) cells when compared to vehicle-treated MCF-12A cells (M–O). An increase in the formation of apoptotic bodies are observed in ESE-16-treated MCF-7 (D–E) and MDA-MB-231 (J–L) cells when compared to vehicle-treated cells (A–C and G–I).</p

Effects of ESE-15-ol on cell division over time.

<p>A) Cell cycle histograms of vehicle-treated and ESE-15-ol-treated MDA-MB-231, MCF-7 and MCF-12A cells after 12 h, 24 h and 48 h exposure. B) Time-dependent change of vehicle-treated and ESE-15-ol-treated MDA-MB-231, MCF-7 and MCF-12A cells in sub G₁ B) Time-dependent change of vehicle-treated and ESE-15-ol-treated MDA-MB-231, MCF-7 and MCF-12A cells in mitosis (G₂/M). A gradual increase in the sub-G₁ fraction is observed over time in ESE-15-ol-treatment across all cell lines, indicating increased cell death. After 48 h, the MCF-12A cells were less affected when compared to the MDA-MB-231 and MCF-7 cells. * indicates a <i>t</i>-test <i>P-</i>value <0.05 for difference between vehicle-treated control and ESE-15-ol-treated samples. † indicates a <i>t</i>-test <i>P-</i>value <0.05 for difference between MCF-7 and MCF-12A ESE-15-ol-treated samples. ‡ indicates a <i>t</i>-test <i>P-</i>value <0.05 for difference between MCF-12A and MDA-MB-231 ESE-15-ol-treated samples.</p

GeneVenn diagram showing of statistically significant differentially expressed genes and proteins in MCF-7, MDA-MB-231 and MCF-12A cells after 24 h exposure.

<p>Common genes (A) affected in MCF-7, MDA-MB-231 and MCF-12A cells, and common proteins (B) affected in MCF-7 and MDA-MB-231 cells exposed to ESE-16 (200 nM) for 24 h.</p

Effects of ESE-15-ol on mitochondrial membrane potential in MDA-MB-231, MCF-7 and MCF-12A cells after 24 h exposure.

<p>A) Flow cytometry histograms indicating relative fluorescence intensity for Mitocapture™ in the red channel (FL3 log). An increase in FL3 log indicates an increase in mitochondrial membrane depolarization and potential apoptosis. B) A statistically significant increase in mitochondrial membrane depolarization in the ESE-15-ol-treated cells compared to the vehicle-treated cells was observed across all three cell lines. * indicates a <i>P-</i>value <0.05 between vehicle-treated cells and ESE-16-treated cells.</p

Docking and kinetics data of ESE-15-ol.

<p>Docking of compound 10 into a mimic of CAIX (A) and the wild-type isoform of CAIX (B) revealed that the double bond of ESE-15-ol at C15 and C16 may act as a nucleophile and interact with the electrophilic hydrogen of His 61. This interaction is posited to be specific of ESE-15-ol over 2EE and explain the isoform specificity of ESE-15-ol towards the CAIX mimic over CAII. Kinetics data (C) demonstrates that ESE-15-ol has an almost 2-fold higher affinity for the CAIX mimic when compared to the wild-type CAII. <i>^a</i> Experimental inhibition constants were determined by Sippel <i>et al.</i> (2011) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052205#pone.0052205-Sippel1" target="_blank">[35]</a>. <i>^b</i> The inhibition of the catalyzed exchange of ¹⁸O between CO₂ and water as measured by membrane-inlet mass spectrometry was used to determine the experimental inhibition constants (K_i) of ESE-15-ol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052205#pone.0052205-Silverman1" target="_blank">[16]</a>. A CA IX mimic was used as characterized previously by Genis <i>et al</i>. 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052205#pone.0052205-Genis1" target="_blank">[15]</a>.</p

Hypothesis for the mechanism of action of ESE-16 on MCF-7 cells.

<p>ESE-16 increases labile iron due to lysosomal rupture and possibly interfering with tubulin/ferritin complexes in actively dividing cells. This in turn contributes towards ROS signaling and activates stress-activated kinases. ESE-16 exposure ultimately leads to the abrogation of Bcl-2 phosporylation and induces apoptosis via mitochondrial membrane depolarization.</p

Effects of ESE-16 on phosphatidylserine externalization and mitochondrial membrane depolarization in MCF-7, MDA-MB-231 and MCF-12A cells over time.

