The Maxi-K (BK) Channel Antagonist Penitrem A as a Novel Breast Cancer-Targeted Therapeutic

Figures

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  Scheme 1. Chemical structures of tested penitrems 1–3.

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  Figure 1. Antiproliferative effects of penitrems 1–3 against BC and non-tumorigenic cells. Viability of (a) BT-474, (b) MDA-MB-231, (c) SK-BR-3, (d) CRL-2765, and (e) MCF-12A cells after incubation with increasing concentrations of 1–3 for 48 h in cell cultures. All values are relative to vehicle control (100% survival). Bars represent mean ± SEM for four replicates within each group.

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  Figure 2. Binding mode of 1 at the Ca2+ binding site of BK channel crystal structure PDB 3MT5. (a) The 2D binding mode of 1 at the Ca2+ binding site. Its C-15 hydroxyl group contributed hydrogen bonding donor interaction with Asp367, which has high affinity for the Ca2+ binding. (b) The overlay of 3D structure of 1 at the Ca2+ binding site of the PDB 3MT5. (c) Shape fitting of 1 within the Ca2+ binding site of BK PDB structure 3MT5.

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  Figure 3. Binding mode of 1 at the Ca2+ binding bowel of the BK channel crystal structure PDB 3MT5. (a) The 2D binding mode of 1 at the Ca2+ binding bowel. Its C-25 hydroxyl group contributed hydrogen bonding donor interaction with ASP895 and accepted hydrogen bonding interaction with Asp897. Both aspartic acid moieties are known to provide direct coordinate for Ca2+ binding at the Ca2+ bowel site. (b) The overlay of 3D structure of 1 at the Ca2+ binding bowel of the PDB 3MT5. (c) Shape fitting of 1 within the Ca2+ binding bowel pocket of BK channel PDB structure 3MT5.

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  Figure 4. Binding mode of 1 at the BK channel crystal structure PDB 3NAF. (a) The 2D binding mode of 1 at the Ca2+ binding bowel of PDB 3NAF. NH-1 of 1 contributed hydrogen bonding donor interaction with Asp894 while the C-25 hydroxyl contributed hydrogen bonding donor interaction with Asp895 at the calcium bowel site. These aspartic acid moieties provide direct coordinate for Ca2+ binding at the Ca2+ bowel site. (b) The overlay of 3D structure of 1 at the Ca2+ binding bowel of the PDB 3NAF. (c) Shape fitting of 1 within the Ca2+ binding bowel pocket PDB structure 3NAF.

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  Figure 5. Binding mode of 3 at the Ca2+ bowel of the PDB crystal structure 3NAF. (a) The 2D binding mode of 3 at the Ca2+ bowel showing its NH-1 hydrogen bonding donor interaction with the critical Asp892. (b) The overlay of 3D structure of 3 at the calcium bowel of the PDB 3NAF crystal structure.

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  Figure 6. Expression of BK channel subunits α-1 (KCNMA1) in BC and non-tumorigenic cell lines. (a) Western blots representing total levels of the BK channel subunits α-1 (KCNMA1) in the different cell lines. (b) Bar graphs indicating densitometric quantitative analysis performed on all blots, in which optical density of each band was normalized with corresponding β-tubulin.

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  Figure 7. In vitro effects of 10 µM treatments of penitrems 1–3 on the expression of BK channel (KCNMA1) and TNF-α (D2D4) in MDA-MB-231 cells using Western blot analysis. (a) Western blots for cells treated with penitrems 1–3. (b) Western blot quantification of the in vitro effects of penitrem 1–3 treatments on the expression of KCNMA1. (c) Western blotting quantification of the effects of 1–3 treatments on the activation of TNF-α. Vertical bars indicate the normalized protein value ± SEM. *: indicate significant differences (p ≤ 0.05).

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  Figure 8. In vitro effects of 10 µM treatments of penitrems 1–3 on the expression of BK channel (KCNMA1) and activation of TNF-α (D2D4) in BT-474 cells using Western blot analysis. (a) Western blot for cells treated with penitrems 1–3. (b) Western blot quantification of the in vitro effects of penitrems 1–3 treatment on the expression of KCNMA1. (c) Western blot quantification of the effects of penitrems 1–3 treatment on the activation of TNF-α. Vertical bars indicate the normalized protein value ± SEM. *: indicate significant differences (p ≤ 0.05).

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  Figure 9. In vitro effects of 10 µM treatments of penitrems 1–3 on the expression of BK channel (KCNMA1) and activation of TNF-α (D2D4) in SK-BR-3 cells using Western blot analysis. (a) Western blot for cells treated with penitrems 1–3. (b) Western blotting quantification of the in vitro effects of penitrem 1–3 treatments on the expression of KCNMA1. (c) Western blot quantification of the effects of 1–3 treatments on the activation of TNF-α. Vertical bars indicate the normalized protein value ± SEM. *: indicate significant differences (p ≤ 0.05).

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  Figure 10. Immunocytochemical fluorescence staining of the total levels of BK channel subunits α-1 (KCNMA1) in MDA-MB-231 and BT-474 BC cells treated with 1 at its IC50 concentration, 9.8 and 10.3 µM, respectively, for 24 h. (a) MDA-MB-231 cells treated with vehicle control. (b) MDA-MB-231 cells treated with 1 at 9.8 µM. (c) BT-474 cells treated with vehicle control. (d) BT-474 cells treated with 1 at 10.3 µM. Red staining indicates positive immunofluorescence signal for KCNMA1 and blue staining indicates cell nuclei counter-stained with DAPI. Magnification of each photomicrograph is 20×. (a) or treatment media containing 1 treatment for 24 h (b). Cells were then fixed in pre-cooled acetone and subjected to immunofluorescence analysis for detection of total KCNMA1 level.

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  Figure 11. Flow cytometry analysis for cell cycle progression in BT-474 cells treated with 1 (PA). Cells in the various treatment groups were synchronized in G1 phase. Histograms were generated using Cell Quest software (PI staining). Column graph shows percentage of BT-474 cells in each phase of the cell cycle. Vertical bars show the average percentage of three independent experiments.