This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zatelli, M. C. Articles by degli Uberti, E. C. Search for Related Content PubMed PubMed Citation Articles by Zatelli, M. C. Articles by degli Uberti, E. C.

Evidence for Differential Effects of Selective Somatostatin Receptor Subtype Agonists on -Subunit and Chromogranin A Secretion and on Cell Viability in Human Nonfunctioning Pituitary Adenomas in Vitro

Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies (M.C.Z., D.P., A.B., M.R.A., A.M., E.C.d.U.), and Department of Experimental and Diagnostic Medicine, Section of Anatomic Pathology (L.C.), University of Ferrara, 44100 Ferrara, Italy; Division of Neurosurgery (R.P.), Hospital of Ferrara, 44100 Ferrara, Italy; Division of Neurosurgery (M.S.), Hospital of Padova, 35100 Padova, Italy; and Biomeasure Incorporated/IPSEN (J.E.T., M.D.C.), Milford, Massachusetts 01757-3650

Address all correspondence and requests for reprints to: Ettore C. degli Uberti, M.D., Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}unife.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Somatostatin (SRIF) analogs interacting with SRIF receptor (SSTR) subtypes SSTR2 and SSTR5 reduce hormone secretion of pituitary adenomas, but their antiproliferative effects are still controversial. We investigated the in vitro effects of SRIF and SSTR-selective agonists interacting with SSTR1 (BIM-23926), SSTR2 (BIM-23120), SSTR5 (BIM-23206), or both SSTR2 and SSTR5 (BIM-23244) on -subunit and chromogranin A secretion and on cell viability of 12 nonfunctioning pituitary adenomas (NFA) expressing SSTR1, SSTR2, and SSTR5, as assessed by RT-PCR. Treatment with SRIF or BIM-23206 did not modify -subunit and chromogranin A secretion, which was significantly inhibited by BIM-23926, BIM-23120, and BIM-23244. SRIF and BIM-23120 did not influence cell viability, which was significantly promoted by BIM-23206 and BIM-23244 and reduced by treatment with BIM-23926.

These results demonstrate that, in the selected NFA, the SSTR1-selective agonist inhibits secretory activity and cell viability, the SSTR2-selective agonist inhibits secretion but not cell viability, and the SSTR5-selective agonist does not influence secretion but promotes cell viability. These data can explain the lack of inhibitory effects of currently used SRIF analogs and suggest that drugs acting potently and preferentially on SSTR1 might be useful for medical treatment of NFA.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
SOMATOSTATIN (SRIF) EXERTS its biological actions, including inhibition of hormone secretion and cell proliferation, through activation of five transmembrane Gcoupled receptors [SRIF receptor (SSTR) subtypes 1–5] (1). SSTR subtypes are expressed in normal and neoplastic human pituitary cells, and there is evidence for an inhibitory role of SSTR2 and SSTR5 activation on hormone secretion in clinically functioning pituitary adenomas (2, 3). Furthermore, an inhibitory effect of a compound interacting with SSTR1 has recently been demonstrated on secretion and proliferation of GH- and prolactin (PRL)-secreting pituitary adenoma cells in vitro (4). These data provide the basis for the successful therapeutic application of SRIF analogs, such as octreotide and lanreotide, which mainly interact with SSTR2 and SSTR5, and of the new SRIF analog, SOM-230, with a unique binding pattern (SSTR1, 2, 3, and 5) (5). The therapeutic efficacy of SRIF analogs is still controversial in the medical approach to patients with clinically nonfunctioning pituitary adenomas (NFA). SSTR expression in NFA has previously been described both in vivo (6, 7, 8, 9) and in vitro (6, 10), without evidence for a correlation with tumor volume (6, 9) or for the therapeutic efficacy of SRIF analogs (7, 8). However, the effects of SSTR-selective agonists on NFA have not yet been studied. Therefore, we investigated the SSTR expression by RT-PCR in a group of 71 NFA, and selected 12 tumors expressing the SSTR1, SSTR2, and SSTR5 subtypes. We then tested, in primary cultures derived from these tumors, the effects of SRIF, SSTR1-selective (BIM-23926), SSTR2-selective (BIM-23120), and SSTR5-selective (BIM-23206) agonists, and of a SSTR2/SSTR5-biselective agonist (BIM-23244) on -subunit and chromogranin A (CgA) secretion and on cell viability in vitro.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human pituitary adenomas

