Schematic diagram illustrating the signaling pathways involved in the development of chemotherapeutic treatment resistance to 5-FU and chemosensitization by curcumin in HCT116 and HCT116+ch3 colon cancer cells.

<p>Schematic diagram illustrating the signaling pathways involved in the development of chemotherapeutic treatment resistance to 5-FU and chemosensitization by curcumin in HCT116 and HCT116+ch3 colon cancer cells.</p

Effect of curcumin and/or 5-FU on apoptotic signaling in HCT116 and HCT116+ch3 colon cancer cells.

<p>HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 1 µM 5-FU for 24 h. HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 0.1 µM 5-FU for 24 h. Whole cell lysates were prepared and analyzed by western blotting for <b>A:</b> expression or cleavage of pro-apoptotic proteins caspase-8, caspase-9, caspase-3, PARP and Bax, and of anti-apoptotic protein BCL-xL <b>B:</b> expression of cyclin D1. The housekeeping protein β-actin served as a positive loading control in all experiments.</p

Effect of curcumin and/or 5-FU on NF-κB and PI-3K/Src activation in HCT116 and HCT116+ch3 colon cancer cells.

<p>HCT116 and HCT116+ch3 cells were either treated with different concentrations of 5-FU (0, 2, 5, 10, 20, 40 µM) alone for 1 h or were pretreated with different concentrations of curcumin (0, 2, 5, 10, 20, 40 µM) for 1 h and then exposed to 1 µM (HCT116) or 0.1 µM (HCT116+ch3) 5-FU for 1 h. <b>A:</b> After preparation of nuclear extracts western blotting was performed with antibodies against NF-κB and PARP as a loading control. <b>B:</b> Cytoplasmic fractions were subsequently examined by western blotting for expression of PI-3K (lane I), Src (lane II) and β-actin (lane III) (loading control).</p

Effect of curcumin and/or 5-FU on the cell cycle of HCT116 and HCT116+ch3 colon cancer cells.

<p>HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin and 1 µM 5-FU for 12 and 24 h (<b>A</b>). HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin and 0.1 µM 5-FU for 12 and 24 h (<b>B</b>). Cell cycle analysis was performed by flow cytometry. These studies were performed in triplicate and the results presented are mean value with standard deviations from three independent experiments. Values are given as mean ± SD (<i>p</i><0.05).</p

Effect of 5-FU and/or curcumin or PI-3K inhibitor wortmannin on activation of IκBα kinase (IKK) in HCT116 and HCT116+ch3 colon cancer cells.

<p>A: HCT116 cells were treated with 5-FU (5 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. B: HCT116+ch3 cells were treated with 5-FU (1 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 0.1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. Cells were lysed and immune complex kinase assays were performed as described in Materials and Methods. Equal amounts of total protein (500 ng protein per lane) were separated by SDS-PAGE under reducing conditions and then analyzed by immunoblotting using antibodies against phosphospecific IκBα (lane I), IKK-α (lane II), and IKK-β (lane III).</p

Effect of curcumin and/or 5-FU on apoptosis in HCT116 and HCT116+ch3 colon cancer cells.

<p>HCT116 and HCT116+ch3 cells were treated with different concentrations of curcumin or 5-FU (0, 1, 5, 10 and 20 µM) or a combination of curcumin (5 µM) and 5-FU (0.1, 1, 2 and 3 µM) for 24 h. Monolayer cultures were stained with Hoechst 33258 (DAPI) to reveal apoptotic changes in the cell nuclei.</p

Ultrastructural evaluation of mitochondrial and apoptotic changes after treatment with curcumin and/or 5-FU in HCT116 and HCT116+ch3 colon cancer cells.

<p> HCT116 cells were treated with curcumin (20 µM), 5-FU (5 µM) or a combination of both (5 µM curcumin and 1 µM 5-FU) for 12, 24, 36, 48, 60 and 72 h. Using a different approach HCT116+ch3 cells were treated with a combination of 5 µM curcumin and 0.1 µM 5-FU for 12, 24, 36, 48, 60 and 72 h. Ultrathin sections were prepared and evaluated by transmission electron microscopy. Micrographs shown are representative of all the cultures evaluated. At the earliest time point when apoptosis was first detected images are highlighted arrows. Mitochondrial changes (arrowheads) are shown. Magnification: x5000, bar = 1 µm.</p

Quantification of mitochondrial and apoptotic changes after treatment with curcumin and/or 5-FU in HCT116 and HCT116+ch3 colon cancer cells.

<p>To quantify the ultrastructural findings, HCT116 (A) and HCT116+ch3 (B) cultures treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057218#pone-0057218-g003" target="_blank">Fig. 3</a> were examined for apoptotic and mitochondrial changes (MC) by counting 100 cells from 20 microscopic fields. The examination was performed in triplicate and the results are provided as mean values with standard deviations SD (<i>p</i><0.05) from three independent experiments.</p

Effect of curcumin and/or 5-FU on mitochondrial damage and cytochrome c release in HCT116 and HCT116+ch3 colon cancer cells.

<p>HCT116 cells were treated with 20 µM curcumin or 5 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 1 µM 5-FU for 24 h. HCT116+ch3 cells were treated with 5 µM curcumin or 1 µM 5-FU or a combination of 5 µM curcumin (4 h pretreatment) and 0.1 µM 5-FU for 24 h. Mitochondrial and cytoplasmic cell fractions were prepared and analyzed by western blotting using antibodies against cytochrome c. The housekeeping protein β-actin served as a loading control.</p

Effect of resveratrol and nicotinamide on association of Sirt-1 proteins with PPAR-γ and NCoR in MSC high-density cultures.

<p>Cultures were treated with 0, 1, 10, 100 mM nicotinamide or pre-treated with 1 µM resveratrol for 4 h followed by co-treatment with nicotinamide over 14 days with osteogenic induction medium. Cultures were lysed and immunoprecipitated with anti-PPAR-γ (a), or anti-Sirt-1 (b, c). The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting using anti-NCoR (a, b) and anti- PPAR-γ (c). The same blots were re-probed with an antibody to anti-PPAR-γ (a), anti-Sirt-1 (b, c). Results shown are representative of three independent experiments.</p