Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Intracellular Ca 2+ mobilization in human primary and tumor cells. (A) Concentration‐dependent release of intracellular Ca 2+ by PAR 2 agonists SLIGKV ‐ NH 2 and trypsin in HUVEC . (B) Concentration‐dependent release of intracellular Ca 2+ by PAR 2 agonists SLIGKV ‐ NH 2 and trypsin and PAR 1 agonists TFLLR ‐ NH 2 and thrombin in A549 cells. (C) Concentration‐dependent release of intracellular Ca 2+ by PAR 2 agonists SLIGKV ‐ NH 2 and trypsin, PAR 1 agonists TFLLR ‐ NH 2 and thrombin, and teleocidin A2 in MDA ‐ MB 231 cells. (D) Kinetics of intracellular Ca 2+ release in HMEC upon stimulation with 10 μ mol/L SLIGKV ‐ NH 2 , 10 nmol/L trypsin and 1 μ mol/L teleocidin A2. Maximum fluorescence was detected by 10 μ mol/L of the ionophore A23187. Results represent data (mean ± SEM ) from at least three independent experiments performed in triplicates. PAR2, proteinase‐activated receptor 2; A549, lung carcinoma cell line; MDA‐MB 231, human breast adenocarcinoma cell line; HUVEC, human umbilical vein endothelial cell; HMEC, human mammary epithelial cells.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Concentration Assay, Fluorescence

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Inhibition of intracellular Ca 2+ mobilization by teleocidin A2 in HUVEC , A549, and MDA ‐ MB 231 cells. (A) Concentration‐dependent curves of PAR 2 ( SLIGKV ‐ NH 2 ) inhibition by teleocidin A2 in HUVEC . (B) Concentration‐dependent curves of PAR 2 ( SLIGKV ‐ NH 2 ) and PAR 1 ( TFLLR ‐ NH 2 ) receptor inhibition by teleocidin A2 in A549 cells. (C, D) Concentration‐dependent curves of PAR 2 ( SLIGKV ‐ NH 2 and trypsin), PAR 1 ( TFLLR ‐ NH 2 and thrombin), and P2Y ( ATP ) receptor inhibition by teleocidin A2 in MDA ‐ MB 231 cells. Results represent means ± SEM , n ≥ 3 ( n , number of replicates). (E) Time dependency of teleocidin A2 induced PAR 2‐mediated Ca 2+ release inhibition. Data shown in bar charts are means ± SEM from three independent experiments performed in triplicates, ns, not statistically significant. (F) Concentration‐dependent activity curves of teleocidin A2 versus varying concentrations of PAR 2 agonist SLIGKV ‐ NH 2 . HUVEC, human umbilical vein endothelial cell; A549, lung carcinoma cell line; MDA‐MB 231, human breast adenocarcinoma cell line; PAR2, proteinase‐activated receptor 2.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Inhibition, Concentration Assay, Activity Assay

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: (A) Biochemical PKC ADP ‐Glo kinase assay. Teleocidin A2 activates purified PKC mix containing isoforms α , β , and ɣ in high micromolar ranges in the presence of phosphatidylserine, but in the absence of diacylglycerol and calcium. Generated ADP during kinase reaction was measured using ADP ‐Glo ™ kinase assay. (B) PKC inhibitor Gö6983 reverses teleocidin A2‐mediated inhibition of PAR 2‐induced Ca 2+ release in MDA ‐ MB 231 cells. Inhibition of PKC with 1, 10, and 100 nmol/L Gö6983 has no impact on PAR 2‐induced intracellular Ca 2+ release. However, the inhibitory effect of 20 nmol/L teleocidin A2 on PAR 2 Ca 2+ release is reversed with increasing Gö6983 concentrations. Gö6983 was preincubated before teleocidin A2 addition and activation of PAR 2 with SLIGKV ‐ NH 2 . (C) PKC activation as detected by phospho‐ MARCKS (Ser152/156) ELISA ; f.i., fold increase. Data shown in bar chart are means ± SEM from three independent experiments performed in triplicates; ns, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001. (D) Inhibition of intracellular Ca 2+ mobilization by PKC activator PMA . Results represent means ± SEM , n ≥ 3 ( n , number of replicates). PKC, protein kinase C; PAR2, proteinase‐activated receptor 2; MDA‐MB 231, human breast adenocarcinoma cell line; PMA, phorbol 12‐myristate 13‐acetate.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Kinase Assay, Purification, Generated, Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Teleocidin A2 interferes with PAR2‐mediated cell migration. (A) Teleocidin A2 reverses SLIGKV ‐ NH 2 or trypsin induced migration in a cell exclusion assay. Microscopy images show representative data of migration into detection zone after 48 h from three independent experiments. Cell culture medium was used as negative control. (B) Cell migration into detection zone shown in (A) was analyzed by particle analysis in ImageJ and basal migration after 48 h (control) was set to 100%. (C) Cell viability of MDA ‐ MB 231 is influenced neither by teleocidin A2 nor by SLIGKV ‐ NH 2 or trypsin. MDA ‐ MB 231 cells were treated at the same conditions as in the migration assay and the amount of viable cells was detected using CellTiter‐Glo ® assay reagent. After a treatment period of 48 h, testing conditions had no statistically significant impact on cell viability when compared to DMSO control. Data shown in bar charts are mean values from three independent experiments with n ≥ 3 ( n , number of replicates); ns, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001. PAR, proteinase‐activated receptor; MDA‐MB 231, human breast adenocarcinoma cell line.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Migration, Exclusion Assay, Microscopy, Cell Culture, Negative Control, Particle Size Analysis, Control, Glo Assay

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Actin cytoskeleton rearrangement. Fluorescent microscopy images of Alexa Fluor 546‐conjugated phalloidin and DAPI ‐stained MDA ‐ MB 231 cells. SLIGKV ‐ NH 2 – proteinase‐activated receptor 2 activation causes stress fiber formation. Cells were treated with either SLIGKV ‐ NH 2 (25 μ mol/L, 30 min) or teleocidin A2 alone (20 nmol/L, 30 min) or teleocidin A2 (20 nmol/L), Y27632 (1 μ mol/L), and EH op‐016 (2 μ mol/L) in the presence of SLIGKV ‐ NH 2 (25 μ mol/L). MDA‐MB 231, human breast adenocarcinoma cell line.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Microscopy, Staining, Activation Assay

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Hypothesis of teleocidin A2‐regulated blockade of PAR 2 signaling pathways. Teleocidin A2 was able to potently inhibit PAR 2‐induced intracellular Ca 2+ mobilization and antagonized PAR 2‐dependent cellular motility, presumably interfering with cancer cell migration by reorganization of the actin cytoskeleton. PAR2, proteinase‐activated receptor 2; PLC , phospholipase C; IP 3, inositol 1,4,5‐trisphosphate; DAG , diacylglycerol; PKC , protein kinase C; ROCK 1/2, rho‐associated, coiled‐coil containing protein kinase 1/2.

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Protein-Protein interactions, Migration

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Summary of EC 50 values of tested functional agonists and IC 50 values of teleocidin A2 inhibiting intracellular Ca 2+ release in the presence of respective agonists

Article Snippet: Teleocidin A2 binding was tested in a PAR2 (agonist radioligand) binding assay which was mandated to CEREP (ref. no. 2424, Celle l'Evescault, France) as described by Kanke et al. ( ).

Techniques: Functional Assay