Twelve clinically NFA (mean maximal diameter, 2.07 ± 0.98 cm; mean volume, 4.2 ± 1.7 cm³) from 12 patients aged 55.4 ± 3 yr (median, 54 yr) were selected for this study from 71 samples derived from patients operated on for NFA on the basis of verification of -subunit, CgA, SSTR1, SSTR2, and SSTR5 mRNA expression by RT-PCR analysis. Table 1 shows patient characteristics and preoperative hormonal values. None of the patients had previously been treated with SRIF analogs or with dopaminergic drugs. All patients underwent transsphenoidal surgery, and immunohistochemical examination for anterior pituitary hormones was performed on all specimens. Of the 12 tumors, one was positive for LH, FSH, and PRL; one was positive for LH and FSH; and two were positive only for FSH. No immunostaining for GH, TSH, and ACTH was detectable in these tumors, and all anterior pituitary hormones were undetectable in the other eight tumors. Therefore, these 12 NFA were histologically diagnosed as gonadotroph cell adenoma or null cell adenoma.

View this table: [in this window] [in a new window]   TABLE 1. Patient characteristics, preoperative hormonal values, and in vitro -subunit and CgA secretion

Tissue samples were collected in accordance with the guidelines of the local committee on human research. A fragment was immediately frozen in liquid nitrogen under ribonuclease-free conditions at the time of surgery and stored at –80 C until RNA isolation was performed. A portion of the fresh tissue was immediately processed for primary culture experiments as previously described (4). Cells were resuspended in high glucose DMEM with 10% fetal bovine serum and antibiotics (EuroClone Ltd, West Yorkshire, UK), seeded in 96-well culture plates (2 x 10⁴ cells/well; 80 wells/tumor), and incubated at 37 C in a humidified atmosphere of 5% CO₂ and 95% air. Fibroblast contamination was excluded by treatment with cis-4-hydroxy-L-proline and morphological examination of the cultured cells (11). After 24 h, medium was changed to serum-free high-glucose DMEM containing 0.2% BSA, 120 nM transferrin, 5 U/liter insulin, 2 mM glutamine, and antibiotics. Cells were then treated with the indicated compounds for evaluation of hormone secretion in conditioned medium that was collected and stored at –20 C for later hormone measurement. Medium was then replaced with the indicated treatments for cell viability experiments.

Isolation of RNA and RT-PCR

To demonstrate the pituitary origin of the samples, RT-PCR analysis for -subunit expression was performed on each specimen. Further expression analysis for CgA, SSTR1, SSTR2, and SSTR5 was performed only in -subunit-expressing tissues, as previously described (4, 12). Briefly, frozen tissues were disrupted and total RNA from the pulverized tumors was extracted with TRIzol reagent (Invitrogen, Milano, Italy), according to the manufacturer’s protocol. To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease (Promega, Milano, Italy). Using a first-strand cDNA synthesis kit (SuperScript Preamplification System for First Strand cDNA Synthesis; Invitrogen), 1 µg total RNA was reverse transcribed with random hexamers according to the manufacturer’s protocol. Reverse transcription (RT) reactions were performed by using the GeneAmp 9700 PCR System (Applera, Monza, Italy) as previously described (4, 13). PCR conditions and oligonucleotide primers for amplification are listed in Table 2. Glyceraldehyde-3-phosphate dehydrogenase amplification was performed as control for RT reaction. PCR products were run on a 2% agarose gel, visualized by ethidium bromide staining, and analyzed with the Fluor-S Multi Imager (Bio-Rad, Milano, Italy). To confirm the correct identification of RT-PCR products, their specificity was verified, after gel purification by Quiaex II (Qiagen, Valencia, CA), by restriction enzyme digestion and direct sequencing (data not shown).

View this table: [in this window] [in a new window]   TABLE 2. Primers and PCR conditions for -subunit and SSTR amplification

To perform QPCR for human SSTR1 mRNA, we used primers and probe for SSTR1 and followed the QPCR procedures described previously (4). All the QPCR reactions were performed, recorded, and analyzed using the ABI 7700 Prism Sequence Detection System (PE Applied Biosystems, Foster City, CA). All samples were carried out in triplicate (100 ng of reverse-transcribed total RNA per well) and repeated at least twice. For each sample, one point of 18S rRNA was loaded to evaluate the retrotranscription efficiency in the same plate and PCR conditions. No template control and RT controls were run in each experiment.

Immunohistochemistry

Hematoxylin and eosin-stained sections from formalin-fixed, paraffin wax-embedded tissues were reviewed to evaluate the morphological features of the disease. Immunohistochemical analysis was performed on formalin-fixed, paraffin wax-embedded sections (4-µm thick) by using standard techniques and the antibodies anti--subunit (human chorionic gonadotropin mouse monoclonal antibody; Novocastra Laboratories Ltd, Newcastle, UK) and anti-CgA (anti-CgA mouse monoclonal antibody; Dako S.p.A., Milano, Italy) at 1:200 dilution in PBS. Visualization was performed with avidin-biotin-peroxidase complex. The cell nuclei were counterstained with hematoxylin. All reagents were from Vector Laboratories (Burlingame, CA).

