28 research outputs found

    Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

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    Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population

    Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

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    Objective: Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment. Methods: Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU. Results: Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (beta 1-integrin, ICAM-1), transforming growth factor-beta signaling molecules (TGF-beta 3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-kappa B, MMP-13), TGF-beta 3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET),thereby sensitizing CSCs to 5-FU treatment. Conclusion: Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-beta and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis

    Bacterial lipopolysaccharides form procollag-enendotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

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    Introduction: We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-kappa B (NF-kappa B), we investigated in detail the effects of LPS on the NF-kappa B pathway and the postulated formation of procollagen-endotoxin complexes. Methods: An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling. Results: Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of I kappa B kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-kappa B, suggesting that the NF-kappa B and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-kappa B correlated with activation of I kappa B alpha kinase, I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4. Conclusion: These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-kappa B and PI-3K signaling pathways and activate proinflammatory activity

    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

    Effect of curcumin and/or 5-FU on mitochondrial damage and cytochrome c release in 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 (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 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

    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

    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
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