Journal: The American journal of pathology

Article Title: Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro.

doi: 10.1016/j.ajpath.2016.01.003

Figure Lengend Snippet: Figure 9 Effects of adipokines on growth (A) and apoptosis (B) of cancer cells alone and of IGF-1 and IGF-1R inhibitor (PPP) on structure (DeI) and apoptosis (C) of cancer cells alone. Immunohistochemistry with the growth marker Ki-67 (A) and the apoptosis marker cleaved caspase-3 (B and C) and morphologic analysis (DeI) are performed in cancer cells treated with IGF-1, leptin, adiponectin, resistin, or PPP, as described in Materials and Methods. IGF-1 significantly promotes Ki-67 expression in both EC-GI-10 and TE-9 cells (A), whereas leptin, adiponectin, and resistin do not affect that in both cell types. IGF-1 significantly inhibits cleaved caspase-3 expression in both EC-GI-10 and TE-9 cells (B), whereas leptin, adiponectin, and resistin do not affect that in both cell types. IGF-1R inhibitor (PPP) significantly promotes cleaved caspase-3 expression in both EC-GI-10 and TE-9 cells (C). Both EC-GI-10 and TE-9 cells treated with IGF-1 (E and H) replicate the ATF-induced structure, in comparison with cell types not treated with IGF-1 (D and G). Interestingly, IGF-1R inhibitor (PPP) drastically inhibits the stratification of EC-GI-10 and TE-9 cells (F and I), showing morphologic apoptosis of them. Data are expressed as means SD. **P < 0.01. Scale bar Z 50 mm. ATF, adipose tissue fragment; IGF, insulin-like growth factor; PPP, picropodophyllin.

Article Snippet: To examine the effects of leptin, adiponectin, resistin, IGF-1, and IGF-1R inhibitor on the structure, proliferation, and apoptosis of ESCC cells, recombinant rat leptin (1 ng/mL; R&D Systems, Inc.), recombinant rat adiponectin (1 mg/mL; Aviscera Bioscience, Inc., Santa Clara, CA), resistin (30 ng/mL; OriGene EU, Herford, Germany), recombinant animal-free human IGF-1 (10 nmol/L; Merck Millipore Corp.), and picropodophyllin (PPP; 1 mmol/L; Merck Millipore Corp.) were added to the culture medium for 3 days.

Techniques: Immunohistochemistry, Marker, Expressing, Comparison

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Human resistin is upregulated in lung adenocarcinoma tissues and promotes migration and invasion in A549 cells. (A) Resistin protein expression in lung adenocarcinoma tissues (T) and paracarcinoma tissues (N) was detected by Western blot. (B) Correlation between MMP 2 protein expression and resistin protein expression in lung adenocarcinoma tissues. (C) A549 cells were seeded at 2000 cells/well in 96‐well plates and treated with different concentrations of resistin (0, 12.5, 25, 50, or 100 ng/mL) for 48 h. Cell proliferation was detected by the MTS assay. OD, optical density. (D) A549 cells were seeded on a 6‐well plate and incubated until confluence, then a cell‐free space was created by scraping. Migration was induced by treatment with different concentrations of resistin (0, 12.5, 25, 50, or 100 ng/mL) for 32 h. (E) 1 × 10 4 A549 cells were seeded in the upper chamber in 100 μl serum‐free medium with different concentrations of resistin (0, 12.5, 25, 50, 100 ng/ml), and 600 ?l 10% FBS RPMI 1640 medium was added to the lower chamber. For invasion assay, the upper insert was pre‐coated with growth factor‐reduced Matrigel. After 12h incubation, migrated cells were fixed for Transwell migration assay while invaded cells were fixed after 24 h incubation for Transwell invasion assay. (F) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Total protein was isolated and expression of MMP 2 and Twist1 was detected by immunoblot assay. β‐Actin was used as an internal control. Data represent mean ± SD . n = 3; * P < 0.05

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Expressing, Western Blot, MTS Assay, Incubation, Invasion Assay, Transwell Migration Assay, Transwell Invasion Assay, Isolation, Control

