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Absence of somatostatin SST2 receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model
European Journal of Pharmacology 644 (2010) 257–262
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Absence of somatostatin SST2 receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model Beatrice Waser a, Renzo Cescato a, Maria-Luisa Tamma b, Helmut R. Maecke b,1, Jean Claude Reubi a,⁎ a b
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland Division of Radiological Chemistry, University Hospital Basel, Basel, Switzerland
a r t i c l e
i n f o
Article history: Received 16 March 2010 Received in revised form 1 July 2010 Accepted 9 July 2010 Available online 16 July 2010 Keywords: Somatostatin receptor Internalization Tumor targeting SOM230 Octreotide Functional selectivity Desensitization
a b s t r a c t Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst2) internalization is a potent mechanism for tumor targeting with sst2 affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst2 internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst2 receptors were injected intravenously with SOM230 or with the [Tyr3, Thr8]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst2 receptors. SOM230-induced sst2 internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst2 internalization triggered by intravenous [Tyr3, Thr8]-octreotide, intravenous SOM230 did not elicit sst2 internalization: immunohistochemically stained sst2 in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5 min, 10 min, 1 h, 6 h, or 24 h after SOM230 injection while sst2 were found intracellularly after [Tyr3, Thr8]-octreotide injection. The inability of stimulating sst2 internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst2 internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst2 internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst2 binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Stable and potent somatostatin analogs such as octreotide and lanreotide are widely used for the successful symptomatic treatment of neuroendocrine tumors (Eriksson and Oberg, 1999; Freda, 2002; Reubi, 2003). More recently, second generation multi-somatostatin analogs, such as KE108 or SOM230 (pasireotide), have been developed having a high affinity for several or all five somatostatin receptor subtypes (Lewis et al., 2003; Reubi et al., 2002; Schmid, 2008). Indeed, KE108 displays high affinity to all 5 somatostatin receptors (ssts), with IC50 values of 2.6± 0.4 nM (sst1), 0.9±0.1 nM (sst2), 1.5±0.2 nM (sst3), 1.6±0.1 nM (sst4), 0.65±0.1 nM (sst5) (Reubi et al., 2002), while SOM230 has high affinity ⁎ Corresponding author. Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, P.O. Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. Tel.: + 41 31 632 3242; fax: + 41 31 632 8999. E-mail addresses: [email protected] (B. Waser), [email protected] (R. Cescato), [email protected] (M.-L. Tamma), [email protected], [email protected] (H.R. Maecke), [email protected] (J.C. Reubi). 1 Present address: Division of Nuclear Medicine, University Hospital Freiburg, Freiburg, Germany. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.07.005
to 4 of the 5 ssts, with IC50 values of 9.3 nM (sst1), 1.0 nM (sst2), 1.5 nM (sst3), N1000 nM (sst4), 0.16 nM (sst5) (Lewis et al., 2003). It is expected that these compounds would better mimic the effects of natural somatostatins than octreotide, that displays respective IC50 values of N10,000 nM (sst1), 2.0±0.7 nM (sst2), 187±55 nM (sst3), N10,000 nM (sst4), and 22±6 nM (sst5) (Reubi et al., 2000). SOM230 for instance is being developed to treat neuroendocrine tumor patients, in particular those that have been poorly sensitive to octreotide treatment (Schmid, 2008). The rationale and the advantage of SOM230 action would be to act not only on somatostatin sst2 receptors but primarily on the non-sst2 receptors expressed in these tumors. Tumors of interest include somatostatin sst5 receptor-expressing adrenocorticotropic hormone (ACTH)-producing pituitary tumors, sst2/sst5 or sst5-expressing growth hormone (GH)-producing pituitary adenomas, and octreotide-resistant carcinoids (Hofland et al., 2005). Recent in vitro data published on KE108 and SOM230 suggest that these compounds do not appear to simply mimic natural somatostatins in selected signal transduction pathways (Ben-Shlomo et al., 2009; Cescato et al., 2009; Lesche et al., 2009; Liu et al., 2005) but to have biased agonistic or functional selectivity properties. While they mimic natural somatostatins in specific signaling systems such as
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the inhibition of cAMP production, they were found to have distinct behavior in others. For instance, neither KE108 nor SOM230 have agonistic properties to stimulate intracellular calcium mobilization and ERK phosphorylation; as opposed to somatostatin or octreotide analogs, they show no effect when applied alone but completely antagonize the somatostatin or octreotide analogs effect, both in sst2-expressing HEK293 cells and in AR42J cells endogenously expressing sst2 (Cescato et al., 2009). Among the clinically relevant properties of somatostatin analogs, agonist-triggered sst2 internalization has been shown to be a potent mechanism to actively transport somatostatin radioligands into tumor cells (Cescato et al., 2006; Liu et al., 2005; Waser et al., 2009). This is relevant for scintigraphic tumor diagnosis and also for targeted tumor radiotherapy. It may therefore be of particular interest to know whether multi-somatostatin analogs such as SOM230 or KE108 are as efficiently internalized in vivo as [Tyr3–Thr8]-octreotide (TATE), in view of a potential development of radiolabeled multisomatostatin analogs for diagnostic or radiotherapeutic purposes. It may also be worth understanding sst2 trafficking triggered by these drugs in order to evaluate their capability of desensitization and escape during long-term treatment in tumor patients. In principle, a compound that does not lead to desensitization would have advantages over a desensitizing agonist. We have recently developed a method permitting to evaluate the in vivo internalization of sst2 in transplanted AR42J tumor cells and pancreatic cells after i.v. injection of the octreotide analog TATE (Waser et al., 2009). The aim of the present study was to use this method to investigate the ability of SOM230 to trigger sst2 internalization in vivo in comparison to TATE. Rats bearing AR42J tumors endogenously expressing the somatostatin sst2 receptors were injected intravenously with SOM230; they were euthanized at various time points and the tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of the somatostatin sst2 receptors. In addition, and for comparison, the sst2 internalization triggered by SOM230 was also evaluated in vitro by immunofluorescence microscopy with the same AR42J cell line used for the in vivo studies.
