Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Relationship between adiponectin expression ratio and demographic characteristics of non-small cell lung cancer (NSCLC) patients.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Survival curve of non-small cell lung cancer patients with high and low expression of adiponectin assessed using Kaplan–Meier analysis.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Multivariate cox regression analysis of mortality.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Samples (lung cancer and the corresponding normal adjacent lung tissues) were analyzed with antibodies to adiponectin, adiponectin receptor 1, and adiponectin receptor 2 by immunohistochemical staining (DAB staining and hematoxylin counterstaining). For negative controls, the antibody was replaced with control IgG. (B-C) Expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 in three pairs of lung cancer and normal tissues analyzed by western blotting. (D–F) Gelatin zymography was used to analyze the activities of MMP-2/MMP-9, MMP-1/MMP-3, and MMP-13/MMP-14. * p < 0.05 vs. normal lung tissue, with representative data from three different patients with NSCLC. T, tumor; N, normal; ADN, Adiponectin; AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Western Blot, Zymography

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Cell viability was analyzed by the MTT assay. The cytotoxic effect of curcumin was obvious at concentrations >75 μM. (B) Expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 in A549 cells were analyzed by western blotting at different curcumin concentrations. (C–D) Exogenous induction and silencing of adiponectin expression in A549 cells. Adiponectin mRNA/protein expression in A549 cells increased significantly after exogenous adiponectin expression as analyzed by real-time PCR. Adiponectin expression decreased significantly after siRNA-mediated adiponectin silencing. NC-siRNA served as a negative control. Representative photos of three independent experiments. * p < 0.05 vs. control. The assays were conducted in triplicate.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: MTT Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Control

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Cellular migration was determined using scratch wound assay and analyzed using the Wimasis WimScratch software. The migratory ability of A549 cells was significantly inhibited by increasing curcumin dosage compared with the control group ( p < 0.05). (B) Invasiveness was determined by Matrigel-coated Boyden chamber assay. Curcumin significantly decreased the invasive ability of A549 cells ( p < 0.05) when the curcumin concentration was >25 uM. (C–D) The application of recombinant adiponectin (rADN) significantly increased the migratory and invasive ability of A549 cells ( p < 0.05). The results of three independent experiments are shown; the assays were conducted in triplicate.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Migration, Scratch Wound Assay Assay, Software, Control, Boyden Chamber Assay, Concentration Assay, Recombinant

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A–C) The expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 were analyzed by western blotting after transfection with adiponectin vector or silencing by siADN, siAdipoR1, and siAdipoR2. (D–E) The migratory and invasive ability of A549 cells was inhibited after silencing of the adiponectin receptor 1 expression by transfection with siAdipoR1.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) A549 cells were treated with various PI3K/AKT and MAP kinase pathway inhibitors {(PI3K (LY294002; 10 μM), AKT (API-59; 3 μM), MAPK inhibitors [PD98059 (10 μM), SB203580 (10 μM), and SP600125 (10 μM) for ERK, p38–MAPK, and JNK, respectively]} for 1 h and later with curcumin (50 μM) for 24 h. Adiponectin expression was analyzed by western blotting. (B) AKT expression was analyzed by western blotting with different concentrations of recombination adiponectin. (C) The activity of p65/p50 of A549 cells was analyzed by EMSA after treatment with recombinant adiponectin or AKT inhibitor (API-59; 3 μM), respectively.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing, Western Blot, Activity Assay, Recombinant

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) A549 cells treated with curcumin, silenced, or transfected with adiponectin were lysed and adiponectin was co-immunoprecipitated using adiponectin antibody. Immunoblotting with the indicated antibodies confirmed the co-precipitation of adiponectin monomer, dimer, and multimer. (B) Co-immunoprecipitation was performed using anti-adiponectin and anti-p65 antibodies. The p65 elute was separated on SDS-PAGE and immunoblotted with the corresponding antibodies, which showed substantial association of adiponectin with the p65 component. (C) Activation of NF-κB by overexpression or silencing of adiponectin was determined by EMSA. Adiponectin increased the nuclear translocation of p65/p50 and NF-κB activation, but silencing adiponectin expression had the opposite effect.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Transfection, Immunoprecipitation, Western Blot, SDS Page, Activation Assay, Over Expression, Translocation Assay, Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Gelatin zymography was used to analyze d the activities of MMP-2/MMP-9, MMP-1/MMP-3, and MMP-13/MMP-14. (A–F) The activities of MMP-9, -1, and -14 were increased after the overexpression of adiponectin and decreased with the silencing of adiponectin. Expression of MMP-3 and -13 did not change regardless of adiponectin augmentation or silencing.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Zymography, Over Expression, Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) The tumor sizes of curcumin-treated mice were decreased significantly compared to those of the control group after 14 days. (B) Tumor adiponectin expression of curcumin-treated mice was decreased significantly compared to that of the control group. (C) Expression of both adiponectin receptor 1 and receptor 2 in the curcumin-treated mice did not change in vivo . (D–F) Expression levels of MMP-2, -9, -3, -13, and -14 were decreased with curcumin treatment in vivo . MMP-1 expression was not altered.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Control, Expressing, In Vivo