Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 1. Elevated amounts of resistin are found in TADCs of lung cancer-bearing patients and mice. A549 and CL1-5-CM increase the expression of resistin in protein levels (A). Cocultures of A549 and CL1-5 cells with DCs increase the expression of resistin protein (B) in DCs. Elevated levels of resistin at mRNA (C) and protein (D) are found in CD11c+ DCs isolated from the lungs of lung cancer-bearing mice. Culture media and LLC were injected into the mice via the tail vein (control group, n = 10; LLC group, n = 8). After 14 days, non-tumorous and tumorous regions of the lungs were harvested. CD11c+ DCs were isolated from fresh lung or tumor tissue, and resistin levels assessed by qRT–PCR. Resistin concentrations in supernatants of TADCs were assessed by enzyme-linked immunosorbent assay (ELISA). (E) Resistinhigh CD11c+ DCs are found to have infiltrated cancer sections. Non-tumorous and tumorous regions (n = 10) were cut, stained and then analyzed by confocal microscopy. (F) High levels of resistin have also been found in the serum of lung cancer patients. Resistin levels in the sera of lung cancer patients (n = 46) and healthy donors (n = 24) were assessed by ELISA. Each value is the mean ± SD of three determinations. The asterisk indicates a significant difference between the two test groups, as analyzed by Student’s t-test (*P < 0.05).

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Expressing, Isolation, Injection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy

Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 2. Resistin increases lung cancer progression and osteoclastogenesis. Resistin increases cell migration, as determined by wound healing (A) and the transwell system (B). Resistin increases cell invasion (C), EMT and Twist expression (D) in A549 and CL1-5 cells. The invasiveness and migration ability of A549 and CL1-5 cells were quantified as described above. Resistin (1 or 10 ng/ml) acts as a chemoattractant of cancer migration and invasion and increases osteoclast differentiation (E) and activity (F). Peripheral blood mononuclear cells were treated with resistin (10 ng/ml) presenting in receptor activator of nuclear factor kappa-B ligand (100 ng/ml) and macrophage colony-stimulating factor (200 ng/ml) for 21 days. Osteoclast cells were stained for TRAP activity, and bone resorption activity determined by bone resorption assay kit. Each value is the mean ± SD of three determinations. The asterisk indicates a significant difference compared with the controls, as analyzed by Student’s t-test (*P < 0.05).

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Migration, Expressing, Activity Assay, Staining

Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 3. Inhibition of resistin by its neutralizing antibody decreases TADC-CM-mediated cancer progression. Resistin antibodies decrease the effects of TADC-CM on cell migration (A), invasion (B), EMT (C) and osteoclastogenesis (D). Lung cancer cells were treated with mdDC-CM or TADC-CM presenting in antihuman resistin antibodies or immunoglobulin G (IgG). A549 or CL1-5 cells were seeded in the upper insert and treated with or without antihuman resistin antibodies (2 μg/ml) or IgG (2 μg/ml), then TADC-CM (20%) was added to the lower well to act as a chemoattractive agent for 24 h. The migratory and invasive cells were quantified as described above. Peripheral blood mononuclear cells were treated with mdDC or TADC-CMs presenting in receptor activator of nuclear factor kappa-B ligand (100 ng/ml) and macrophage colony-stimulating factor (200 ng/ml) for 21 days. Osteoclast cells were stained for TRAP activity. Each value is the mean ± SD of three determinations. The asterisk indicates a significant difference between the two test groups, as analyzed by Student’s t-test (*P < 0.05).

