Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide

Recently, we developed a technique that allows the in vivo visualization in man of somatostatin receptor-positive neuroendocrine tumors after i.v. injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide. Radiotherapy of such tumors using somatostatin analogs coupled to alpha- or beta-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, [125I-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 internalize (acid-resistant) radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of the dose of radioligand added were obtained. Binding and internalization of [125I-Tyr3]octreotide 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 [125I-Tyr3]octreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [125I-Tyr3]octreotide 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 [125I-Tyr3]octreotide to a broken cell preparation of AtT20 cells was displaced in a dose-dependent manner by unlabeled octreotide, with an IC50 of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTP-binding protein-dependent manner by mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found.(ABSTRACT TRUNCATED AT 400 WORDS

Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas

Interferon-alpha (IFN alpha) may exert direct inhibitory effects on cell proliferation and on the production of different peptide hormones. We investigated the effect of IFN alpha on hormone production by 15 GH-secreting pituitary adenomas, 4 clinically nonfunctioning or gonadotroph pituitary adenomas, and 4 prolactinomas in vitro. In the GH-secreting pituitary adenoma cultures, a short term (72-h) incubation with IFN alpha (50-100 U/mL) significantly inhibited GH secretion in 3 of 7 cases and PRL secretion in 6 of 7 cultures. During prolonged incubation (14 days) with IFN alpha, GH and/or PRL secretion was significantly inhibited in 7 of 8 cultures (GH, 17-78% inhibition; PRL, 39-88% inhibition). In the clinically nonfunctioning or gonadotroph cultures

Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary- and islet cell tumors

The effects of somatostatin (SS-14 and/or SS-28) and of the three octapeptide SS-analogs that are available for clinical use (octreotide, BIM-23014 and RC-160) on hormone release by primary cultures of 15 clinically nonfunctioning pituitary adenomas (NFA), 7 prolactinomas, and 2 insulinomas were investigated. In the pituitary adenoma cultures, a comparison was made with the effects of the dopamine (DA) agonists bromocriptine and/or quinagolide. In 5 NFAs, 2 prolactinomas and 1 insulinoma somatostatin receptor (subtype) expression was determined by ligand binding studies and by in situ hybridization to detect sst1, sst2, and sst3 messenger RNAs (mRNAs). Four NFA cultures did not secrete detectable amounts of alpha-subunit, FSH, and/or LH. In the other cultures, hormone and/or subunit release was inhibited by DA-agonists (10 nM) in 9 of 11, by SS (10 nM) in 7 of 11, and by octapeptide SS-analogs (10 nM) in 3 of 10 cultures. In three NFA cultures, hormone release was sensitive to SS but not to SS-analogs. In all cultures, except for one, DA-agonists were the most effective in inhibiting hormone release. In the prolactinoma cultures, PRL release was inhibited by DA-agonists (10 nM) in 7 of 7, by SS in 4 of 4, and by octapeptide SS-analogs in 3 of 7 cultures. A dissociation between the effects of SS and SS-analogs was found in 3 cases. In the cultures sensitive to both bromocriptine and SS-28, bromocriptine was the most potent compound in 2 out of 4 cultures. In the 2 other cultures, both compounds were equally effective. In 2 insulinoma cultures, insulin release was inhibited by SS, and by octapeptide SS-analogs in only one. The presence or absence of an inhibitory effect by octreotide was in all cases in parallel with the presence or absence of the inhibitory effect by BIM-23014 and RC-160. Autoradiographic studies using [125I-Tyr0]SS28 showed specific binding in 4 of 5 NFAs, 1 of 2 prolactinomas, and 1 of 1 insulinoma. Specific [125I-Tyr3]octreotide binding was found in 2 of 5 NFAs, in 1 of 2 prolactinomas, and in the insulinoma. Two NFAs showed binding of SS28, but not of the sst2.5 specific ligand octreotide. The tumors showed variable sst1 and/or sst3 mRNA expression, whereas no sst2 expression was found. In conclusion, a dissociation between the inhibitory effects of SS on the one hand and of the octapeptide SS-analogs octreotide, BIM-23014 and RC-160 on the other hand, is observed in a small subgroup of NFAs, prolactinomas, and insulinomas, suggesting that novel sst subtype specific SS-analogs might be of benefit in the treatment of selected patients with somatostatin receptor positive secreting tumors not resp

The multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somatostatin receptor type 5.