<p>A) Measurement of phosphatidylserine externalization in MCF-7, MDA-MB-231 and MCF-12A cells. Apoptosis induction increases in a time-dependent manner in ESE-16-treated cells. The effect is more pronounced in MCF-7 and MDA-MB-231 cells. B) Comparison of differences in mitochondrial membrane depolarization in MCF-7, MDA-MB-231 and MCF-12A cells exposed to ESE-16 (200 nM) over time (6 h –48 h). Mitochondrial membrane depolarization increased in a time-dependent manner in ESE-17-treated cells. The effect is more pronounced in MCF-7 and MDA-MB-231 cells. C) The effects of JNK and p38 inhibitors on ESE-16-induced mitochondrial membrane depolarization. D) The effects of an antioxidant (NAC) and the iron chelators (DFO and 1,10-Phenanthroline) on ESE-16-induced mitochondrial membrane depolarization. * indicates a <i>P-</i>value <0.05 between vehicle-treated cells and ESE-16-treated (200 nM) cells. † indicates a <i>P-</i>value <0.05 between MCF-7 and MCF-12A cells exposed to ESE-16. ‡ indicates a <i>P-</i>value <0.05 between MDA-MB-231 and MCF-12A cells exposed to ESE-16. ¤ indicates a <i>P-</i>value <0.05 between ESE-16-treated cells and ESE-16-treated cells in combination with JNK inhibitor. ¤¤ indicates a <i>P-</i>value <0.05 between ESE-16-treated cells and ESE-16-treated cells in combination with p38 inhibitor. § indicates a <i>P-</i>value <0.05 between ESE-16-treated and other treated samples.</p

Effects of ESE-16 on Bcl-2 phosphorylation and expression in MCF-7, MDA-MB-231 and MCF-12A cells after 24 h exposure.

<p>A) Flow cytometry histograms of total Bcl-2 content (FL1 Log) and Bcl-2 phosphorylated at Ser70 (FL3 log) in MCF-7, MDA-MB-231 and MCF-12A cells after 24 h exposure to ESE-16 (200 nM). Fluorescence Intensity units = FI units. B) Bar-chart demonstrating the distribution of fluorescence intensity (FI) units of Bcl-2 (Ser 70) (FL3 Log) labeled MCF-7, MBA-MB-231 and MCF-12A cells after 24 h exposure to ESE-16 (200 nM). C) Comparison of differences in distribution of fluorescence intensity (FI) units of Bcl-2 (Ser 70) (FL3 Log) labeled MCF-7, MDA-MB-231 and MCF-12A cells after 24 h exposure to ESE-16 (200 nM). D) B) Total Bcl-2 expression after 24 h for MCF-7, MDA-MB-231 and MCF-12A cells after 24 h exposure to ESE-16 (200 nM).</p

Hoechst 33342 and acridine orange-stained MCF-7, MDA-MB-231 and MCF-12A cells at 400× magnification after 24 h exposure.

<p>Cells completing cell division are observed in vehicle-treated cells (A–C, G–I and M–O) while an increase in the number of cells blocked in metaphase is observed in and ESE-16-treated (200 nM) cells (D–E, J–L and <i>P–</i>R). Acridine orange staining appears to be more concentrated in actively dividing cells in both treated and untreated cells.</p

Synthesis of ESE-15-ol, inhibition of carbonic anhydrase activity by ESE-15-ol and the effects of ESE-15-ol on extracellular acidity.

<p>A) Synthesis of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol from 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen- 17-one. Reagents and conditions: (a) 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (0.11 mmol) and CeCl₃ (0.12 mmol) in MeOH :THF (9∶2 <i>v</i>/<i>v</i>) at ambient temperature for 1hr. (b) After 1 hr, reaction was cooled to 0°C and NaBH₄ (0.21 mmol) was added and stirred for 2 hr. B) Reaction velocity (R₁/[E]) of wild-type CAII and a mimic of CAIX as determined by the catalysis of ¹⁸O exchange. Wild-type CAII K_i = 167±19 nM and CAIX mimic K_i = 89±23 nM, calculated using the Henderson method for tight-binding inhibitors. C) Changes in extracellular pH of confluent MDA-MB-231 cells treated with the CAIX inducer, DFO, and ESE-15-ol and a combination of DFO and ESE-15-ol. ESE-15-ol inhibited DFO-induced reduction in extracellular pH. * indicates a <i>t</i>-test <i>P-</i>value <0.05 for difference between vehicle-treated control and the DFO-treated samples.</p