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Internalization of sst₂, sst₃, and sst₅ Receptors: Effects of Somatostatin Agonists and Antagonists

¹ Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland; ² Institute of Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg, Germany; ³ Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California; ⁴ Nuklearmedizinische Klinik und Poliklinik, Technical University of Munich, Munich, Germany; ⁵ Endocrine Research, Beaufour-Ipsen Group, Milford, Massachusetts; ⁶ Division of Radiological Chemistry, University Hospital, Basel, Switzerland; and ⁷ Department of Integrative Biology and Pharmacology, University of Texas Health Science Center–Houston, Houston, Texas

Correspondence: For correspondence or reprints contact: Jean Claude Reubi, MD, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, P.O. Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. E-mail: reubi{at}patho.unibe.ch

The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst₂), somatostatin receptor subtype 3 (sst₃), and somatostatin receptor subtype 5 (sst₅) in vitro at the protein level. Methods: Human embryonic kidney 293 (HEK293) cells expressing the sst₂, sst₃, or the sst₅ were used in a morphologic immunocytochemical internalization assay using specific sst₂, sst₃ and sst₅ antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst₂-expressing CHO-K1 cells using an ELISA. Results: Agonists with a high sst₂-binding affinity were able to induce sst₂ internalization in the HEK293 and CHO-K1 cell lines. New sst₂ agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr³, Thr⁸]-octreotide; NOC is [1-NaI³]-octreotide; and BOC-ATE is [BzThi³, Thr⁸]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst₂ than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst₃ affinity such as KE108 were able to induce sst₃ internalization. In sst₂- or sst₃-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst₂- or sst₃-selective antagonists, respectively. Antagonists alone had no effect on sst₂ or sst₃ internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst₅ internalization. Unexpectedly, however, potent sst₅ agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst₅ internalization under the same conditions. Conclusion: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst₂, sst₃, and sst₅ internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst₂ and sst₃ internalization, sst₅ internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst₅ agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.

Key Words: somatostatin receptors • receptor internalization • antagonist • tumor targeting • receptor immunocytochemistry

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