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Inhibition, Migration, Staining, Activity Assay

Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 4. Resistin induces histone modification of Twist promoter by increasing WHSC1 expression. (A) The profile of gene expression in resistin-treated A549 cells. Cells were treated with resistin (10 ng/ml) for 6 h, and gene expression was assessed by microarray. (B) Resistin changes WHSC1 expression and the methylation modification of histone proteins. Cells were treated with resistin (1 or 10 ng/ml) for 6 h, and various protein levels were assessed by immunoblot assays. (C) The binding of WHSC1 on promoter regions of Twist. The change of H3K27me3 (D) and H3K36me2 (E) on promoter regions of Twist in both A549 and CL1-5 cells. Blockade of WHSC1 prevents resistin-mediated Twist upregulation (F), cell migration (G) and invasion (H). Cells were transfected with either control siRNA or WHSC1 siRNA, and the efficacy of siRNA was assessed by qRT–PCR. siRNA-transfected cells were treated with resistin (10 ng/ml) for the specified times (Twist and histone methylation, 6 h; cell migration, 48 h; invasion, 48 h). Protein levels were assessed by immunoblot assays. Cell migration and invasion were assessed by QCM™ 24-well Cell Migration and Invasion Assay kits. Dimethylation and trimethylation of histone H3 and H4 were assessed by chromatin immunoprecipitation assay. Each value is the mean ± SD of three determinations. The asterisk indicates a significant difference between the two test groups, as analyzed by Student’s t-test (*P < 0.05).

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Modification, Expressing, Gene Expression, Microarray, Methylation, Western Blot, Binding Assay, Migration, Transfection, Control, Quantitative RT-PCR, Invasion Assay, Chromatin Immunoprecipitation

Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 5. The role of Twist on resistin-mediated EMT. (A) Twist1 siRNA decreased Twist1 mRNA in lung cancer cells. (B) The effect of Twist1 siRNA on E-cadherin expression. Twist1 siRNA decreased resistin-mediated cell migration (C) and invasion (D). Cells were transfected with control siRNA or Twist1 siRNA and then treated with resistin (10 ng/ml) for the indicated times (24 h for E-cadherin, and 48 h for cell migration and invasion assay). Twist1 mRNA levels were assessed by RT–PCR after 48 h transfection. The invasiveness and migration ability of A549 and CL1-5 cells were quantified by QCM™ 24-well Cell Migration and Invasion assay. The asterisk indicates a significant difference with the control, as analyzed by analysis of variance with Student’s t-test post hoc (*P < 0.05). All experiments were performed independently at least three times.

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Expressing, Migration, Transfection, Control, Invasion Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf-Hirschhorn syndrome candidate 1/Twist pathway.

doi: 10.1093/carcin/bgt281

Figure Lengend Snippet: Fig. 6. Neutralization of resistin decreases the occurrence and invasiveness of LLC in mice. Resistin increases metastasis of LLC (A) and enhances invasiveness of LLC (B) in the lungs of mice. Resistin increases WHSC1 and Twist expression, as well as H3K36 dimethylation in tumor of the lungs of mice. The mice were dosed every 5 days with intraperitoneal injections of phosphate-buffered saline (n = 5) or recombinant mouse resistin protein (0.33 mg/kg) (n = 5). After 24 days, non-tumorous and tumorous regions of the lungs were harvested. The levels of various proteins were assessed by immunoblot assay (C). Anti-mouse resistin antibodies reduce the tumor nodules (D) and decrease the invasiveness (E) of LLC in the lungs of the mice. LLC were injected into mice via the tail vein. The mice were dosed every 7 days with intraperitoneal injections immunoglobulin G (n = 7) or anti-mouse resistin antibodies (33 μg per mice) (n = 7). After 24 days, non-tumorous and tumorous regions of the lungs were harvested. (F) Scheme of proposed interactions between cancer and TADCs. Lung cancer greatly increases DCs’ secretion of resistin, which in turn enhances lung cancer cells’ migration, invasion and EMT. TADCs-derived resistin triggers WHSC1 expression, which in turn enhances transcription of Twist by increasing H3K36me2. The asterisk indicates a significant difference with the controls, as analyzed by analysis of variance with Student’s t-test post hoc (*P < 0.05).

Article Snippet: For blocking experiment, isotype control immunoglobulin G and goat polyclonal anti-mouse resistin antibodies (33 μg/mice) (Fitzgerald Industries International, North Acton, MA) were intraperiotenally injected three times, once every 7 days, after the administration of LLC.

Techniques: Neutralization, Expressing, Saline, Recombinant, Western Blot, Injection, Migration, Derivative Assay