Objective: Currently, there is no effective medical treatment for patients with pituitary-dependent Cushing’s disease. A novel somatostatin (SS) analogue, named SOM230, with high binding affinity to SS receptor subtypes sst1, sst2, sst3 and sst5 was recently introduced. We compared the in vitro effects of the sst2-preferring SS analogue octreotide (OCT) and the multi-ligand SOM230 on ACTH release by human and mouse corticotroph tumour cells. Methods: By quantitative RT-PCR the sst subtype expression level was determined in human corticotroph adenomas. In vitro, the inhibitory effect of OCT and SOM230 on ACTH release by dispersed human corticotroph adenoma cells and mouse AtT20 corticotroph adenoma cells was determined. In addition, the influence of dexamethasone on the responsiveness to OCT and SOM230 was studied. Results: Corticotroph adenomas expressed predominantly sst5 mRNA (six out of six adenomas), whereas sst2 mRNA expression was detected at significantly lower levels. In a 72 h incubation with 10 nmol/l SOM230, ACTH release was inhibited in three out of five cultures (range –30 to –40%). Ten nmol/l OCT slightly inhibited ACTH release in only one of five cultures (– 28%). In AtT20 cells, expressing sst2, sst3 and sst5, SOM230 inhibited ACTH secretion with high potency (IC50 0.2 nmol/l). Dexamethasone (10 nmol/l) pre-treatment did not influence the sensitivity of the cells to the inhibitory effect of SOM230, suggesting that sst5 is relatively resistant to negative control by glucocorticoids. Conclusions: The selective expression of sst5 receptors in corticotroph adenomas and the preferential inhibition of ACTH release by human corticotroph adenoma cells by SOM230 in vitro, suggest that SOM230 may have potential in the treatment of patients with pituitary-dependent Cushing’s disease

The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro.

To determine the inhibitory profile of the novel somatostatin (SRIF) analog SOM230 with broad SRIF receptor binding, we compared the in vitro effects of SOM230, octreotide (OCT), and SRIF-14 on hormone release by cultures of different types of secreting pituitary adenomas. OCT (10 nM) significantly inhibited GH release in seven of nine GH-secreting pituitary adenoma cultures (range, -26 to -73%), SOM230 (10 nM) in eight of nine cultures (range, -22 to -68%), and SRIF-14 (10 nM) in six of six cultures (range, -30 to -75%). The sst analysis showed predominant but variable levels of somatostatin receptor (sst)(2) and sst(5) mRNA expression. In one culture completely resistant to OCT, SOM230 and SRIF-14 significantly inhibited GH release in a dose-dependent manner with an IC(50) value in the low nanomolar range. In the other cultures, SOM230 showed a lower potency of GH release inhibition (IC(50), 0.5 nM), compared with OCT (IC(50), 0.02 nM) and SRIF-14 (IC(50), 0.02 nM). A positive correlation was found between sst(2) but not sst(5) mRNA levels in the adenoma cells and the inhibitory potency of OCT on GH release in vivo and in vitro, and the effects of SOM230 and SRIF-14 in vitro. In three prolactinoma cultures, 10 nM OCT weakly inhibited prolactin (PRL) release in only one (-28%), whereas 10 nM SOM230 significantly inhibited PRL release in three of three cultures (-23, -51, and -64.0%). The inhibition of PRL release by SOM230 was related to the expression level of sst(5) but not sst(2) mRNA. Several conclusions were reached. First, SOM230 has a broad profile of inhibition of tumoral pituitary hormone release in the low nanomolar range, probably mediated via both sst(2) and sst(5) receptors. The higher number of responders of GH-secreting pituitary adenoma cultures to SOM230, compared with OCT, suggest that SOM230 has the potency to increase the number of acromegalic patients which can be biochemically controlled. Second, compared with OCT, SOM230 is more potent in inhibiting PRL release by mixed GH/PRL-secreting adenoma and prolactinoma cells

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland

The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D2 receptor. D2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland

Crucial role for somatostatin receptor subtype 2 in determining the uptake of [¹¹¹In-DTPA-D-Phe¹]octreotide in somatostatin receptor-positive organs

