Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists.
Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists.
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发表时间：
2006-03
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通讯作者：
R. Cescato;S. Schulz;B. Waser;Véronique Eltschinger;J. Rivier;H. Wester;M. Culler;M. Ginj;Qisheng Liu;A. Schonbrunn;J. Reubi
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作者：
R. Cescato;S. Schulz;B. Waser;Véronique Eltschinger;J. Rivier;H. Wester;M. Culler;M. Ginj;Qisheng Liu;A. Schonbrunn;J. Reubi
肿瘤细胞通过受体介导的内化摄取放射性标记的生长抑素类似物是体内靶向肿瘤生长抑素受体的关键过程。在本研究中，生长抑素受体内化诱导的各种生长抑素类似物进行了测量与新的免疫细胞化学方法，允许贩运的生长抑素受体亚型2（SST 2），生长抑素受体亚型3（SST 3），生长抑素受体亚型5（SST 5）在体外的蛋白质水平的特征。方法采用表达sst 2、sst 3和sst 5的人胚肾293细胞（HEK 293），用特异性sst 2、sst 3和sst 5抗体进行形态学免疫细胞化学内化试验，定性和定量测定生长抑素激动剂或拮抗剂诱导生长抑素受体内化的能力。此外，已经使用ELISA在表达sst 2的CHO-K1细胞中比较和定量了这些激动剂的选择的内化性质。结果具有高sst 2结合亲和力的激动剂能够诱导HEK 293和CHO-K1细胞系中的sst 2内化。新的sst 2激动剂，如Y-DOTA-TATE、Y-DOTA-NOC、Lu-DOTA-BOC-ATE（其中DOTA是1，4，7，10-四氮杂环十二烷-1，4，7，10-四乙酸; TATE是[Tyr 3，Thr 8]-奥曲肽; NOC是[1-NaI 3]-奥曲肽;和BOC-ATE是[BzThi 3，Thr 8]-奥曲肽），含碘化糖的奥曲肽类似物，或BIM-23244在内化sst 2方面比DTPA-奥曲肽（其中DTPA是二亚乙基三胺五乙酸）有效得多。类似地，具有高sst 3亲和力的化合物如KE 108能够诱导sst 3内化。在表达Sst 2或Sst 3的细胞系中，激动剂诱导的受体内化分别被Sst 2或Sst 3选择性拮抗剂有效地废除。单独的拮抗剂对SST 2或SST 3内化没有影响。我们还发现生长抑素-28和生长抑素-14可以诱导sst 5内化。然而，出乎意料的是，有效的sst 5激动剂如KE 108、BIM-23244和L-817，818在相同条件下不能诱导sst 5内化。结论采用灵敏和重复性好的免疫细胞化学方法，定性和定量测定了各种生长抑素类似物诱导SST 2、SST 3和SST 5内化的能力。而所有的激动剂触发SST 2和SST 3的内化，SST 5的内化诱导天然生长抑素肽，但不是合成的高亲和力SST 5激动剂。这样的分析将是相当大的帮助，为未来的特性的配体预见核医学应用。
UNLABELLED 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.