2. Material and methods 2.1. Reagents All reagents were of the best grade available and were purchased from common suppliers. The sst2-specific antibody R2-88 was provided by Dr. Agnes Schonbrunn (Houston, TX, USA) and the rabbit monoclonal antibody UMB-1 (SS-8000RM) was purchased from Biotrend GmbH, Germany. The secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H + L) was from Molecular Probes, Inc. (Eugene, OR, USA). [Tyr3–Thr8]-octreotide (TATE) was provided by Dr. H.R. Mäcke (Basel, Switzerland). SOM230 (pasireotide) and the two sst2 antagonists DOTA-Bass (DOTA-[4-NO2–Phe-c(DCys–Tyr– DTrp–Lys–Thr–Cys)-DTyr–NH2]) (Ginj et al., 2006) and BIM-23A180 (Cpa-c(DCys–Tyr–DTrp–NMeLys–Thr–Cys)-Nal–NH2) (Rajeswaran et al., 2001) were provided by Dr. J.E. Rivier (La Jolla, CA, USA). The purity of the compounds was N95%.
2.2. Cell line The rat pancreatic tumor cell line AR42J (CRL-1492) was obtained from ATCC (LGC Standards, Teddington, Middlesex, UK) and cultured at 37 °C and 5% CO2 in Ham's F12K containing 2 mM L-glutamine and supplemented with 20% (vol/vol) fbs, 100 U/ml penicillin and 100 μg/ml streptomycin. All culture reagents were from Gibco BRL, Life Technologies (Grand Island, NY).
2.3. Receptor autoradiography Cell membrane pellets of AR42J cells were prepared and receptor autoradiography was performed on 20 μm thick pellet sections (mounted on microscope slides), as described in detail previously for pellets from other cell lines (Cescato et al., 2008; Erchegyi et al., 2009). For each of the tested compounds, complete displacement experiments were performed with the universal somatostatin radioligand [125I]-[Leu8, D-Trp22, Tyr25]-somatostatin-28 (2000 Ci/mmol; Anawa, Wangen, Switzerland) using 6000 cpm/100μl and increasing concentrations of the unlabeled compounds ranging from 0.1 to 1000 nmol/l. Somatostatin-28 was run in parallel as control using the same increasing concentrations. The slides were exposed to Biomax MR film (Kodak) for 7 days at 4 °C. IC50 values were calculated after quantification of the data using a computer-assisted image processing system (Cescato et al., 2008). Tissue standards containing known amounts of isotopes, cross-calibrated to tissue-equivalent ligand concentrations, were used for quantification (Cescato et al., 2008). 2.4. Animal tumor models Animals were kept, treated, and cared for in compliance with the guidelines of the Swiss regulations (approval 789). A total of 10 to 12 million AR42J cells, freshly suspended in sterile phosphate-buffered saline (PBS), were subcutaneously implanted in one flank of Lewis rats (49–55 g). 11–15 days after inoculation, the rats, weighing 120–150 g, showed solid palpable tumor masses (tumor weight 70–150 mg) as reported previously (Ginj et al., 2008; Waser et al., 2009) and were used for the in vivo internalization experiments. Peptides were solubilized in sodium chloride solution (0.9%, 0.1% bovine serum albumin) and injected into the rats in a total volume of 0.2 ml under isoflurane anesthesia. Two rats were used for each experimental condition. A first set of rats was injected with 0.21 mg/animal SOM230 into the lateral tail vein and euthanized 2.5 min, 10 min, 1 h, 6 h and 24 h after injection. As a positive control for somatostatin sst2 receptor internalization, 0.21 mg/animal TATE was injected in rats that where then euthanized after 1 h. Untreated rats injected only with PBS were used as negative controls. To test for antagonism a second set of rats was first injected with SOM230 (a 100-fold excess compared to the amount of TATE) into the lateral tail vein, followed 5 min later by a second injection with 0.0021 mg/animal TATE again into the lateral tail vein. This 0.0021 mg/ animal low dose of TATE, still able to induce a complete sst2 internalization (Waser et al., 2009), was used to permit the use of a lower dose of SOM230 (but still in 100 times excess of TATE) in the antagonist test. As positive control for antagonism, the sst2 antagonist DOTA-Bass (Ginj et al., 2006), was applied instead of SOM230 in a similar experimental setting in 100-fold excess compared to TATE. The animals were then euthanized 1 h after the second injection. The application of 0.0021 mg/animal of TATE alone was used as positive control for agonism. The tumors and pancreas of each animal were collected. All samples were cut in half. One half of the samples were immersed in a 4% formalin solution for 24 h and paraffin-embedded for immunohistochemical investigations. The other half was frozen in dry ice and stored at −80 °C for further in vitro receptor autoradiography. 2.5. Immunohistochemistry of somatostatin sst2 receptors Sst2 immunohistochemistry was performed as described before (Waser et al., 2009). The samples were either tested with the sst2specific polyclonal antibodies R2-88 or the rabbit monoclonal antibody UMB-1 (Fischer et al., 2008; Korner et al., 2005). Formalin-fixed, paraffin-embedded tissue sections (4 μm-thick) were used. The best antigen-retrieval method for R2-88 and UMB-1 immunohistochemistry was boiling in the microwave in 5% urea buffer (pH 9.5). R2-88 was applied in a 1:1000 dilution and UMB-1 in a 1:100 dilution. The secondary antibody was a biotinylated goat anti-rabbit
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immunoglobulin. Antibody binding was visualized using the VECTASTAIN Elite ABC Kit (Vector). Staining was performed with diaminobenzidine and counterstaining with hemalum. For negative control, the primary antibodies were preabsorbed with 100 nM of the corresponding antigen peptide. In all experiments, a well-characterized gastroenteropancreatic neuroendocrine tumor strongly expressing sst2 as determined by receptor autoradiography, was included as positive control. 2.6. In vitro sst2 internalization assay based on immunofluorescence microscopy AR42J cells were grown on poly-D-lysine (20 μg/ml) (Sigma-Aldrich, St. Louis, MO) coated 35 mm four-well plates (Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany) for 24 h at 37 °C and with 5% CO2. Cells were then treated at 37 °C in growth medium for 30 min with the somatostatin analogs as indicated and then processed for immunofluorescence microscopy as described previously (Cescato et al., 2006; Liu et al., 2005) using as primary antibodies the sst2-specific antibody R2-88 or the rabbit monoclonal antibody UMB-1 (Fischer et al., 2008; Korner et al., 2005; Waser et al., 2009). Alexa Fluor 488 goat antirabbit IgG (H + L) was used as secondary antibody. The cells were