SRIF and SSTR-selective agonists

SRIF (Stilamin 250) was purchased from Serono Pharma (Roma, Italy). The SRIF analogs used in this study and their respective affinities to the different SSTRs are listed in Table 3. Each compound, provided by Biomeasure Incorporated (Milford, MA), was resuspended in 0.01 N acetic acid containing 0.1% BSA, as previously described (13). Specificity and selectivity of the analogs were determined by radioligand binding assay on CHO-K1 cells stably transfected with each of the SSTR subtypes, as previously described (2). The biological activity of each agonist was evaluated as previously described (13, 14).

-Subunit secretion

The effects of SRIF and its analogs on -subunit secretion were analyzed by measuring human -subunit immunoreactivity in the culture medium from primary cultured pituitary cells incubated for 8 h with or without 10^–8 M SRIF or each SSTR agonist (conditioned media) with a RIA kit (Free Subunit RIA kit; DRG Instruments GmbH, Marburg, Germany) as previously described (12, 15). The detection limit was 0.05 U/liter, with intra- and interassay coefficients of variation of 7.1 and 11.1%, respectively. Hormone assays were performed in duplicate after appropriate sample dilutions of conditioned medium from treated cells. Results were obtained by determining the mean value among eight replicates.

CgA secretion

The effects of SRIF and its analogs on CgA secretion were analyzed by measuring human CgA immunoreactivity in the culture medium from primary cultured pituitary cells incubated for 8 h with or without 10^–8 M SRIF or each SSTR agonist, as previously described, with an ELISA kit (Dako S.p.A.) (12, 15). The detection limit was 2.0 U/liter, with intra- and interassay coefficients of variation of 5.8 and 8.6%, respectively. Hormone assays were performed in duplicate after appropriate sample dilutions of conditioned medium from treated cells. Results were obtained by determining the mean value among eight replicates.

Cell viability

The effect of SRIF and of each selective agonist on cell viability of pituitary adenomas in vitro was assessed by the CellTiter 96 Aq_ueous Non-Radioactive Cell Proliferation Assay (Promega), as previously described (14), after incubation for 48 h in medium with or without 10^–8 M SRIF or each SRIF analog. MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromid) is a colorimetric method for determining the number of viable cells that is widely accepted and used for cell viability assessment (4) because the recorded absorbance strongly correlates to the cell number, independently of whether or not they are proliferating. Treatments were renewed after the first 24 h of incubation. At the end of the incubation period, the plates were incubated for an additional 4 h at 37 C in a humidified 5% CO₂ atmosphere with the staining solution. The absorbance at 490 nm was then recorded using an ELISA plate reader (EASIA Reader; Medgenix, Springfield, MO). Results (absorbance at 490 nm) were obtained by determining the mean value of at least six experiments in eight replicates.

Statistical analysis

Results of hormone assays and cell viability experiments are expressed as the mean ± SE. A preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), the Student’s paired or unpaired t test was used to evaluate individual differences between means. To measure the strength of association between pairs of variables without specifying dependencies, Spearman order correlations were performed. P < 0.05 was considered significant in all tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SSTR mRNA expression

Seventy-one samples derived from patients operated on for NFA underwent RT-PCR analysis for -subunit, CgA, SSTR1, SSTR2, and SSTR5 mRNA expression, and 12 tumors (16.9%) were selected on the basis of the expression of all five mRNAs (Fig. 1). No correlation was found between SSTR expression pattern, patients’ clinical characteristics, and preoperative hormonal values. The absolute levels of SSTR1 mRNA were also investigated in the selected samples, showing a mean ± SE level of SSTR1 mRNA of 32.9 ± 8.9 x 10⁴ molecules/µg of reverse-transcribed total RNA, ranging from 0.4–68.3 x 10⁴ molecules/µg of reverse-transcribed total RNA. SSTR1 mRNA levels did not correlate to patients’ preoperative -subunit plasma levels, immunohistochemical findings, age, or sex.

View larger version (121K): [in this window] [in a new window]   FIG. 1. -Subunit, CgA, SSTR1, SSTR2, SSTR5, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in NFA. Extracted RNA (1 µg/reaction) was treated with deoxyribonuclease and subjected to RT. Aliquots from the generated cDNA were subjected to subsequent PCR amplification of -subunit, CgA, SSTR1, SSTR2, SSTR5, and GAPDH using the primers indicated in Table 2. PCR products were resolved on a 2% agarose gel. The expected PCR products are indicated with arrows (lane M, 100-bp PCR marker; lane +, positive control).

