Internalization of the Radioiodinated Somatostatin

Recently, we developed a technique that allows the in uiuo visualization in man of somatostatin receptor-positive neuroendocrine tumors after iv injection of [iZ31-Tyr3]octreotide or [“‘In-DTPA-nPhe’loctreotide. Radiotherapy of such tumors using somatostatin analogs coupled to OLor P-emitting radionuclides has been proposed as an application for radiolabeled somatostatin analogs. To develop this concept further, it is of importance to know whether the above-mentioned radiolabeled somatostatin analogs are internalized by the tumor cells, and whether it might be possible to manipulate the degree of internalization. In the present study we investigated the internalization of a stable somatostatin analog, [‘251-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells and primary cultures of human GH-secreting pituitary tumor cells. Treatment of the cells with low pH was used to distinguish between membrane-bound (acid-releasable) and internalized (acid-resistant) radioligand. [‘““I-Ty?Joctreotide showed a time-dependent increasing accumulation in AtTPO cells; after 4 h of incubation, values up to 6-S% of the dose of radioligand added were obtained. Binding and internalization of [‘“51-Tyr310ctreotide were temperature dependent and inhibited by pertussis toxin. Inhibitors of lysosomal degradation did not increase the amount of internalized radioligand. After 4 h of incubation, 88% of the radioactivity present in the cells was still peptide bound, suggesting a low intracellular breakdown of this radioligand. Six of seven human GH-secreting adenoma cell cultures also internalized [iz51Tyr3Joctreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [lz51Tyr3Joctreotide by unlabeled octreotide showed a bell-shaped curve in AtT20 cells. At low concentrations (0.1 and 1 nM), binding and internalization were increased, whereas at higher concentrations, saturation occurred. In contrast to this, binding of [‘251-Tyr3Joctreotide to a broken cell preparation of AtT20 cells was displaced in a dosedependent manner by unlabeled octreotide, with an IC,, of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [ i2”I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTPbinding protein-dependent manner by mouse AtT20 and human GHsecreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [i2”ITyr3Joctreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found. Because membrane binding was simultaneously increased, this is suggested to be related to a rapid recruitment of somatostatin receptors at the outer tumor cell membrane. (Endocrinology 136: 3698-3706, 1995) S OMATOSTATIN receptors (SS-R) are present in all normal target tissues of the peptide, such as brain, anterior pituitary gland, and pancreas. In a variety of human tumors, frequently originating from normal somatostatin (SS) target tissues, high numbers of SS-R can be detected by classical biochemical binding techniques as well as by in vitro autoradiography. These tumors include those with amine precursor uptake and decarboxylation characteristics (pituitary tumors, endocrine pancreatic tumors, carcinoids, paragangliomas, small cell lung cancers, medullary thyroid carcinomas, and pheochromocytomas) as well as meningiomas, well differentiated brain tumors (astrocytomas), neuroblastomas, lymphomas, and some human breast cancers (1). Recently, we developed a technique that allows the in viva visualizaReceived March 1, 1995. Address all correspondence and requests for reprints to: Dr. L. J. Hofland, Department of Internal Medicine III, University Hospital Dijkzigt, 40 Doctor Molewaterplein, 3015 GD Rotterdam, The Netherlands. + This work was supported by a grant from the Dutch Cancer Foundation (EUR.94-807). tion in man of the above-mentioned SS-R-positive tumors after iv injection of [iz31-Tyr3]octreotide (2,3) or [“‘In-DTPAu-Phe’loctreotide (4). Using this technique, we showed that certain tumors, especially those with a high number of SS-R, could be visualized 48 h after injection (l-4). This rather long residence time of radioactivity on human tumors in viuo suggests that the radioligand is internalized by the tumor cells. Internalization of radioligand is of special importance when radiotherapy of certain SS-R-positive human cancers with CXor p-emitting isotopes coupled to SS analogs is considered (5, 6). At present, equivocal data have been reported with respect to internalization of SS. Receptor-mediated endocytosis of SS has been demonstrated in rat anterior pituitary cells and rat islet cells (7-14), whereas other investigators found that [lz51Tyr’]SS-14 and [‘251-Tyr”]SS-14 are not rapidly internalized by GH,C, rat pituitary cells and RINm5F insulinoma cells, respectively, probably due to degradation of these radioligands at the cell surface (15, 16). As data with respect to internalization of SS may have been influenced by the susceptibility to degradation of the SS ligands used in the above-