Journal: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: The N ‐acylethanolamine acid amidase (NAAA) selective inhibitor AM9053 affects tumour cell proliferation. (a) Cell viability assay in HCT116 and (b) in healthy human colonic epithelial cells (HCEC) alone or in the presence of AM9053 (0.1–3 μM, 24 h) ( n = 5). Results are expressed as mean ± SD. (c) Cell proliferation rate of HCT116 cells incorporating bromodeoxyuridine (BrdU), alone or in the presence of AM9053 (0.1–3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 5 independent experiments). Values are expressed as means ± SD; * P < 0.05 versus control, as assessed by one‐way ANOVA followed by Dunnett's multiple comparisons test. (d) Cell proliferation rate of HCEC incorporating BrdU, alone or in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 6). Values are expressed as means ± SD; n.s., not significant as assessed by unpaired Student's t ‐test. (e) The antiproliferative effect of AM9053 (3 μM) was also evaluated in the presence of GW6471 (3 μM, PPAR‐α antagonist), 5′‐iodoresiniferatoxin (I‐RTX 0.1 μM, TRPV1 antagonist), AM251 (1 μM, CB 1 antagonist) and AM630 (1 μM, CB 2 antagonist). All results are expressed as percentage of cell proliferation ( n = 5 independent experiments) and as means ± SD; * P < 0.05 versus control and/or AM9053 as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Viability Assay

Journal: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: The inhibitor AM9053 blocks tumoural cell cycle. (a) Cell cycle analysis by flow cytometry of HCT116 cells treated or not with AM9053 (3 μM, 24 h). Results are expressed as fold change of the cells in each cell cycle phase ( n = 8 independent experiments) in order to uniform the % values of the cells in each phase of the cell cycle ( n = 1 outlier has been removed by ROUT test). Values are expressed as means ± SD; * P < 0.05 versus control, as assessed by unpaired Student's t test and/or Mann–Whitney test. (b) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells treated (right figure) or not (left figure) with AM9053 (3 μM, 24 h). Gene expression, in HCT116 cells, alone or in the presence of AM9053 (3 μM, 24 h), of cyclin A2 (c), cyclin‐dependent kinase 2 (CDK2) (d), cyclin B1 (e) and cyclin‐dependent kinase 1 (CDK1) (f). Gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula ( n = 6 independent experiments). Values are expressed as means ± SD; * P < 0.05 versus control and analysed by Student's t ‐test; n.s., not significant

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Cell Cycle Assay, Flow Cytometry, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: Transient knock‐down of N ‐acylethanolamine acid amidase (NAAA) is associated with an antiproliferative effect. (a) NAAA gene expression in HCT116 cells alone (untransfected) or in the presence of scramble small interfering (si)RNA and/or NAAA siRNA ( n = 6 independent experiments). NAAA gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula. Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA and/or NAAA siRNA as assessed by one‐way ANOVA followed by Kruskal–Wallis comparisons test; n.s., not significant. (b) Cell proliferation rate of HCT116 cells incorporating BrdU in the following conditions: alone (untransfected), untransfected in the presence of AM9053 (3 μM, 24 h), scramble siRNA, NAAA siRNA and NAAA siRNA in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 5 independent experiments) and as means ± SD; * P < 0.05 versus untransfected and/or NAAA siRNA, as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test; n.s., not significant. (c) Cell cycle analysis by flow cytometry of HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA. Results are expressed as fold change of the cells in each cell cycle phase ( n = 6) in order to uniform the % values of the cells in each phase of the cell cycle. Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA, as assessed by unpaired Student's t ‐test. (d) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells in the presence of scramble siRNA (upper figure) and/or NAAA siRNA (lower figure). Gene expression, in HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA, of cyclin A2 (e), CDK2 (f), cyclin B1 (g) and CDK1 (h). Gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula ( n = 6) ( n = 1 outlier has been removed by ROUT test for cyclin B1 analysis). Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA, as assessed by Student's t ‐test; n.s., not significant

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Expressing, Quantitative RT-PCR, Cell Cycle Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/Polyps with Dysplasia

doi: 10.3390/ijms21113840

Figure Lengend Snippet: Effect of CPE on EMT phenotype in HT29 and HCM116 human colon cancer cells. ( A ) Effect of CPE on cell growth in the two cell lines. ( B , C ) Effect of CPE pretreatment on cell growth. HT29 cells ( B ) and HCT116 cells were treated with CPE of IC5 for 24 h. ( C ). ( D ) Effect of CPE on cell invasion. ( E ) Effect of CPE on protein levels of factors associated with BRAF, Hippo pathway, and CLDN4. ( F ) Effect of CPE on protein levels of factors associated with EMT and stemness. ( G ) BRAF mutation, nuclear YAP and protein levels of factors associated with EMT were examined in 4 CRC cases with or without CPE expression. Error bar, standard deviation from 3 independent trials. CLDN4 M, membranous CLDN4; CLDN4 C, cytosolic CLDN4; EMT, epithelial-mesenchymal-transition; IC, inhibitory concentration.

Article Snippet: HT29 and HCT116 human OSCC cell lines were purchased from Dainihon Pharmaceutical Co. (Tokyo, Japan).

Techniques: Mutagenesis, Expressing, Standard Deviation, Concentration Assay