Immunostaining for -subunit was detected in all examined NFA throughout the cytoplasm, being sparsely distributed in cell clusters among the sections, with variable cell number and staining intensity. Moreover, all cases showed a strong and diffuse positive cytoplasmic staining for CgA (Fig. 2).

View larger version (124K): [in this window] [in a new window]   FIG. 2. Immunohistochemistry. Immunostaining for -subunit (A) and CgA (B) in a NFA (magnification, x400).

Effects of SRIF and SSTR-selective agonists on -subunit secretion by NFA

To determine the effects of SRIF and of the SSTR-selective agonists on -subunit secretion by dispersed NFA cells, we assessed -subunit concentrations in conditioned medium from the 12 selected NFA primary cultures treated with SRIF or each SRIF analog. The basal -subunit level in the culture medium of 12 x 10⁴ cells cultured for 12 h was 2.5 ± 0.5 U/liter. No detectable concentrations of PRL, GH, TSH, and ACTH were found in the culture medium. Moreover, no -subunit immunoreactivity was detected in fresh culture medium.

As indicated in Fig. 3, SRIF and the selective SSTR5 agonist, BIM-23206, did not significantly modify -subunit secretion by NFA. On the contrary, treatment with the SSTR1-selective agonist, BIM-23926, significantly inhibited -subunit secretion by NFA (38.1%, P < 0.05), as did the SSTR2-selective agonist, BIM-23120 (37.8%, P < 0.05), and the SSTR2/SSTR5-biselective agonist, BIM-23244 (29.4%, P < 0.05). No statistically significant difference was observed between the extent of -subunit secretion inhibition induced by BIM-23926, BIM-23120, or BIM-23244.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Effects of SRIF and SSTR-selective agonists on -subunit secretion by NFA. Pituitary cells were incubated in 96-well plates for 8 h in culture medium supplemented with either SRIF or each SRIF analog at 10–8 M, and control cells were treated with vehicle solution. -Subunit secretion by each primary culture was then measured by RIA. Data from NFA primary cultures were evaluated independently with eight replicates each and were expressed as the mean ± SE percent -subunit secretion inhibition vs. untreated control cells. *, P < 0.05 vs. untreated control cells.

Effects of SRIF and SSTR-selective agonists on CgA secretion by NFA

To determine the effects of SRIF and of the SSTR-selective agonists on CgA secretion by dispersed NFA cells, we assessed CgA concentrations in conditioned medium from the 12 selected NFA primary cultures treated with SRIF or each SRIF analog. The basal CgA level in the culture medium of 12 x 10⁴ cells cultured for 12 h was 4.5 ± 1.4 U/liter. No CgA immunoreactivity was detected in fresh culture medium.

As indicated in Fig. 4, neither SRIF nor the selective SSTR5 agonist, BIM-23206, significantly modified CgA secretion by NFA. On the contrary, treatment with the SSTR1-selective agonist, BIM-23926, significantly inhibited CgA secretion by NFA (30.8%, P < 0.05), as did the SSTR2-selective agonist, BIM-23120 (22.3%, P < 0.05), and the SSTR2/SSTR5-biselective agonist, BIM-23244 (24.3%, P < 0.05). No statistically significant difference was observed between the extent of CgA secretion inhibition induced by BIM-23926, BIM-23120, or BIM-23244.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Effects of SRIF and SSTR-selective agonists on CgA secretion by NFA. Pituitary cells were incubated in 96-well plates for 8 h in culture medium supplemented with either SRIF or each SRIF analog at 10–8 M, and control cells were treated with vehicle solution. CgA secretion by each primary culture was then measured by ELISA. Data from NFA primary cultures were evaluated independently with eight replicates each and were expressed as the mean ± SE percent CgA secretion inhibition vs. untreated control cells. *, P < 0.05 vs. untreated control cells.

Effects of SRIF and SSTR-selective agonists on NFA cell viability

To determine the effects of SRIF and of the SSTR-selective agonists on cell viability of dispersed NFA cells, we assessed cell number after treatment with SRIF or each SRIF analog. As indicated in Fig. 5, SRIF and BIM-23120 did not significantly influence cell viability of NFA in primary culture, which was significantly (P < 0.05) promoted by treatment with the SSTR5-selective agonist, BIM-23206, or with the biselective SSTR2/SSTR5 agonist, BIM-23244 (25% and 13%, respectively; P < 0.05). No statistical difference was observed between the effect of BIM-23206 and BIM-23244. On the contrary, the SSTR1-selective agonist, BIM-23926, significantly reduced NFA cell viability in vitro (16.5%, P < 0.05).

View larger version (13K): [in this window] [in a new window]   FIG. 5. Effects of SRIF and SSTR-selective agonists on cell viability of NFA. Pituitary cells were incubated in 96-well plates for 48 h in culture medium supplemented with either SRIF or each SRIF analog at 10–8 M, and control cells were treated with vehicle solution. Cell viability of each primary culture was measured as absorbance at 490 nm. Data from NFA primary cultures were evaluated independently with eight replicates each and were expressed as the mean ± SE percent cell viability inhibition vs. untreated control cells. *, P < 0.05 vs. untreated control cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study is the first to provide evidence that SSTR-selective agonists may differentially influence secretory activity and cell viability of human NFA in vitro. Previous studies have shown that human NFA express multiple SSTR (6, 10, 16, 17, 18), but application of the current clinically used SRIF analogs has provided inconclusive results in terms of reduction in tumor mass (19, 20, 21). On the other hand, there are experimental data indicating that SRIF and its analogs, mainly interacting with SSTR2 and SSTR5, can indeed inhibit basal (22) or phorbol myristate acetate-induced (10) cell proliferation of NFA primary cultures in vitro. The discrepancies between in vivo and in vitro results may be due to the different SSTR expression pattern displayed by human NFA, which has been documented as highly variable (10). Our findings show that SSTR are differently involved in the modulation of secretory activity and cell viability of NFA in vitro.

Production of gonadotropin subunits is found in the majority of NFA. The -subunit is the most commonly produced monomer subunit (23), and immunoreactivity for this protein could be demonstrated also in our samples. However, -subunit plasma levels are found to be elevated only in 20% of cases (24). Previous studies have shown evidence for -subunit secretion by NFA in vitro but failed to demonstrate an inhibitory effect of SRIF on this parameter (25). Moreover, it has been shown that administration of octreotide reduces -subunit plasma levels in two of six patients with -subunit-secreting pituitary adenomas (26) and in six of 10 patients with NFA (27). Lanreotide has also been shown to induce a significant reduction in -subunit secretion by a mixed LH-, PRL-, and -subunit-secreting pituitary adenoma, both in vivo and in vitro (15). However, no evidence for tumor reduction was shown in any case. CgA has been suggested as a valuable marker of NFA because immunoreactivity for this protein could be demonstrated in NFA cells (28), where it mainly colocalizes in secretory granules (29), which is in agreement with the data shown here. Moreover, immunoscintigraphy with an anti-CgA monoclonal antibody was capable of detecting the majority of NFA in a series of 11 patients, with concordant immunohistochemical findings (30). We show that CgA secretion, as well as -subunit, can be significantly reduced by treatment of NFA primary cultures with compounds interacting with SSTR1 or SSTR2. These data confirm the previously described antisecretory effects of SSTR1- and SSTR2-selective agonists on pituitary adenoma cells in vitro (2, 4). On the contrary, the SSTR5-selective agonist did not influence -subunit or CgA secretion in any of the examined NFA primary cultures. It has previously been shown that SSTR5 mediates the inhibition of PRL secretion induced by SRIF and its analogs in pure PRL- and mixed GH/PRL-secreting adenomas, as well as the suppression of GH secretion in somatotroph cells, together with SSTR2 (2, 31). Moreover, SSTR2- and SSTR5-selective agonists have additive effects (3), and a biselective SSTR2/SSTR5 agonist has enhanced efficacy in suppressing GH secretion from octreotide-resistant human GH-secreting adenomas (32). Recently, a functional association between SSTR2 and SSTR5 in inhibiting GH secretion has been suggested (33). In our study, the dual SSTR2/SSTR5 agonist, BIM-23244, inhibited NFA secretory activity to the same extent as the SSTR2-selective agonist, BIM-23120, in accordance with the lack of any antisecretory effect of the SSTR5-selective agonist, BIM-23206. Therefore, BIM-23244 is more active than SRIF most likely because SRIF interacts with all of the SSTR subtypes, whereas, in this assay, BIM-23244 would act as a selective SSTR2. It has been previously demonstrated that combined activation of SSTR subtypes could produce either synergistic (2) or antagonistic results (13). In many assays, it is typical for SRIF to be less effective than an agonist analog that is selective for a specific subtype that is involved in the response or to produce a bell-shaped dose-response curve in which higher concentrations become less efficacious. The most reasonable explanation is that SRIF is activating multiple SSTR subtypes, some of which are antagonistic to the response. Moreover, our study shows that treatment with SRIF and the SSTR2-selective agonist did not influence cell viability, which was promoted by the SSTR5-selective agonist and the SSTR2/SSTR5-biselective agonist and reduced by treatment with the SSTR1-selective agonist. These data are in agreement with previous reports (10) showing a lack of inhibitory effect on basal cell viability of NFA for SRIF and its analogs, which mainly interact with SSTR2 and/or SSTR5. On the other hand, our results show that treatment with a SSTR5-selective agonist significantly promoted NFA cell viability. This effect might account for the lack of NFA volume reduction observed in patients treated with octreotide or lanreotide, which mainly interact with SSTR2 and SSTR5 (7, 19, 20, 21). Moreover, our findings could explain the increase in the tumor volume observed in some cases during such therapy (19). These results suggest that indication for medical therapy with octreotide or lanreotide in NFA patients should be critically discussed until clinical evidence for beneficial effects has clearly been demonstrated.

The SSTR5-selective agonist (BIM-23206) has previously been demonstrated to exert a positive effect on cell proliferation in a medullary thyroid carcinoma cell line (13), but the same compound has been shown to inhibit cell proliferation in GH-secreting pituitary adenomas in primary culture (11). Therefore, it is likely that SSTR agonists differently affect hormone secretion and cell proliferation depending not only on the specific activated receptor subtype but also on the specific tissue. The main finding of our work is the demonstration that selective SSTR1 agonist is effective in reducing cell viability of NFA. In our study, the inhibitory effects on secretory activity and cell viability induced by BIM-23926 treatment of NFA cultures did not correlate with SSTR1 mRNA levels, suggesting that even low levels of SSTR1 expression may warrant an inhibitory activity of compounds binding to this receptor. Accordingly, SSTR1-selective activation results in decreased secretory activity and reduced cell viability both in pituitary adenomas (4) and in medullary thyroid carcinoma (14), suggesting that SSTR1-selective agonists may be effective on a wide range of endocrine cells. Indeed, almost all human tumors express SSTR1 mRNA (34), suggesting that compounds targeting SSTR1 might represent a good tool for the control of neoplastic growth. Moreover, recent immunohistochemical studies have shown that SSTR1 is one of the most expressed SSTR subtypes in NFA (18). Therefore, stable SRIF analogs binding with high affinity to SSTR1, whether selectively or together with other SSTR subtypes, may indeed open a new frontier in the treatment of NFA.

In conclusion, our results demonstrate that, in vitro, a SSTR1-selective agonist inhibits both secretory activity and cell viability in a group of NFA. Moreover, a SSTR2-selective agonist reduces secretion but fails to affect cell viability, whereas a SSTR5-selective agonist does not influence secretion but seems to stimulate cell viability. These data suggest that SSTR1 agonists could be considered potential pharmacological tools for the treatment of NFA. Further studies on a greater number of pituitary adenomas are needed to clarify the potential applications of SSTR1-selective agonists on NFA medical therapy.

	Footnotes

 
This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (60%, 2002; MIUR 2002067251-003), Fondazione Cassa di Risparmio di Ferrara, Associazione Italiana per la Ricerca sul Cancro, IPSEN Italia S.p.A., and the Associazione Ferrarese dell’Ipertensione Arteriosa.

Abbreviations: CgA, Chromogranin A; NFA, nonfunctioning pituitary adenomas; PRL, prolactin; QPCR, quantitative PCR; RT, reverse transcription; SRIF, somatostatin; SSTR, somatostatin receptor.

Received November 11, 2003.

