Article Figures & Data
Figures
- FIGURE 1.
sst2 internalization is induced by various agonists and is abolished by antagonist Coy-14. HEK-sst2 cells were treated for 30 min either with vehicle (no peptide [A]) or with various agonists at concentrations inducing submaximal internalization effect (100 nmol/L [B and C] and 10 nmol/L [D–F]). (H–L) Cells treated with same agonists at same concentrations as in B–F but in presence of excess of specific sst2-antagonist Coy-14 (5 [H], 10 [I], and 1 [J–L] μmol/L). Effect of antagonist alone (10 μmol/L [G]) is also shown. After incubation with peptides, cells were processed for immunocytochemistry. Clear punctate perinuclear staining is detectable for all tested agonists. This punctate staining is efficiently abolished by excess of antagonist Coy-14. Antagonist alone has no effect on internalization.
- FIGURE 2.
Dose response of agonist-induced sst2 internalization. HEK-sst2 cells were treated with 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, or 10 μmol/L of various agonists. Also included are cells treated with 100 nmol/L TOC plus 5 μmol/L Coy-14 or with 1 μmol/L TOC plus 50 μmol/L Coy-14. After 30 min of incubation with peptides, cells were processed for immunocytochemistry, and internalized sst2 was quantified. (A) Representative experiment with dose-response curves for various agonists inducing sst2 internalization. TOC, Y-DOTA-TATE, and I-Gal-S-TATE are considerably more potent in inducing sst2 internalization than is DTPA-octreotide. TOC-induced internalization is efficiently abolished by excess of Coy-14. (B) Representative immunofluorescence images of agonist-induced internalized sst2 using DTPA-octreotide and TOC. TOC is almost 2 orders of magnitude more potent in inducing sst2 internalization than is DTPA-octreotide.
- FIGURE 3.
sst3 internalization is induced by various agonists and is efficiently abolished by sst3-specific antagonist sst3-ODN-8. HEK-sst3 cells were treated either with vehicle (no peptide [A]) or with various agonists at concentrations inducing submaximal internalization effect (100 nmol/L [B–D]). Cells treated with same agonists at same concentrations as in B–D but in presence of excess of specific sst3-antagonist sst3-ODN-8 (5 μmol/L [F–H]). Effect of antagonist alone (50 μmol/L [E]) is also illustrated. After incubation with peptides, cells were processed for immunocytochemistry. Clear perinuclear staining is detectable for all agonists tested. This staining is efficiently abolished by 50-fold excess of antagonist sst3-ODN-8. Antagonist alone has no effect on internalization.
- FIGURE 4.
Dose response of agonist-induced sst3 internalization. HEK-sst3 cells were treated with 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, or 10 μmol/L of somatostatin-28 or KE108. Also included are cells treated with 100 nmol/L somatostatin-28 plus 5 μmol/L sst3-ODN-8 or with 1 μmol/L somatostatin-28 plus 50 μmol/L sst3-ODN-8. After incubation with peptides, cells were processed for immunocytochemistry, and internalized sst3 was quantified. This experiment shows that somatostatin-28 and KE108 are of similar potency in inducing sst3 internalization. Somatostatin-28–induced internalization effect is efficiently abolished by excess of sst3-ODN-8
- FIGURE 5.
sst5 internalization is induced by native somatostatin-28 but not by high-affinity synthetic sst5 agonists. HEK-sst5 cells were treated either with vehicle (no peptide); with 1 nmol/L, 10 nmol/L, 100 nmol/L, or 1 μmol/L somatostatin-28; or with 1 μmol/L KE108, 1 μmol/L BIM-23244, or 1 μmol/L L-817,818. After incubation with peptides, cells were processed for immunocytochemistry. Dose-response experiment with somatostatin-28 shows that it can elicit sst5 internalization. This is, however, not true for sst5 agonists KE108, BIM-23244, and L-817,818. Note also presence of intracellular sst5 even when cells were treated with vehicle alone (no peptide). All images are composed of 2 single pictures.
Tables
- TABLE 1
sst2, sst3, and sst5 Binding Affinities and Internalization of Somatostatin and Somatostatin Analogs