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imaged using a Leica DM RB immunofluorescence microscope and an Olympus DP10 camera. 3. Results It has been reported previously that SOM230 shows high binding affinities to sst1, sst2, sst3, and sst5 (Lewis et al., 2003; Schmid, 2008). We confirm the high binding affinities of SOM230 for sst2 in our own cellular model system, the AR42J cells endogenously expressing the somatostatin sst2 receptor. The following IC50 values (mean ± S.E.M.; n = 3) were obtained in the sst2 binding assay: SOM230: 1.6 ± 0.4 nM; the reference compound TATE: 0.8 ± 0.03 nM; the antagonist DOTA-Bass: 1.2 ± 0.1 nM. 3.1. SOM230 fails to induce somatostatin sst2 receptor internalization As has been described previously by Waser et al. (Waser et al., 2009) in untreated rats bearing an AR42J-tumor, the somatostatin sst2 receptors are localized at the plasma membrane of both, the AR42J-tumor cells (Fig. 1A, control) and the cells of the pancreas (Fig. 1B, control), detected immunohistochemically using the two distinct sst2 specific antibodies R2-88 and UMB-1. The intravenous application of TATE (0.21 mg/animal)
Fig. 1. Time course of somatostatin sst2 receptor internalization in AR42J tumors (A) and rat pancreas (B) as determined by R2-88 or UMB-1 immunohistochemistry. Animals euthanized 2.5 min, 10 min, 1 h, 6 h, or 24 h after the injection of 0.21 mg/animal SOM230 show with both antibodies, R2-88 and UMB-1, a complete absence of sst2 internalization in AR42J tumor cells as well as in pancreatic cells, comparable to cells of untreated animals (control). However, 1 h after the injection of 0.21 mg/animal TATE a massive sst2 internalization is detectable in AR42J tumor cells and pancreatic cells. Bars = 0.01 mm.
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in AR42J-tumor bearing rats euthanized 1 h after injection triggers a massive and rapid internalization of the somatostatin sst2 receptors into the tumor cells (Fig. 1A) and the pancreatic cells (Fig. 1B). Conversely, the injection of the same dose of SOM230 (0.21 mg/animal) fails to trigger sst2 internalization in the AR42J-tumor cells (Fig. 1A) as well as in the pancreatic cells (Fig. 1B) in a time course study ranging from 2.5 min up to 24 h. Moreover, this in vivo result is confirmed by an in vitro immunofluorescence microscopy based internalization assay with AR42J cells using the sst2-specific antibodies R2-88 and UMB-1. Fig. 2 illustrates that the treatment of AR42J cells with 1 μM SOM230 for 30 min fails to stimulate somatostatin sst2 receptor internalization, while treating the cells with 1 μM TATE for the same time elicits a pronounced relocation of the somatostatin sst2 receptors from the plasma membrane to intracellular compartments resembling endosomes, as has been shown previously with other cellular systems (Liu et al., 2005). An identical staining pattern was obtained with both antibodies, R2-88 and UMB-1. 3.2. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization Since SOM230 is not able to trigger sst2 internalization in our animal model system, even at high doses, we determined whether SOM230 may exhibit antagonistic properties in vivo. To this end rats bearing the AR42J-tumor were injected first with SOM230 in a 100-fold excess compared to TATE, followed by a second injection of TATE at a dose (0.0021 mg/animal) known to elicit somatostatin sst2 receptor internalization, and then euthanized 1 h post-injection. Fig. 3 shows that, in this experimental setting, SOM230 is not able to antagonize TATEinduced sst2 internalization in AR42J tumors and pancreas, since the receptors are localized intracellularly in both tissues when analyzed immunohistochemically using the R2-88 antibody. However, when an established sst2-antagonist (DOTA-Bass) is used as a positive control to test for antagonism instead of SOM230, the TATE-induced sst2 internalization is completely abolished and the receptors remain at the plasma membrane in both tissues. The above presented in vivo result is confirmed in vitro by immunofluorescence microscopy based internalization assay with AR42J cells using the R2-88 antibody. Fig. 4 shows that applying a
100-fold excess of SOM230 does not antagonize sst2 internalization induced by 100 nM TATE, while using the established sst2-antagonist BIM-23A180 (Rajeswaran et al., 2001) completely abolishes TATEinduced sst2 internalization resulting in a pronounced membrane staining. 4. Discussion We show here that SOM230 has a different behavior than an octreotide analog in triggering sst2 internalization in vivo, although both compounds have a comparable sst2 binding affinity. Even very high doses of SOM230 are unable to stimulate sst2 internalization at a wide range of times between 2.5 min and 24 h post-injection, while this is achieved with TATE even at lower doses (Waser et al., 2009). In vitro data using different cellular systems have shown previously the poor capacity of SOM230 to stimulate somatostatin sst2 receptor internalization (Lesche et al., 2009; Liu et al., 2005), fully confirmed by the present in vivo and in vitro data. The lack of effect of SOM230 to stimulate sst2 internalization may be related to its previously reported strong functional selectivity properties (Cescato et al., 2009) that make it quite different from octreotide in various second messenger systems. It is worth to notice, however, that although SOM230 lacks the ability to stimulate sst2 internalization, it is also unable to produce a measurable antagonistic effect since it cannot inhibit agonist-induced sst2 internalization. This is at difference to the previously reported lack of effect of SOM230 to stimulate intracellular calcium mobilization and ERK phosphorylation in AR42J cells, that is, however, accompanied by a competitive antagonistic behavior at blocking the effect of octreotide analogs on these two parameters (Cescato et al., 2009). The results showing the incapacity of SOM230 to trigger the internalization of sst2 in vivo and in vitro may have two important consequences. The first is related to the development of SOM230-based imaging procedures for tumors of patients to be selected for long-term SOM230 therapy. The second relates to the distinct effects of octreotide and SOM230 on desensitization and escape from therapy in neuroendocrine tumor patients. There is increasing need in medicine and particularly in oncology for individualized therapy. This means among others that a drug shall be given to a tumor patient only if there is molecular biological evidence that the considered tumor is an adequate target for that
