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

Long-term treatment with the dopamine agonist quinagolide of patients with clinically non-functioning pituitary adenoma

OBJECTIVE: This study was performed to evaluate the effect of prolonged treatment with the dopamine agonist quinagolide on serum gonadotropin and alpha-subunit concentrations and tumor volume in patients with clinically non-functioning pituitary adenomas (CNPA). DESIGN: Ten patients with CNPA were treated with quinagolide (0.3 mg daily). The median duration of treatment was 57 months (range 36-93 months). Blood samples for measurement of serum gonadotropin and alpha-subunit concentrations were drawn before treatment, after 5 days, and at each outpatient visit. Computerized tomography or magnetic resonance imaging of the pituitary region and Goldmann perimetry were done before and at regular intervals during treatment. RESULTS: A significant decrease of serum FSH, LH or alpha-subunit concentrations was found in nine patients. The levels remained low during the entire treatment period. In two out of three patients with pre-existing visual field defects a slight improvement was shown during the first months of treatment, but eventually deterioration occurred in all three patients. A fourth patient developed unilateral ophthalmoplegia dur

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

Relative potencies of the somatostatin analogs octreotide, BIM-23014, and RC-160 on the inhibition of hormone release by cultured human endocrine tumor cells and normal rat anterior pituitary cells

textabstractIn the present study we investigated the effects of the somatostatin (SS) analogs octreotide, RC-160, and BIM-23014 on GH release by cultured cells of human GH-secreting pituitary tumors, in normal rat anterior pituitary cells, and on gastrin release by cultured cells from a human gastrinoma. In all GH-secreting adenomas and in rat anterior pituitary cells, RC-160 was the most potent compound. RC-160 significantly inhibited GH-, PRL, and/or alpha-subunit release by human GH-secreting pituitary adenoma cells in concentrations as low as 10(-12)-10(-14) M, whereas at the same concentrations, octreotide and BIM-23014 did not inhibit or were significantly less effective in inhibiting GH release (P < 0.01, RC-160 vs. octreotide and BIM-23014). In rat anterior pituitary cell cultures, the IC50 values for inhibition of GH release were, in rank order of potency, 0.1, 5.3, 47, 48, and 99 pM for RC-160, SS-14, BIM-23014, octreotide, and SS-28, respectively. Maximal inhibitory effects by the three analogs were the same in the human GH adenoma cell cultures and the rat anterior pituitary cell cultures (-60%). On the basis of these data, RC-160 appears to be about 500 times more potent than octreotide and BIM-23014 in inhibiting GH release by rat anterior pituitary cells in vitro. Forskolin (100 microM) as well as pretreatment of the cells with pertussis toxin significantly diminished the inhibitory effects of the three SS analogs and those of SS-14 and SS-28 to the same extent. The latter data suggest that octreotide, RC-160, and BIM-23014 act mainly via a pertussis toxin-sensitive G-protein and an adenylyl cyclase-dependent mechanism. In the human gastrinoma culture, RC-160 inhibited gastrin release significantly more than octreotide at 10(-12)- and 10(-14)-M concentrations (P < 0.01). In conclusion, the SS analogs octreotide, RC-160, and BIM-23014 may have significant different potencies of inhibition of hormone release in vitro, with RC-160 being the most potent SS analog and octreotide and BIM-23014 having similar potencies. Depending on the pharmacokinetic properties of these three octapeptide SS analogs, these observations may have consequences for the medical therapy of patients with SS receptor-positive endocrine tumors

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

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side-chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4-, and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis.</p

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

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side-chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4-, and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis.</p