Effects of niclosamide on Wnt/β-catenin signaling in cancer cells.

<p>(A) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 6-well plates were treated with niclosamide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin were then examined by GST-E-cadherin binding assay. (B) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 24-well plates were transiently transfected with the TOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with 2.4 µM niclosamide. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells.</p

Effects of niclosamide on LRP6 expression and degradation.

<p>(A) PC-3 cells were incubated with 10 µg/ml of cycloheximide in the presence of Niclosamide (1.2 µM) or vehicle for 0, 3, 6, 10 or 24 h. Cells were then harvested, and the level of endogenous LRP6 was examined by Western blotting. Samples were also probed with anti-actin antibody to verify equal loading. The pixels for each band were measured, normalized and plotted. Data are mean values of three independent experiments with the SD values indicated by error bars. *P<0.05, **P<0.01 versus corresponding control value. (B) PC-3 and MDA-MB-231 cells in 6-well plates were treated with niclosamide for 24 h. The cells were then harvested and LRP6 mRNA levels were determined by real-time RT-PCR and normalized to the message levels of GAPDH mRNA. All the values are the average of triple determinations with the s.d. indicated by error bars.</p

Effects of niclosamide on Wnt3A and LRP6-induced Wnt/β signaling in HEK293 cells.

<p>(A) HEK293 cells in 6-well plates were treated with Wnt3A CM (20%) and niclosamide at indicated concentrations for 24 h. The levels of cytosolic free β-catenin, total cellular β-catenin, LRP6 and phospho-LRP6 (p-LRP6) were examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) HEK293 cells in 24-well plates were transiently transfected with LRP6 plasmid or the corresponding control vector, along with TOPFlash construct and β-galactosidase-expressing vector in each well. After being incubated for 24 h, cells were treated with niclosamide or niclosamide plus Wnt3A CM at indicated concentrations for 24 h. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are averages of three determinations with the standard deviations indicated by error bars. **P<0.01 compared to the control cells without niclosamide treatment.</p

Effects of niclosamide on expression of LRP6, phosph-LRP6 (p-LRP6), Dvl2, Axin2 and Cyclin D1 in cancer cells.

<p>(A) DU145 and PC3 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The total cellular levels of LRP6, p-LRP6, Dvl2, Axin2 and Cyclin D1were then examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) PC-3 and DU145 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The cytosolic levels of Dvl2 were then examined. All the samples were also probed with anti-actin antibody to verify equal loading.</p

New hydrazonoindolin-2-ones: Synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into PLGA microspheres - Fig 9

<p>FT-IR spectrum of a) PLGA, b) Pure 7e, c) Physical mixture of the PLGA + 7e, d) Plain microspheres, and e) 7e-loaded microspheres.</p

Dose-dependent changes in cell cycle.

<p>Distribution of cells within the cell cycle as well as total cell numbers were quantitated by fluorescent staining of nuclear DNA with DAPI. Phosphorylated Rb protein was detected by indirect immunofluorescence followed by automated image acquisition and analysis.</p

Effects of niclosamide on cancer cell apoptosis and proliferation.

<p>(A) Cancer cells were treated with niclosamide at the indicated concentrations for 24 h. Floating and attached cells were combined for apoptosis detection by the Cell Death ELISA kit from Roche Diagnostics Corporation for histone-associated DNA fragments as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029290#s4" target="_blank">Materials and Methods</a>. (B) Cancer cells in 96-well plates were treated with niclosamide for 72 h. Cell viability was measured by the Cell Titer Glo Assay system. All the values are the average of triplicate determinations with the s.d. indicated by error bars. **P<0.01 versus corresponding control value.</p

Cumulative <i>in vitro</i> release of 7e from PLGA microspheres.

<p>Each point represents the mean ± SD obtained from triplicate samples.</p

IC₅₀ of the anti-proliferative activity of compounds 7b, 7d, 7e and sunitinib against HT-29, ZR-75 and A-549 cell lines.

<p>IC₅₀ of the anti-proliferative activity of compounds 7b, 7d, 7e and sunitinib against HT-29, ZR-75 and A-549 cell lines.</p

Anti-proliferative (cell growth inhibitory activity at 30 μM concentration) activity of the target compounds 4a-o, 7a-e and sunitinib against HT-29, ZR-75 and A-549 cell lines.

<p>Anti-proliferative (cell growth inhibitory activity at 30 μM concentration) activity of the target compounds 4a-o, 7a-e and sunitinib against HT-29, ZR-75 and A-549 cell lines.</p