Fig. 2. SOM230 fails to induce somatostatin sst2 receptor internalization in AR42J cells as determined by immunofluorescence microscopy using the antibodies R2-88 or UMB-1. AR42J cells endogenously expressing the somatostatin sst2 receptor were either treated with vehicle alone, or with 1 μM SOM230 or 1 μM TATE for 30 min at 37 °C. In contrast to TATE, which elicits a pronounced sst2 internalization, SOM230 completely fails to trigger sst2 internalization in AR42J cells as detected with both antibodies.
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Fig. 3. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization in AR42J cells as determined by R2-88 immunohistochemistry. Animals were injected with 0.0021 mg/animal TATE in the lateral tail vein 5 min after they were injected with a 100-fold excess of SOM230. The animals were then euthanized 1 h post-injection. In AR42J tumor cells as well as in the pancreatic cells the somatostatin sst2 receptor is localized intracellularly indicating that SOM230 is unable to antagonize TATE-stimulated receptor internalization, as shown in the control when 0.0021 ml/animal TATE is applied alone. However, when a 100-fold excess of the sst2 antagonist DOTA-Bass (sst2-Antag.) is used together with TATE instead of SOM230, the TATE-induced sst2 internalization is completely abolished. Bars = 0.01 mm.
particular drug. For SOM230 therapy indication, it means that it would be advantageous to be able to predict the success of treatment. One way to reach this goal is to have radiolabeled SOM230 injected into the patient that would be then taken up preferably by the
Fig. 4. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization in AR42J cells as determined by immunofluorescence microscopy using the R2-88 antibody. AR42J cells were either treated with 100 nM TATE, with 10 μM SOM230 or with 100 nM TATE in the presence of 10 μM SOM230 for 30 min at 37 °C. At a high concentration of 10 μM, SOM230 is not able to stimulate sst2 internalization, but it is also not able to antagonize TATE-stimulated sst2 internalization. However, when TATE is applied together with the established sst2 antagonist BIM-23A180 (sst2-Antag.) (Rajeswaran et al., 2001) the TATE-induced sst2 internalization is completely abolished.
somatostatin receptors expressed in the tumor. The degree of its uptake, in particular in comparison to the Octreoscan uptake, would give information about a potential successful therapeutical application of SOM230 in this patient, as compared to an octreotide treatment. Since, however, no correlation can be found between the affinity binding of SOM230 (as a mean of its final action at the cellular level) and its capability to trigger internalization for the most important somatostatin receptor subtype expressed in tumors, sst2, there is little probability to successfully develop a SOM230-based radiotracer for that purpose. The present data seem to indicate that SOM230 and analogs thereof are inadequate for such a diagnostic imaging. It should be emphasized that these negative internalization data do not preclude to other functional parameters positively influenced by SOM230, such as its ability to potently inhibit hormone release. This is a further example of functional selectivity of SOM230 (Cescato et al., 2009; Liu et al., 2005). The fact that the somatostatin sst2 receptors are not internalized and remain at the cell surface even after the application of large doses of SOM230, while all receptors are internalized after comparable TATE treatment, may suggest that SOM230 could be of particular benefit for long-term hormone-inhibitory treatment of GEP tumors, by preventing desensitization of the somatostatin sst2 receptors and therefore preventing escape from therapy (Ben-Shlomo et al., 2009; Elberg et al., 2002; Reisine, 1984). Our data indicate indeed that during a long-term SOM230 therapy there are always a large proportion of sst2 remaining at the cell membrane, possibly receptive to further continuous drug therapy. The present data may be a molecular explanation for preliminary reports of clinical studies showing less desensitization in neuroendocrine tumors after SOM230 treatment compared to octreotide therapy (Hofland et al., 2005). References Ben-Shlomo, A., Schmid, H., Wawrowsky, K., Pichurin, O., Hubina, E., Chesnokova, V., Liu, N.A., Culler, M., Melmed, S., 2009. Differential ligand-mediated pituitary somatostatin receptor subtype signaling: implications for corticotroph tumor therapy. J. Clin. Endocrinol. Metab. 94, 4342–4350.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Absence of somatostatin SST2 receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model Beatrice Waser a, Renzo Cescato a, Maria-Luisa Tamma b, Helmut R. Maecke b,1, Jean Claude Reubi a,⁎ a b
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland Division of Radiological Chemistry, University Hospital Basel, Basel, Switzerland
a r t i c l e
i n f o
Article history: Received 16 March 2010 Received in revised form 1 July 2010 Accepted 9 July 2010 Available online 16 July 2010 Keywords: Somatostatin receptor Internalization Tumor targeting SOM230 Octreotide Functional selectivity Desensitization
