20 research outputs found

    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

    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

    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

    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

    Toxicity of 5-FU, curcumin and the combination treatment on HCT116/MRC-5 cells and cellular uptake of curcumin in these cells in high density and monolayer tumor microenvironment co-culture.

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    <p>A: Quantification of the number of colonosheres was achieved by counting the number of spheroid colonies from 10 microscopic fields in the high density microenvironment co-cultures. Cultures were either left untreated (Co) or were treated with 5-FU (0.1, 1, 5 or 10µM), curcumin (0.1, 1, 5 or 10µM) or were pretreated with curcumin (5µM) for 4 h, and then exposed to 5-FU (0.1, 1, 5 or 10µM) for 10 days and evaluated by light microscopy. 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 (**). Toluidine blue staining profile (2B/C, a) and cellular curcumin uptake (2B/C, b) of HCT116 (B) in high density and in MRC-5 (C) in monolayer co-culture. Tumor microenvironment co-cultures were either left untreated (Co) or were treated with 5-FU (5µM) (5-FU), curcumin (5µM) (Cur) or were pretreated with curcumin (5µM) for 4 h, and then exposed to 5-FU (0.1µM) (Cur+5−FU) for 10 days and evaluated under a light or fluorescent microscope. Images shown are representative of three independent experiments. F =  Filter. (*) =  HCT116 colonosheres. Magnification 4B, 4Ca: 200x, 4Cb: 400x; 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

    Curcumin, 5-FU and the combinational treatment suppress TGF-β-mediated synergistic crosstalk between CRC-cells and fibroblasts in tumor microenvironment co-cultures.

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    <p>A-B: 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 TGF-β3 (A) or p-Smad2 (B). C-D: 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 neutralizing pan-TGF-β antibody (10, 20, 30 ng/ml). After 10 days of culture, total cell lysates of HCT116 high density cultures were prepared and immunoblotting performed for TGF-β3 (C) or p-Smad2 (D). 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 (*).</p

    Effects of 5-FU, curcumin and the combinational treatment on proliferation-, metastatic gene products and NF-κB expression in the high density tumor microenvironment co-culture.

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    <p>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 analyzed by western blotting and quantitative densitometry for MMP-13 (a) and NF-κB (b). 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 (*).</p

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

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    <p>A: High density cultures of HCT116 and HCT116+ch3 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 a transmission 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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