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4) is the functional receptor of human resistin in A549 lung adenocarcinoma cells for migration and invasion. (A) A549 cells (1 × 10 4 ) were seeded in the upper Transwell chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 5 μmol/L TAK ‐242 ( TLR 4 inhibitor). Migrated or invaded cells were photographed in at least five randomly chosen fields. A549 cells pretreated with or without 5 μmol/L TAK ‐242 for 1 h were treated with 50 ng/mL resistin for 24 h. The relative mRNA (B) and protein (C) expression of MMP 2 and Twist1 was quantitated. (D) A549 cells were seeded in a 6‐well plate and transfected with negative control (NC) mimic or TLR 4 si RNA at 70% confluence. After 24 h of transfection, cells were seeded in the upper chamber (1 × 10 4 cells per well) treated with or without 50 ng/mL resistin. Migrated or invaded cells were counted. (E) A549 cells were seeded in a 6‐well plate and transfected with NC mimic or TLR 4 si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, total protein was isolated and expression of MMP 2, TLR 4, and Twist1 was detected. (F) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Total protein was isolated and expression of TLR 4 was detected. (G) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Expression of TLR 4 in A549 membrane was detected by flow cytometry. (H) A549 cells were treated with human recombinant resistin with His‐tag (500 ng/mL) for 1 h and then lysed and immunoprecipitated with agarose beads‐conjugated anti‐His mouse clonal antibody. IP, immunoprecipitation. (I) Five domains of TLR 4 and GST were purified and incubated with U937 cell lysates. Immunoprecipitation assay was carried out. Data represent mean ± SD . n = 3; * P < 0.05

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Functional Assay, Migration, Incubation, Expressing, Transfection, Negative Control, Isolation, Membrane, Flow Cytometry, Recombinant, Immunoprecipitation, Purification

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Epidermal growth factor receptor ( EGFR ) is involved in resistin‐induced migration and invasion of lung adenocarcinoma cells. (A) A549 cells (1 × 10 4 ) were seeded in the upper chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 1 μmol/L erlotinib HC l ( EGFR inhibitor). Migrated or invaded cells were counted. (B,C) A549 cells pretreated with or without 1 μmol/L erlotinib HC l for 1 h were treated with 50 ng/mL resistin for 24 h. Relative mRNA (B) and protein (C) expression of MMP 2 and Twist1 was quantitated. (D) A549 cells were seeded in a 6‐well plate and transfected with negative control (NC) mimic or EGFR si RNA at 70% confluence. After 24 h of transfection, cells were seeded in the upper chamber (1 × 10 4 cells per well) treated with or without 50 ng/mL resistin. Migrated or invaded cells were counted. (E) A549 cells were seeded in a 6‐well plate and transfected with NC mimic or EGFR si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, total protein was isolated and expression of MMP 2, EGFR , and Twist1 were detected. (F) A549 cells pretreated with or without 5 μmol/L TAK ‐242 and/or 10 μmol/L PP 1 (Src inhibitor) for 1 h were treated with 50 ng/mL resistin for 15 min. Total protein was isolated and expression of phosphorylated (p‐)Src, Src, p‐ EGFR , and EGFR was detected by Western blot

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Incubation, Expressing, Transfection, Negative Control, Isolation, Western Blot

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Phosphatidylinositol 3‐kinase ( PI 3K)/Akt/nuclear factor‐κB ( NF ‐κB) are the downstream signals mediating resistin‐induced migration and invasion of lung adenocarcinoma cells. (A) A549 cells (1 × 10 4 ) were seeded in the upper Transwell chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 10 μmol/L LY 294002 ( PI 3K inhibitor) or 10 μmol/L BAY 11‐7082 ( NF ‐κB inhibitor). The migrated or invaded cells were counted. (B) A549 cells pretreated with or without 10 μmol/L LY 294002 or 10 μmol/L BAY 11‐7082 for 1 h were treated with 50 ng/mL resistin for 24 h. Total protein was isolated and expression of MMP 2 and Twist1 was detected by Western blot. (C) A549 cells pretreated with or without 5 μmol/L TAK ‐242, 10 μmol/L PP 1, 1 μmol/L erlotinib HC l, or 10 μmol/L LY 294002 for 1 h were treated with 50 ng/mL resistin for 15 min. Total protein was isolated and expression of phosphorylated (p‐)p85, p85, p‐Akt, and Akt was detected by Western blot. (D) A549 cells pretreated with or without 5 μmol/L TAK ‐242, 10 μmol/L PP 1, 1 μmol/L erlotinib HC l, 10 μmol/L LY 294002, or 10 μmol/L BAY 11‐7082 for 1 h were treated with 50 ng/mL resistin for 2 h. Cells were fixed and blocked and then incubated with primary antibody against p65 and Alexa Fluor 488‐conjugated secondary antibody. Stained cells were photographed with a fluorescence microscope. (E) A549 cells were seeded in a 6‐well plate and transfected with negative control mimic, Toll‐like receptor 4 ( TLR 4) si RNA , or epidermal growth factor receptor ( EGFR ) si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, cells were fixed and blocked and then incubated with primary antibody against p65 and Alexa Fluor 488‐conjugated secondary antibody. Stained cells were photographed with a fluorescence microscope

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Incubation, Isolation, Expressing, Western Blot, Staining, Fluorescence, Microscopy, Transfection, Negative Control

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Schematic illustration of the molecular mechanism underlying the migration and invasion induced by resistin in A549 lung adenocarcinoma cells. EGFR , epidermal growth factor receptor; NF ‐κB, nuclear factor‐κB; TLR 4, Toll‐like receptor 4

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration