Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Expression levels of DR5 and c-FLIP proteins in lung ADC, lung SCC and Non-CLT were detected by IHC. (A and B) Strong positive staining of DR5 was observed in the cell nucleus and cytoplasm of lung ADC and SCC. (C) Positive staining of DR5 was also found in Non-CLT. (D and E) Positive staining of c-FLIP was observed in the cytoplasm of lung ADC and SCC. (F) Negative control staining of c-FLIP was found in Non-CLT. (IHC, DAB staining; original magnification ×400 and ×50). ADC, adenocarcinoma; SCC, squamous cell carcinoma; non-CLT, non-cancerous control lung tissues; IHC, immunohistochemistry.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing, Staining, Negative Control, Control, Immunohistochemistry

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Comparison of the expression of DR5 and c-FLIP in lung SCC and lung ADC compared to the noncancerous tissues. The percentages of positive expression of DR5 in the lung SCC and lung ADC were significantly higher than these in the noncancerous tissues (both P<0.001). The percentages of positive expression of c-FLIP in lung SCC and lung ADC were significantly higher than these in the noncancerous tissues (both P<0.001). Positive expression of DR5 exhibited a higher percentage in lung ADC than SCC and positive expression of c-FLIP had a higher percentage in lung SCC compared to ADC, and the differences were statistically significant (both P<0.001). ADC, adenocarcinoma; SCC, squamous cell carcinoma.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Comparison, Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Association between expression of DR5 and c-FLIP proteins and clinicopathological features of the NSCLC patients (n=227).

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Pairwise association between expression of DR5 and c-FLIP proteins in lung SCC and ADC.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Kaplan-Meier curves for overall survival of NSCLC patients as assessed using the log-rank test (all tests were 2-sided). (A) High expression of DR5 was significantly associated with a prolonged overall survival (P=0.029). (B) Negative expression of c-FLIP was significantly associated with a more favorable prognosis (P=0.046), as well as (C) without lymph node metastasis (P=0.001), (D) well and moderate differentiation (P=0.002) and (E) early-stage disease (P<0.001). (F) Histological type was not significantly associated with overall survival (P=0.453). NSCLC, non-small cell lung cancer.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Summary of the univariate and multivariate analyses for overall survival in the 227 NSCLC cases.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: CD13 inhibition enhances the effects of TRAIL in decreasing cell survival in tumor cells. (A) Expression levels of CD13, DR4, DR5, ERK1/2, and p-ERK1/2 in the indicated tumor cell lines, determined by Western blot analysis. (B) Analysis of the purification of TRAIL protein. M: Molecular weight protein markers (kDa); 1: TRAIL protein; 2: anti-TRAIL Western blot. (C) The tumor cell lines (A549, MCF-7, HT1080, and PANC-1) in 96-well plates were exposed to bestatin (0.01, 0.1, 0.5, 1, 1.25, 2.5, or 5 mg/mL) or WM15 (4, 12, or 20 μg/mL) for 48 h. (D) The indicated cell lines in 96-well plates were co-treated with TRAIL and bestatin for 48 h: A549, TRAIL 1.56–200 nmol/L, bestatin 0.01 or 0.1 mg/mL; MCF-7, TRAIL 1.56–200 nmol/L, bestatin 0.01 or 0.1 mg/mL; HT1080, TRAIL 0.039–5 μmol/L, bestatin 0.01 or 0.1 mg/mL; and PANC-1, TRAIL 0.039–5 μmol/L, bestatin 0.01 or 0.1 mg/mL. (E) The indicated cell lines were pretreated with WM15 (5 μg/mL) for 8 h and then co-treated with TRAIL (10 nmol/L) for an additional 48 h. The survival rates were normalized to the control group values (untreated cells). Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs . control, ## P < 0.01 vs . TRAIL.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Western Blot, Purification, Molecular Weight, Control