Accepted June 29, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Patel YC 1999 Somatostatin and its receptor family. Front Neuroendocrinol 20:157–198[CrossRef][Medline]
2. Shimon I, Taylor JE, Dong JZ, Bitonte RA, Kim S, Morgan B, Coy DH, Culler MD, Melmed S 1997 Somatostatin receptor subtype specificity in human fetal pituitary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid stimulating hormone, and prolactin regulation. J Clin Invest 99: 789–798
3. Jaquet P, Saveanu A, Gunz G, Fina F, Zamora AJ, Grino M, Culler MD, Moreau JP, Enjalbert A, Ouafik LH 2000 Human somatostatin receptor subtypes in acromegaly: distinct patterns of messenger ribonucleic acid expression and hormone suppression identify different tumoral phenotypes. J Clin Endocrinol Metab 85:781–792[Abstract/Free Full Text]
4. Zatelli MC, Piccin D, Tagliati F, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Culler MD, degli Uberti EC 2003 Somatostatin receptor subtype 1 selective activation in human growth hormone- and prolactin-secreting pituitary adenomas: effects on cell viability, growth hormone and prolactin secretion. J Clin Endocrinol Metab 88:2797–2802[Abstract/Free Full Text]
5. Bruns C, Briner U, Lewis G, Meno-Tetang G, Weckbecker G 2002 SOM230: a new somatostatin peptidomimetic with broad SRIF receptor binding and a unique inhibitory profile. Eur J Endocrinol 146:707–716[Abstract]
6. Nielsen S, Mellemkjaer S, Rasmussen LM, Ledet T, Olsen N, Bojsen-Moller M, Astrup J, Weeke J, Jorgensen JO 2001 Expression of somatostatin receptors on human pituitary adenomas in vivo and ex vivo. J Endocrinol Invest 24:430–437[Medline]
7. Oppizzi G, Cozzi R, Dallabonzana D, Orlandi P, Benini Z, Petroncini M, Attanasio R, Milella M, Banfi G, Possa M 1998 Scintigraphic imaging of pituitary adenomas: an in vivo evaluation of somatostatin receptors. J Endocrinol Invest 21:512–519[Medline]
8. Broson-Chazot F, Houzard C, Ajzenberg C, Nocaudie M, Duet M, Mundler O, Marchandise X, Epelbaum J, Gomez De Alzaga M, Schafer J, Meyerhof W, Sassolas G, Warnet A 1997 Somatostatin receptor imaging in somatotroph and non-functioning pituitary adenomas: correlation with hormonal and visual responses to octreotide. Clin Endocrinol (Oxf) 47:589–598[CrossRef][Medline]
9. Plockinger U, Bader M, Hopfenmuller W, Saeger W, Quabbe HJ 1997 Results of somatostatin receptor scintigraphy do not predict pituitary tumor volume- and hormone-response to octreotide therapy and do not correlate with tumor histology. Eur J Endocrinol 136:369–376[Abstract]
10. Florio T, Thellung S, Arena S, Corsaro A, Spaziante R, Gussoni G, Acuto C, Giusti M, Giordano G, Schettini G 1999 Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro. Eur J Endocrinol 141:396–408[Abstract]
11. Danila D, Haidar JN, Zhang X, Katznelson L, Culler MD, Klibanski A 2001 Somatostatin receptor-specific analogs: effects on cell proliferation and growth hormone secretion in human somatotroph tumors. J Clin Endocrinol Metab 86:2976–2981[Abstract/Free Full Text]
12. Zatelli MC, Piccin D, Bondanelli M, Tagliati F, De Carlo E, Culler MD, degli Uberti EC 2003 An in vivo OctreoScan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro. Horm Metab Res 35:349–354[CrossRef][Medline]
13. Zatelli MC, Tagliati F, Taylor JE, Rossi R, Culler MD, degli Uberti EC 2001 Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line TT. J Clin Endocrinol Metab 86:2161–2169[Abstract/Free Full Text]
14. Zatelli MC, Tagliati F, Piccin D, Taylor JE, Culler MD, Bondanelli M, degli Uberti EC 2002 Somatostatin receptor subtype 1 selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 297:821–827
15. Saveanu A, Morange-Ramos I, Gunz G, Dufour H, Enjalbert A, Jaquet P 2001 A luteinizing hormone, -subunit- and prolactin-secreting pituitary adenoma responsive to somatostatin analogs: in vivo and in vitro studies. Eur J Endocrinol 145:35–41[Abstract]
16. Miller GM, Alexander JM, Bikkal HA, Katznelson L, Zervas NT, Klibanski A 1995 Somatostatin receptor subtype gene expression in pituitary adenomas. J Clin Endocrinol Metab 80:1386–1392[Abstract]
17. Panetta R, Patel YC 1995 Expression of mRNA for all five human somatostatin receptors (hSSTR1–5) in pituitary tumors. Life Sci 56:333–342[CrossRef][Medline]
18. Pawlikowski M, Pisarek H, Kunert-Radek J, Radek A 2003 Immunohistochemical detection of somatostatin receptor subtypes in "clinically nonfunctioning" pituitary adenomas. Endocr Pathol 14:231–238[CrossRef][Medline]