a b s t r a c t Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst2) internalization is a potent mechanism for tumor targeting with sst2 affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst2 internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst2 receptors were injected intravenously with SOM230 or with the [Tyr3, Thr8]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst2 receptors. SOM230-induced sst2 internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst2 internalization triggered by intravenous [Tyr3, Thr8]-octreotide, intravenous SOM230 did not elicit sst2 internalization: immunohistochemically stained sst2 in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5 min, 10 min, 1 h, 6 h, or 24 h after SOM230 injection while sst2 were found intracellularly after [Tyr3, Thr8]-octreotide injection. The inability of stimulating sst2 internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst2 internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst2 internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst2 binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Stable and potent somatostatin analogs such as octreotide and lanreotide are widely used for the successful symptomatic treatment of neuroendocrine tumors (Eriksson and Oberg, 1999; Freda, 2002; Reubi, 2003). More recently, second generation multi-somatostatin analogs, such as KE108 or SOM230 (pasireotide), have been developed having a high affinity for several or all five somatostatin receptor subtypes (Lewis et al., 2003; Reubi et al., 2002; Schmid, 2008). Indeed, KE108 displays high affinity to all 5 somatostatin receptors (ssts), with IC50 values of 2.6± 0.4 nM (sst1), 0.9±0.1 nM (sst2), 1.5±0.2 nM (sst3), 1.6±0.1 nM (sst4), 0.65±0.1 nM (sst5) (Reubi et al., 2002), while SOM230 has high affinity ⁎ Corresponding author. Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, P.O. Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. Tel.: + 41 31 632 3242; fax: + 41 31 632 8999. E-mail addresses: [email protected] (B. Waser), [email protected] (R. Cescato), [email protected] (M.-L. Tamma), [email protected], [email protected] (H.R. Maecke), [email protected] (J.C. Reubi). 1 Present address: Division of Nuclear Medicine, University Hospital Freiburg, Freiburg, Germany. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.07.005
to 4 of the 5 ssts, with IC50 values of 9.3 nM (sst1), 1.0 nM (sst2), 1.5 nM (sst3), N1000 nM (sst4), 0.16 nM (sst5) (Lewis et al., 2003). It is expected that these compounds would better mimic the effects of natural somatostatins than octreotide, that displays respective IC50 values of N10,000 nM (sst1), 2.0±0.7 nM (sst2), 187±55 nM (sst3), N10,000 nM (sst4), and 22±6 nM (sst5) (Reubi et al., 2000). SOM230 for instance is being developed to treat neuroendocrine tumor patients, in particular those that have been poorly sensitive to octreotide treatment (Schmid, 2008). The rationale and the advantage of SOM230 action would be to act not only on somatostatin sst2 receptors but primarily on the non-sst2 receptors expressed in these tumors. Tumors of interest include somatostatin sst5 receptor-expressing adrenocorticotropic hormone (ACTH)-producing pituitary tumors, sst2/sst5 or sst5-expressing growth hormone (GH)-producing pituitary adenomas, and octreotide-resistant carcinoids (Hofland et al., 2005). Recent in vitro data published on KE108 and SOM230 suggest that these compounds do not appear to simply mimic natural somatostatins in selected signal transduction pathways (Ben-Shlomo et al., 2009; Cescato et al., 2009; Lesche et al., 2009; Liu et al., 2005) but to have biased agonistic or functional selectivity properties. While they mimic natural somatostatins in specific signaling systems such as
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the inhibition of cAMP production, they were found to have distinct behavior in others. For instance, neither KE108 nor SOM230 have agonistic properties to stimulate intracellular calcium mobilization and ERK phosphorylation; as opposed to somatostatin or octreotide analogs, they show no effect when applied alone but completely antagonize the somatostatin or octreotide analogs effect, both in sst2-expressing HEK293 cells and in AR42J cells endogenously expressing sst2 (Cescato et al., 2009). Among the clinically relevant properties of somatostatin analogs, agonist-triggered sst2 internalization has been shown to be a potent mechanism to actively transport somatostatin radioligands into tumor cells (Cescato et al., 2006; Liu et al., 2005; Waser et al., 2009). This is relevant for scintigraphic tumor diagnosis and also for targeted tumor radiotherapy. It may therefore be of particular interest to know whether multi-somatostatin analogs such as SOM230 or KE108 are as efficiently internalized in vivo as [Tyr3–Thr8]-octreotide (TATE), in view of a potential development of radiolabeled multisomatostatin analogs for diagnostic or radiotherapeutic purposes. It may also be worth understanding sst2 trafficking triggered by these drugs in order to evaluate their capability of desensitization and escape during long-term treatment in tumor patients. In principle, a compound that does not lead to desensitization would have advantages over a desensitizing agonist. We have recently developed a method permitting to evaluate the in vivo internalization of sst2 in transplanted AR42J tumor cells and pancreatic cells after i.v. injection of the octreotide analog TATE (Waser et al., 2009). The aim of the present study was to use this method to investigate the ability of SOM230 to trigger sst2 internalization in vivo in comparison to TATE. Rats bearing AR42J tumors endogenously expressing the somatostatin sst2 receptors were injected intravenously with SOM230; they were euthanized at various time points and the tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of the somatostatin sst2 receptors. In addition, and for comparison, the sst2 internalization triggered by SOM230 was also evaluated in vitro by immunofluorescence microscopy with the same AR42J cell line used for the in vivo studies.