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: CD13 inhibition increases the expression of DR4 protein and cell surface DR4, and suppresses DR4 degradation. (A) The indicated cell lines were treated with bestatin (A549: 2 mg/mL, MCF-7: 1 mg/mL) for 4, 8, 12, or 24 h; (B) The indicated cell lines were treated with bestatin (A549: 1, 2, or 4 mg/mL, MCF-7: 0.5, 1, or 2 mg/mL) for 24 h. (C) The indicated cell lines were exposed to WM15 (5 μg/mL) for 24 h. (D) The indicated cell lines were exposed to CD13 siRNA (50 nmol/L) for 24 h. (E) The indicated cell lines were treated with bestatin (A549: 4 mg/mL, MCF-7: 2 mg/mL) for 24 h and then harvested for staining of DRs and subsequent flow cytometric analysis of cell surface DR4. The control cells were stained with a matched control FITC-conjugated IgG isotype antibody or FITC-conjugated anti-DR4 antibody, and the bestatin-treated cells were stained with FITC-conjugated anti-DR4 antibody. The MFI for each sample is indicated. (F) The indicated cell lines were exposed to bestatin (A549: 2 mg/mL, MCF-7: 1 mg/mL) for 24 h, and this was followed by the addition of 50 μg/mL CHX at different times. (G) Cells were pretreated with MG132 (10 μM) for 2 h and then received the indicated treatments for different times. Whole cell lysates were prepared from these cells and used to detect the levels of the indicated proteins with Western blot analysis. The results are plotted as the DR4 levels relative to those at time 0 of CHX treatment. The indicated proteins were quantified, and each was previously normalized to the level of GAPDH. Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs. control.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Staining, Control, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Loss of DR4 protects tumor cells against enhanced killing effects of TRAIL together with CD13 inhibition. (A) A549 and MCF-7 cells were exposed to 48 h pretreatment with 30 nmol/L DR4 siRNA, followed by 24 h treatment with TRAIL (50 nmol/L) and bestatin (0.5 mg/mL). (B) The expression of the indicated proteins was assessed by Western blot analysis after 36 h pretreatment with DR4 siRNA followed by 24 h treatment with TRAIL and bestatin at the indicated concentrations. Data are means ± SD for 3 independent experiments. * P < 0.05 and ** P < 0.01 vs. TRAIL + bestatin + siCtrl.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Expressing, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Inhibition of ERK1/2 phosphorylation augments the cell death caused by TRAIL without up-regulating DR4. (A) The indicated cell lines in 96-well plates were pretreated with PD98059 (20 μmol/L) for 6 h followed by treatment with TPA (200 nmol/L) for 30 min and co-treatment with TRAIL (10 nmol/L) for an additional 48 h. The survival rates were normalized to the control group values (untreated cells). ** P < 0.01 vs. TRAIL + PD98059. (B) The indicated cell lines were treated with PD98059 (20 μmol/L) for 24 h, and then the whole cell lysates were prepared from the cells and used to detect the levels of phosphorylated and total ERK1/2 and DR4 by Western blot analysis. The indicated proteins were quantified, and each was previously normalized to the level of GAPDH. Data are means ± SD for 3 independent experiments. * P < 0.05, ** P < 0.01 vs. control.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

Techniques: Inhibition, Phospho-proteomics, Control, Western Blot

Journal: Cancer Biology & Medicine

Article Title: CD13 inhibition augments DR4-induced tumor cell death in a p-ERK1/2-independent manner

doi: 10.20892/j.issn.2095-3941.2020.0196

Figure Lengend Snippet: Immunohistochemical staining to determine the expression of Ki67, DR4, and p-ERK1/2 in tumor tissues of nude mice treated with bestatin and/or TRAIL. (A) Cells with brownish yellow particles were considered Ki67 positive in HT1080 xenografts. The percentage of cells with positive staining for Ki67 expression was normalized to that in the control group. n = 6. ** P < 0.01 vs . control, ## P < 0.01 vs. TRAIL. The scale bar corresponds to 100 μm. The harvested tumors were lysed, and Western blot analysis was performed to detect the expression of DR4 (B), and phosphorylated and total ERK1/2 (C). The indicated proteins were quantified, and each was previously normalized to the level of tubulin. n = 3. * P < 0.05 vs. control. (D) Schematic presentation of the synergistic inhibitory mechanism.

Article Snippet: Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS.

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