Receptor internalization Binding affinity sst2 sst3 sst5 Compound sst2 sst3 sst5 HEK* CHO† Antagonized HEK* Antagonized HEK* Natural somatostatins Somatostatin-28 2.3 ± 0.1 (39) 3.7 ± 0.3 (38) 2.4 ± 0.2 (36) + + (0.71 ± 0.05) Yes + Yes + Somatostatin-14 0.7 ± 0.2 (5) 3.3 ± 1.7 (4) 10 ± 4 (4) + ND Yes + ND + Various synthetic analogs [Tyr3]-octreotide (TOC) 2.8 ± 0.6 (4) 225 ± 82 (4) 9.9 ± 1.8 (4) + + (0.51 ± 0.07) Yes − ND − Vapreotide acetate (RC160) 1.8 ± 0.3 (5) 233 ± 34 (4) 4.8 ± 0.9 (5) + + (4.3 ± 1.4) ND − ND − Lanreotide (BIM-23014) 1.6 ± 0.4 (2) 438 ± 39 (2) 7.4 ± 1.2 (2) + + (1.6 ± 0.1) ND − ND − KE108 0.9 ± 0.1 (7) 1.5 ± 0.2 (7) 0.7 ± 0.1 (7) + ND Yes + Yes − NOC-ATE 3.6 ± 1.6 (3) 302 ± 137 (3) 17 ± 10 (3) + ND ND − ND − BIM-23244 0.18 ± 0.02 (3) 53 ± 7 (3) 0.5 ± 0.2 (3) + + (0.08 ± 0.01) ND − ND − L-779,976 0.6 ± 0.1 (4) >1,000 (4) >1,000 (4) + + (0.73 ± 0.18) ND − ND − L-817,818 >1,000 (3) 164 ± 17 (2) 1.6 ± 0.4 (4) − — ND − ND − Chelated analogs DTPA-octreotide (MP2321) 13 ± 2 (5) 376 ± 84 (5) 299 ± 51 (6) + + (23 ± 1) Yes − ND − DOTA-lanreotide (MP2353) 26 ± 3 (6) 771 ± 229 (6) 73 ± 12 (6) + + (4.5 ± 0.4) Yes − ND − Y-DOTA-TOC 11 ± 2 (6) 389 ± 135 (5) 114 ± 29 (5) + + (0.72 ± 0.14) Yes − ND − Y-DOTA-lanreotide 23 ± 5 (4) 290 ± 105 (4) 16 ± 3 (4) + ND Yes − ND − Y-DOTA-NOC 3.3 ± 0.2 (3) 26 ± 2 (3) 10 ± 2 (3) + + (0.41 ± 0.01) Yes + Yes − Y-DOTA-TATE 1.6 ± 0.4 (3) >1,000 (3) 187 ± 50 (3) + + (0.61 ± 0.08) Yes − ND − Lu-DOTA-BOC-ATE 2.4 ± 0.3 (2) 11 ± 1 (2) 8.3 ± 0.4 (2) + + (0.46 ± 0.11) Yes + ND − Lu-DOTA-NOC-ATE 3.6 ± 0.3 (2) 31 ± 2 (2) 15 ± 1 (2) + + (1.0 ± 0.3) Yes + ND − I-Gluc/Gal-compounds I-Gluc-TOC 2.2 ± 0.7 (3) 357 ± 22 (3) 64 ± 24 (3) + + (0.78 ± 0.12) Yes − ND − I-Gluc-TATE 2.0 ± 0.5 (3) >1,000 (3) 521 ± 269 (3) + ND Yes − ND − I-Gluc-S-TATE 2.0 ± 0.7 (3) 398 ± 19 (3) 310 ± 156 (3) + + (0.73 ± 0.21) Yes − ND − I-Gal-S-TATE 2.0 ± 0.8 (3) 491 ± 63 (3) 413 ± 167 (3) + + (0.89 ± 0.31) Yes − ND − Antagonists Coy-14 (BIM-23A760) 10 ± 4 (4) 61 ± 14 (3) 53 ± 19 (2) — — ND ND ND ND sst3-ODN-8 >1,000 (3) 6.7 ± 2.6 (3) >1,000 (3) ND ND ND − ND ND ↵* For immunofluorescent internalization assay in HEK cells, + = internalized at agonist dose of at least 100 nmol/L, − = not internalized at agonist dose of greater than 100 nmol/L, and ND = not determined.
↵† For internalization measured with ELISA in CHO-sst2 cells, + = internalized, − = not internalized, and ND = not determined. Values in parentheses are median effective concentration, EC50 (±SEM, n ≥ 2).
Binding affinities were measured using in vitro receptor autoradiography, as reported previously. Values were taken from previous reports (4,18,25–27,38,39) or represent our unpublished data. Binding affinity values are inhibitory concentration of 50% (IC50), in nmol/L (mean ± SEM); number of independent studies is given in parentheses. Antagonized means abolition of receptor internalization in presence of excess concentrations of receptor-specific antagonist, either Coy-14 for sst2 or sst3-ODN-8 for sst3, performed with immunofluorescent internalization assay in HEK-sst2 or HEK-sst3 cells.
Jump to section
Related Articles
Cited By...
- International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature
- Long-Acting Somatostatin Analog Therapy Differentially Alters 68Ga-DOTATATE Uptake in Normal Tissues Compared with Primary Tumors and Metastatic Lesions
- Upregulation of Key Molecules for Targeted Imaging and Therapy
- Hallmarks of gastrointestinal neuroendocrine tumours: implications for treatment
- The Somatostatin Analog 188Re-P2045 Inhibits the Growth of AR42J Pancreatic Tumor Xenografts
- Unexpected Sensitivity of sst2 Antagonists to N-Terminal Radiometal Modifications
- Positron-emission Tomography (PET) Imaging Agents for Diagnosis of Human Prostate Cancer: Agonist vs. Antagonist Ligands
- Evaluation of 177Lu-DOTA-sst2 Antagonist Versus 177Lu-DOTA-sst2 Agonist Binding in Human Cancers In Vitro
- Treatment with Octreotide Does Not Reduce Tumor Uptake of 68Ga-DOTATATE as Measured by PET/CT in Patients with Neuroendocrine Tumors
- PET of Somatostatin Receptor-Positive Tumors Using 64Cu- and 68Ga-Somatostatin Antagonists: The Chelate Makes the Difference
- 18F-Labeled Bombesin Analog for Specific and Effective Targeting of Prostate Tumors Expressing Gastrin-Releasing Peptide Receptors
- Radiolabeled Bicyclic Somatostatin-Based Analogs: A Novel Class of Potential Radiotracers for SPECT/PET of Neuroendocrine Tumors
- Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery
- Highly Efficient In Vivo Agonist-Induced Internalization of sst2 Receptors in Somatostatin Target Tissues
- Peptide-Based Probes for Cancer Imaging
- New Pansomatostatin Ligands and Their Chelated Versions: Affinity Profile, Agonist Activity, Internalization, and Tumor Targeting
- Bombesin Receptor Antagonists May Be Preferable to Agonists for Tumor Targeting
- Novel insights in somatostatin receptor physiology
- Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors
- Receptor Radionuclide Therapy of Tumors: A Road from Basic Research to Clinical Applications