2. Material and methods 2.1. Reagents All reagents were of the best grade available and were purchased from common suppliers. The sst2-specific antibody R2-88 was provided by Dr. Agnes Schonbrunn (Houston, TX, USA) and the rabbit monoclonal antibody UMB-1 (SS-8000RM) was purchased from Biotrend GmbH, Germany. The secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H + L) was from Molecular Probes, Inc. (Eugene, OR, USA). [Tyr3–Thr8]-octreotide (TATE) was provided by Dr. H.R. Mäcke (Basel, Switzerland). SOM230 (pasireotide) and the two sst2 antagonists DOTA-Bass (DOTA-[4-NO2–Phe-c(DCys–Tyr– DTrp–Lys–Thr–Cys)-DTyr–NH2]) (Ginj et al., 2006) and BIM-23A180 (Cpa-c(DCys–Tyr–DTrp–NMeLys–Thr–Cys)-Nal–NH2) (Rajeswaran et al., 2001) were provided by Dr. J.E. Rivier (La Jolla, CA, USA). The purity of the compounds was N95%.
2.2. Cell line The rat pancreatic tumor cell line AR42J (CRL-1492) was obtained from ATCC (LGC Standards, Teddington, Middlesex, UK) and cultured at 37 °C and 5% CO2 in Ham's F12K containing 2 mM L-glutamine and supplemented with 20% (vol/vol) fbs, 100 U/ml penicillin and 100 μg/ml streptomycin. All culture reagents were from Gibco BRL, Life Technologies (Grand Island, NY).
2.3. Receptor autoradiography Cell membrane pellets of AR42J cells were prepared and receptor autoradiography was performed on 20 μm thick pellet sections (mounted on microscope slides), as described in detail previously for pellets from other cell lines (Cescato et al., 2008; Erchegyi et al., 2009). For each of the tested compounds, complete displacement experiments were performed with the universal somatostatin radioligand [125I]-[Leu8, D-Trp22, Tyr25]-somatostatin-28 (2000 Ci/mmol; Anawa, Wangen, Switzerland) using 6000 cpm/100μl and increasing concentrations of the unlabeled compounds ranging from 0.1 to 1000 nmol/l. Somatostatin-28 was run in parallel as control using the same increasing concentrations. The slides were exposed to Biomax MR film (Kodak) for 7 days at 4 °C. IC50 values were calculated after quantification of the data using a computer-assisted image processing system (Cescato et al., 2008). Tissue standards containing known amounts of isotopes, cross-calibrated to tissue-equivalent ligand concentrations, were used for quantification (Cescato et al., 2008). 2.4. Animal tumor models Animals were kept, treated, and cared for in compliance with the guidelines of the Swiss regulations (approval 789). A total of 10 to 12 million AR42J cells, freshly suspended in sterile phosphate-buffered saline (PBS), were subcutaneously implanted in one flank of Lewis rats (49–55 g). 11–15 days after inoculation, the rats, weighing 120–150 g, showed solid palpable tumor masses (tumor weight 70–150 mg) as reported previously (Ginj et al., 2008; Waser et al., 2009) and were used for the in vivo internalization experiments. Peptides were solubilized in sodium chloride solution (0.9%, 0.1% bovine serum albumin) and injected into the rats in a total volume of 0.2 ml under isoflurane anesthesia. Two rats were used for each experimental condition. A first set of rats was injected with 0.21 mg/animal SOM230 into the lateral tail vein and euthanized 2.5 min, 10 min, 1 h, 6 h and 24 h after injection. As a positive control for somatostatin sst2 receptor internalization, 0.21 mg/animal TATE was injected in rats that where then euthanized after 1 h. Untreated rats injected only with PBS were used as negative controls. To test for antagonism a second set of rats was first injected with SOM230 (a 100-fold excess compared to the amount of TATE) into the lateral tail vein, followed 5 min later by a second injection with 0.0021 mg/animal TATE again into the lateral tail vein. This 0.0021 mg/ animal low dose of TATE, still able to induce a complete sst2 internalization (Waser et al., 2009), was used to permit the use of a lower dose of SOM230 (but still in 100 times excess of TATE) in the antagonist test. As positive control for antagonism, the sst2 antagonist DOTA-Bass (Ginj et al., 2006), was applied instead of SOM230 in a similar experimental setting in 100-fold excess compared to TATE. The animals were then euthanized 1 h after the second injection. The application of 0.0021 mg/animal of TATE alone was used as positive control for agonism. The tumors and pancreas of each animal were collected. All samples were cut in half. One half of the samples were immersed in a 4% formalin solution for 24 h and paraffin-embedded for immunohistochemical investigations. The other half was frozen in dry ice and stored at −80 °C for further in vitro receptor autoradiography. 2.5. Immunohistochemistry of somatostatin sst2 receptors Sst2 immunohistochemistry was performed as described before (Waser et al., 2009). The samples were either tested with the sst2specific polyclonal antibodies R2-88 or the rabbit monoclonal antibody UMB-1 (Fischer et al., 2008; Korner et al., 2005). Formalin-fixed, paraffin-embedded tissue sections (4 μm-thick) were used. The best antigen-retrieval method for R2-88 and UMB-1 immunohistochemistry was boiling in the microwave in 5% urea buffer (pH 9.5). R2-88 was applied in a 1:1000 dilution and UMB-1 in a 1:100 dilution. The secondary antibody was a biotinylated goat anti-rabbit
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immunoglobulin. Antibody binding was visualized using the VECTASTAIN Elite ABC Kit (Vector). Staining was performed with diaminobenzidine and counterstaining with hemalum. For negative control, the primary antibodies were preabsorbed with 100 nM of the corresponding antigen peptide. In all experiments, a well-characterized gastroenteropancreatic neuroendocrine tumor strongly expressing sst2 as determined by receptor autoradiography, was included as positive control. 2.6. In vitro sst2 internalization assay based on immunofluorescence microscopy AR42J cells were grown on poly-D-lysine (20 μg/ml) (Sigma-Aldrich, St. Louis, MO) coated 35 mm four-well plates (Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany) for 24 h at 37 °C and with 5% CO2. Cells were then treated at 37 °C in growth medium for 30 min with the somatostatin analogs as indicated and then processed for immunofluorescence microscopy as described previously (Cescato et al., 2006; Liu et al., 2005) using as primary antibodies the sst2-specific antibody R2-88 or the rabbit monoclonal antibody UMB-1 (Fischer et al., 2008; Korner et al., 2005; Waser et al., 2009). Alexa Fluor 488 goat antirabbit IgG (H + L) was used as secondary antibody. The cells were
