23 research outputs found

    Curcumin or 5-FU suppresses TGF-β3 and TGF-βR expression in CRC cells in high density tumor microenvironment co-culture.

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    <p>A: High density mono-cultures of HCT116 cells were left untreated, high density tumor microenvironment co-cultures of HCT116/MRC-5 cells were either left untreated, or treated with 5-FU (5µM), or with curcumin (5µM) or pre-treated with curcumin (5µM) for 4 h, and then exposed to 5-FU (0.1µM) for 10 days. The cultures were subjected to immunofluorescence labeling with primary antibodies for TGF-β3 (a-e) and TGF-β3R (f-j) followed by incubation with rhodamine- or FITC-coupled secondary antibodies. Images shown are representative of three different experiments. Magnification 400×. bar 30 nm. B: To quantify the amount of TGF-β3 and TGF-βR-positive cells in high density cultures described above, 200 cells from 15 microscopic fields within the stained slides were counted. The results are provided as the mean values with S.D. from three independent experiments. Values were compared with the control and statistically significant values with <i>p<</i>0.05 were designated by an asterisk (*) and <i>p<</i>0.01 were designated by an asterisk (**).</p

    Schematic demonstrating the crosstalk between CRC-cells and fibroblasts in high density tumor microenvironment co-cultures.

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    <p>A 10µl drop of cell suspension containing around 1 million HCT116 cells is placed on a nitrocellulose filter on top of a steelnet bridge and the cells are nurtured by diffusion. MRC-5 cells are grown in monolayer on the bottom of the petri dish. This model mimics a three dimensional <i>in vivo</i> situation and allows the exchange between resident components and the cancer cells in the tumor microenvironment on the air medium interphase. Addition of therapeutic agents such as curcumin, 5-FU or neutralizing pan-TGF-β3 antibody can interact and influence cell signaling in both cell types influencing tumor cell and tumor stem cell proliferation, malignity and EMT.</p

    High density tumor microenvironment co-cultures induce EMT with loss of epithelial markers and increase of mesenchymal markers.

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    <p>A-C: HCT116 high density mono-cultures were either left untreated (HCT, Co.) or were co-cultured with MRC-5 in monolayer. Tumor microenvironment co-cultures were either left untreated (Co.), treated with curcumin alone (5µM), 5-FU alone (1, 5, and 10µM) or were pretreated for 4 h with curcumin (5µM) followed by treatment with 5-FU (0.1, 1, 2, 3µM). After 10 days of culture, total cell lysates of HCT116 high density cultures were prepared and immunoblotting performed for vimentin (A) or E-cadherin (B) or Slug (C). D-I: HCT116 high density mono-cultures were either left untreated (HCT, Co.) or were co-cultured with MRC-5 in monolayer. Tumor microenvironment co-cultures were either left untreated (Co.), or treated with curcumin (1, 5, 10, 20 µM). After 10 days of culture, total cell lysates of HCT116 HD-cultures were prepared for immunoblotting (D-F) or immunofluorescence performed on sections (G-I) for vimentin (D, G) or E-cadherin (E, H) or Slug (F, I). Densitometric evaluation of protein expression as revealed by western blot analysis was performed in triplicate. Housekeeping protein β-actin served as a loading control in all experiments. Values were compared to the control and statistically significant values with <i>p</i><0.05. Significant values are marked with (*). F =  Filter. (>) =  HCT116 cells. Magnification G-I: 200×, bar = 30 nm.</p

    Cytotoxicity of 5-FU, curcumin and the combination treatment on colon cancer cells in high density cultures.

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    <p>High density cultures of HCT116, HCT116+ch3 (A), or HCT116R and HCT116+ch3R (B) were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and stained with Hoechst 33258 (DAPI) to reveal apoptotic changes of the cell nuclei. Pictures are representative of three individual experiments.</p

    Colon cancer stem cell marker expression in high density cultures as shown by western blotting evaluation.

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    <p>High density cultures of HCT116 and HCT116+ch3 (left lanel, A, B, C) and of HCT116R and HCT116+ch3R (right panel, A, B, C) cells were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 3 days and whole cell lysates prepared and analyzed by western blotting and quantitative densitometry for expression of CD133, CD44 and ALDH1. Western blots shown are representative of three independent experiments. The housekeeping protein β-actin served as a positive loading control in all experiments. Significant values are marked with (*).</p

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

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    <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.

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    <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 NF-κB and PI-3K/Src activation in HCT116 and HCT116+ch3 colon cancer cells.

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    <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

    Ultrastructural evaluation of cytotoxicity of 5-FU, curcumin and the combinational treatment on HCT116 5-FU-chemoresistant and HCT116+ch3 5-FU-chemoresistant cell lines in high density cultures.

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    <p>A: High density cultures of HCT116R and HCT116+ch3R were either left untreated or were treated with 5-FU (5 µM), curcumin (20 µM), or 5-FU/curcumin in combination (0.1/5 µM). Cultures were evaluated after 1, 3, 7, and 10 days, and evaluated ultrastructurally with an electron microscope. At the earliest time point when apoptosis (arrows) was first detected images are highlighted in red boxes. Micrographs shown are representative of three individual experiments. Magnification: x5000, bar = 1 µm. B: Mitochondrial changes (MC) and apoptosis were quantified by counting 100 cells with morphological features of apoptotic cell death from 25 different microscopic fields and results presented are mean values with standard deviations from three independent experiments. Significant values are marked with (*).</p
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