Human somatostatin (SS) receptor (sst)-positive tumors can be visualized by gamma camera scintigraphy after the injection of [111In- diethylenetriaminepentaacetic acid (DTPA)-D-Phe1] octreotide. Uptake of [111In-DTPA-D-Phe1]octreotide is dependent on sst-mediated internalization of the radioligand by the tumor cells. Human sst-positive tumors frequently express multiple sst subtypes. In vitro studies have demonstrated that the 5 sst subtypes (ssti1-5) differentially internalize sst-bound ligand. The present study was performed to evaluate the role of sst2 in vivo in determining the uptake of [ 111In-DTPA-D-Phe1]octreotide, as well as of the more "universal" ligand [111In-DTPA]SS-14, by sst-positive organs expressing multiple sst subtypes. Methods: Wild-type and sst2 knockout mice (n = 4 per treatment group) were injected intravenously with 1 MBq (0.1 μg) [111In-DTPA-D-Phe1]octreotide or [ 111In-DTPA]SS-14. After 24 h, the animals were sacrificed and radioactivity in the organs under investigation was determined. In addition, the sst subtype messenger RNA (mRNA) expression pattern in these organs was determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Results: RT-PCR analysis demonstrated the presence of all 5 sst subtype mRNAs in the adrenals and pituitary of wild-type mice but no sst2 in the knockout mice. The thymus expressed mRNA for sst2 and sst4 mRNA in wild-type mice, whereas no sst2 was detected in knockout mice. In wildtype mice, the in vivo uptake values (in percentage injected dose per gram of tissue) of [111In-DTPA-D-Phe1]octreotide for the pituitary, adrenals, pancreas, and thymus amounted to 1.2 ± 0.2, 0.26 ± 0.03, 0.18 ± 0.03, and 0.30 ± 0.05, respectively, in wild-type mice. Compared with wild-type mice, sst2 knockout mice had dramatically lower uptake values in these organs - lower by 97%, 83%, 96%, and 94%, respectively (P &lt; 0.01 vs. wild type). Comparable differences in the uptake of radioactivity between wild-type and knockout mice were found using [111In-DTPA]SS-14 as the radiotracer. Interestingly, in some organs expressing sst2 mRNA (liver, muscle, and peripheral blood mononuclear cells), no specific binding of [111In-DTPA-D-Phe1] octreotide or [111In-DTPA]SS-14 to sst in vivo was found, suggesting that the sst2 protein expression level was very low in these tissues. Conclusion: The uptake of [111In-DTPA-D-Phe1]octreotide and [111In-DTPA]SS-14 in sst-positive organs is determined predominantly by sst2.</p

Crucial role for somatostatin receptor subtype 2 in determining the uptake of [¹¹¹In-DTPA-D-Phe¹]octreotide in somatostatin receptor-positive organs

Human somatostatin (SS) receptor (sst)-positive tumors can be visualized by gamma camera scintigraphy after the injection of [111In- diethylenetriaminepentaacetic acid (DTPA)-D-Phe1] octreotide. Uptake of [111In-DTPA-D-Phe1]octreotide is dependent on sst-mediated internalization of the radioligand by the tumor cells. Human sst-positive tumors frequently express multiple sst subtypes. In vitro studies have demonstrated that the 5 sst subtypes (ssti1-5) differentially internalize sst-bound ligand. The present study was performed to evaluate the role of sst2 in vivo in determining the uptake of [ 111In-DTPA-D-Phe1]octreotide, as well as of the more "universal" ligand [111In-DTPA]SS-14, by sst-positive organs expressing multiple sst subtypes. Methods: Wild-type and sst2 knockout mice (n = 4 per treatment group) were injected intravenously with 1 MBq (0.1 μg) [111In-DTPA-D-Phe1]octreotide or [ 111In-DTPA]SS-14. After 24 h, the animals were sacrificed and radioactivity in the organs under investigation was determined. In addition, the sst subtype messenger RNA (mRNA) expression pattern in these organs was determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Results: RT-PCR analysis demonstrated the presence of all 5 sst subtype mRNAs in the adrenals and pituitary of wild-type mice but no sst2 in the knockout mice. The thymus expressed mRNA for sst2 and sst4 mRNA in wild-type mice, whereas no sst2 was detected in knockout mice. In wildtype mice, the in vivo uptake values (in percentage injected dose per gram of tissue) of [111In-DTPA-D-Phe1]octreotide for the pituitary, adrenals, pancreas, and thymus amounted to 1.2 ± 0.2, 0.26 ± 0.03, 0.18 ± 0.03, and 0.30 ± 0.05, respectively, in wild-type mice. Compared with wild-type mice, sst2 knockout mice had dramatically lower uptake values in these organs - lower by 97%, 83%, 96%, and 94%, respectively (P &lt; 0.01 vs. wild type). Comparable differences in the uptake of radioactivity between wild-type and knockout mice were found using [111In-DTPA]SS-14 as the radiotracer. Interestingly, in some organs expressing sst2 mRNA (liver, muscle, and peripheral blood mononuclear cells), no specific binding of [111In-DTPA-D-Phe1] octreotide or [111In-DTPA]SS-14 to sst in vivo was found, suggesting that the sst2 protein expression level was very low in these tissues. Conclusion: The uptake of [111In-DTPA-D-Phe1]octreotide and [111In-DTPA]SS-14 in sst-positive organs is determined predominantly by sst2.</p

The role of mTOR inhibitors in the inhibition of growth and cortisol secretion in human adrenocortical carcinoma cells

Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 coul