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imaged using a Leica DM RB immunofluorescence microscope and an Olympus DP10 camera. 3. Results It has been reported previously that SOM230 shows high binding affinities to sst1, sst2, sst3, and sst5 (Lewis et al., 2003; Schmid, 2008). We confirm the high binding affinities of SOM230 for sst2 in our own cellular model system, the AR42J cells endogenously expressing the somatostatin sst2 receptor. The following IC50 values (mean ± S.E.M.; n = 3) were obtained in the sst2 binding assay: SOM230: 1.6 ± 0.4 nM; the reference compound TATE: 0.8 ± 0.03 nM; the antagonist DOTA-Bass: 1.2 ± 0.1 nM. 3.1. SOM230 fails to induce somatostatin sst2 receptor internalization As has been described previously by Waser et al. (Waser et al., 2009) in untreated rats bearing an AR42J-tumor, the somatostatin sst2 receptors are localized at the plasma membrane of both, the AR42J-tumor cells (Fig. 1A, control) and the cells of the pancreas (Fig. 1B, control), detected immunohistochemically using the two distinct sst2 specific antibodies R2-88 and UMB-1. The intravenous application of TATE (0.21 mg/animal)
Fig. 1. Time course of somatostatin sst2 receptor internalization in AR42J tumors (A) and rat pancreas (B) as determined by R2-88 or UMB-1 immunohistochemistry. Animals euthanized 2.5 min, 10 min, 1 h, 6 h, or 24 h after the injection of 0.21 mg/animal SOM230 show with both antibodies, R2-88 and UMB-1, a complete absence of sst2 internalization in AR42J tumor cells as well as in pancreatic cells, comparable to cells of untreated animals (control). However, 1 h after the injection of 0.21 mg/animal TATE a massive sst2 internalization is detectable in AR42J tumor cells and pancreatic cells. Bars = 0.01 mm.
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in AR42J-tumor bearing rats euthanized 1 h after injection triggers a massive and rapid internalization of the somatostatin sst2 receptors into the tumor cells (Fig. 1A) and the pancreatic cells (Fig. 1B). Conversely, the injection of the same dose of SOM230 (0.21 mg/animal) fails to trigger sst2 internalization in the AR42J-tumor cells (Fig. 1A) as well as in the pancreatic cells (Fig. 1B) in a time course study ranging from 2.5 min up to 24 h. Moreover, this in vivo result is confirmed by an in vitro immunofluorescence microscopy based internalization assay with AR42J cells using the sst2-specific antibodies R2-88 and UMB-1. Fig. 2 illustrates that the treatment of AR42J cells with 1 μM SOM230 for 30 min fails to stimulate somatostatin sst2 receptor internalization, while treating the cells with 1 μM TATE for the same time elicits a pronounced relocation of the somatostatin sst2 receptors from the plasma membrane to intracellular compartments resembling endosomes, as has been shown previously with other cellular systems (Liu et al., 2005). An identical staining pattern was obtained with both antibodies, R2-88 and UMB-1. 3.2. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization Since SOM230 is not able to trigger sst2 internalization in our animal model system, even at high doses, we determined whether SOM230 may exhibit antagonistic properties in vivo. To this end rats bearing the AR42J-tumor were injected first with SOM230 in a 100-fold excess compared to TATE, followed by a second injection of TATE at a dose (0.0021 mg/animal) known to elicit somatostatin sst2 receptor internalization, and then euthanized 1 h post-injection. Fig. 3 shows that, in this experimental setting, SOM230 is not able to antagonize TATEinduced sst2 internalization in AR42J tumors and pancreas, since the receptors are localized intracellularly in both tissues when analyzed immunohistochemically using the R2-88 antibody. However, when an established sst2-antagonist (DOTA-Bass) is used as a positive control to test for antagonism instead of SOM230, the TATE-induced sst2 internalization is completely abolished and the receptors remain at the plasma membrane in both tissues. The above presented in vivo result is confirmed in vitro by immunofluorescence microscopy based internalization assay with AR42J cells using the R2-88 antibody. Fig. 4 shows that applying a
100-fold excess of SOM230 does not antagonize sst2 internalization induced by 100 nM TATE, while using the established sst2-antagonist BIM-23A180 (Rajeswaran et al., 2001) completely abolishes TATEinduced sst2 internalization resulting in a pronounced membrane staining. 4. Discussion We show here that SOM230 has a different behavior than an octreotide analog in triggering sst2 internalization in vivo, although both compounds have a comparable sst2 binding affinity. Even very high doses of SOM230 are unable to stimulate sst2 internalization at a wide range of times between 2.5 min and 24 h post-injection, while this is achieved with TATE even at lower doses (Waser et al., 2009). In vitro data using different cellular systems have shown previously the poor capacity of SOM230 to stimulate somatostatin sst2 receptor internalization (Lesche et al., 2009; Liu et al., 2005), fully confirmed by the present in vivo and in vitro data. The lack of effect of SOM230 to stimulate sst2 internalization may be related to its previously reported strong functional selectivity properties (Cescato et al., 2009) that make it quite different from octreotide in various second messenger systems. It is worth to notice, however, that although SOM230 lacks the ability to stimulate sst2 internalization, it is also unable to produce a measurable antagonistic effect since it cannot inhibit agonist-induced sst2 internalization. This is at difference to the previously reported lack of effect of SOM230 to stimulate intracellular calcium mobilization and ERK phosphorylation in AR42J cells, that is, however, accompanied by a competitive antagonistic behavior at blocking the effect of octreotide analogs on these two parameters (Cescato et al., 2009). The results showing the incapacity of SOM230 to trigger the internalization of sst2 in vivo and in vitro may have two important consequences. The first is related to the development of SOM230-based imaging procedures for tumors of patients to be selected for long-term SOM230 therapy. The second relates to the distinct effects of octreotide and SOM230 on desensitization and escape from therapy in neuroendocrine tumor patients. There is increasing need in medicine and particularly in oncology for individualized therapy. This means among others that a drug shall be given to a tumor patient only if there is molecular biological evidence that the considered tumor is an adequate target for that
Fig. 2. SOM230 fails to induce somatostatin sst2 receptor internalization in AR42J cells as determined by immunofluorescence microscopy using the antibodies R2-88 or UMB-1. AR42J cells endogenously expressing the somatostatin sst2 receptor were either treated with vehicle alone, or with 1 μM SOM230 or 1 μM TATE for 30 min at 37 °C. In contrast to TATE, which elicits a pronounced sst2 internalization, SOM230 completely fails to trigger sst2 internalization in AR42J cells as detected with both antibodies.
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Fig. 3. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization in AR42J cells as determined by R2-88 immunohistochemistry. Animals were injected with 0.0021 mg/animal TATE in the lateral tail vein 5 min after they were injected with a 100-fold excess of SOM230. The animals were then euthanized 1 h post-injection. In AR42J tumor cells as well as in the pancreatic cells the somatostatin sst2 receptor is localized intracellularly indicating that SOM230 is unable to antagonize TATE-stimulated receptor internalization, as shown in the control when 0.0021 ml/animal TATE is applied alone. However, when a 100-fold excess of the sst2 antagonist DOTA-Bass (sst2-Antag.) is used together with TATE instead of SOM230, the TATE-induced sst2 internalization is completely abolished. Bars = 0.01 mm.
particular drug. For SOM230 therapy indication, it means that it would be advantageous to be able to predict the success of treatment. One way to reach this goal is to have radiolabeled SOM230 injected into the patient that would be then taken up preferably by the
Fig. 4. SOM230 fails to antagonize TATE-stimulated somatostatin sst2 receptor internalization in AR42J cells as determined by immunofluorescence microscopy using the R2-88 antibody. AR42J cells were either treated with 100 nM TATE, with 10 μM SOM230 or with 100 nM TATE in the presence of 10 μM SOM230 for 30 min at 37 °C. At a high concentration of 10 μM, SOM230 is not able to stimulate sst2 internalization, but it is also not able to antagonize TATE-stimulated sst2 internalization. However, when TATE is applied together with the established sst2 antagonist BIM-23A180 (sst2-Antag.) (Rajeswaran et al., 2001) the TATE-induced sst2 internalization is completely abolished.
somatostatin receptors expressed in the tumor. The degree of its uptake, in particular in comparison to the Octreoscan uptake, would give information about a potential successful therapeutical application of SOM230 in this patient, as compared to an octreotide treatment. Since, however, no correlation can be found between the affinity binding of SOM230 (as a mean of its final action at the cellular level) and its capability to trigger internalization for the most important somatostatin receptor subtype expressed in tumors, sst2, there is little probability to successfully develop a SOM230-based radiotracer for that purpose. The present data seem to indicate that SOM230 and analogs thereof are inadequate for such a diagnostic imaging. It should be emphasized that these negative internalization data do not preclude to other functional parameters positively influenced by SOM230, such as its ability to potently inhibit hormone release. This is a further example of functional selectivity of SOM230 (Cescato et al., 2009; Liu et al., 2005). The fact that the somatostatin sst2 receptors are not internalized and remain at the cell surface even after the application of large doses of SOM230, while all receptors are internalized after comparable TATE treatment, may suggest that SOM230 could be of particular benefit for long-term hormone-inhibitory treatment of GEP tumors, by preventing desensitization of the somatostatin sst2 receptors and therefore preventing escape from therapy (Ben-Shlomo et al., 2009; Elberg et al., 2002; Reisine, 1984). Our data indicate indeed that during a long-term SOM230 therapy there are always a large proportion of sst2 remaining at the cell membrane, possibly receptive to further continuous drug therapy. The present data may be a molecular explanation for preliminary reports of clinical studies showing less desensitization in neuroendocrine tumors after SOM230 treatment compared to octreotide therapy (Hofland et al., 2005). References Ben-Shlomo, A., Schmid, H., Wawrowsky, K., Pichurin, O., Hubina, E., Chesnokova, V., Liu, N.A., Culler, M., Melmed, S., 2009. Differential ligand-mediated pituitary somatostatin receptor subtype signaling: implications for corticotroph tumor therapy. J. Clin. Endocrinol. Metab. 94, 4342–4350.
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