19. Andersen M, Bjerre P, Schroder HD, Edal A, Hoilund-Carlsen PF, Pedersen PH, Hagen C 2001 In vivo secretory potential and the effect of combination therapy with octreotide and cabergoline in patients with clinically nonfunctioning pituitary adenomas. Clin Endocrinol (Oxf) 54:23–30[CrossRef][Medline]
20. Colao A, Di Sarno A, Marzullo P, Di Somma C, Cerbone G, Landi ML, Faggiano A, Merola B, Lombardi G 2000 New medical approaches in pituitary adenomas. Horm Res 53(Suppl 3):76–87
21. Gasperi M, Petrini L, Pilosu R, Nardi M, Marcello A, Mastio F, Bartalena L, Martino E 1993 Octreotide treatment does not affect the size of most non-functioning pituitary adenomas. J Endocrinol Invest 16:541–543[Medline]
22. Renner U, Mojto J, Lange M, Muller OA, von Werder K, Stalla GK 1994 Effect of bromocriptine and SMS 201–995 on growth of human somatotrophic and non-functioning pituitary adenoma cells in vitro. Eur J Endocrinol 130:80–91[Abstract]
23. Shomali M, Katznelson L 2002 Medical therapy of gonadotropin-producing and non-functioning pituitary adenomas. Pituitary 5:89–98[CrossRef][Medline]
24. Demura R, Jibiki K, Kubo O, Odagiri E, Demura H, Kitamura K, Shizume K 1986 The significance of -subunit as a tumor marker for gonadotropin-producing pituitary adenomas. J Clin Endocrinol Metab 63:564–569[Abstract]
25. Klibanski A, Alexander JM, Bikkal HA, Hsu DW, Swearingen B, Zervas NT 1991 Somatostatin regulation of glycoprotein hormone and free subunit secretion in clinically nonfunctioning and somatotroph adenomas in vitro. J Clin Endocrinol Metab 73:1248–1255[Abstract]
26. Katznelson L, Oppenheimer DS, Coughlin JF, Kliman B, Schoenfeld DA, Klibanski A 1992 Chronic somatostatin analog administration in patients with -subunit-secreting pituitary tumors. J Clin Endocrinol Metab 75:1318–1325[Abstract]
27. Merola B, Colao A, Ferone D, Selleri A, Di Sarno A, Marzullo P, Biondi B, Spaziante R, Rossi E, Lombardi G 1993 Effects of a chronic treatment with octreotide in patients with functionless pituitary adenomas. Horm Res 40:149–155[Medline]
28. Heaney AP, Curry WJ, Pogue KM, Armstrong VL, Mirakhur M, Sheridan B, Johnston CF, Buchanan KD, Atkinson AB 2000 Immunohistochemical evaluation of the post-translational processing of chromogranin A in human pituitary adenomas. Pituitary 3:67–75[CrossRef][Medline]
29. Rosa P, Bassetti M, Weiss U, Huttner WB 1992 Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas. J Histochem Cytochem 40:523–533[Abstract]
30. Colombo P, Siccardi AG, Paganelli G, Magnani P, Songini C, Buffa R, Faglia G, Fazio F 1996 Three-step immunoscintigraphy with anti-chromogranin A monoclonal antibody in tumours of the pituitary region. Eur J Endocrinol 135:216–221[Abstract]
31. Shimon I, Yan X, Taylor JE, Weiss MH, Culler MD, Melmed S 1997 Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors. J Clin Invest 100:2386–2392[Medline]
32. Saveanu A, Gunz G, Doufour H, Caron P, Fina F, Ouafik L, Culler MD, Moreau JP, Enjalbert A, Jaquet P 2001 BIM-23244, a somatostatin receptor subtype 2- and 5-selective analog with enhanced efficacy in suppressing growth hormone (GH) from octreotide-resistant human GH-secreting adenomas. J Clin Endocrinol Metab 86:140–145[Abstract/Free Full Text]
33. Ren S-G, Taylor JT, Dong J, Yu R, Culler MD, Melmed S 2003 Functional association of somatostatin receptor subtypes 2 and 5 in inhibiting human growth hormone secretion. J Clin Endocrinol Metab 88:4239–4245[Abstract/Free Full Text]
34. Patel YC 1997 Molecular pharmacology of somatostatin receptor subtypes. J Endocrinol Invest 20:348–367[Medline]

This article has been cited by other articles:

				T. Florio, F. Barbieri, R. Spaziante, G. Zona, L. J Hofland, P. M van Koetsveld, R. A Feelders, G. K Stalla, M. Theodoropoulou, M. D Culler, et al. Efficacy of a dopamine-somatostatin chimeric molecule, BIM-23A760, in the control of cell growth from primary cultures of human non-functioning pituitary adenomas: a multi-center study Endocr. Relat. Cancer, June 1, 2008; 15(2): 583 - 596. [Abstract] [Full Text] [PDF]

				A. Fusco, G. Gunz, P. Jaquet, H. Dufour, A. L. Germanetti, M. D Culler, A. Barlier, and A. Saveanu Somatostatinergic ligands in dopamine-sensitive and -resistant prolactinomas Eur. J. Endocrinol., May 1, 2008; 158(5): 595 - 603. [Abstract] [Full Text] [PDF]

				L. A. Nolan, H. A. Schmid, and A. Levy Octreotide and the Novel Multireceptor Ligand Somatostatin Receptor Agonist Pasireotide (SOM230) Block the Adrenalectomy-Induced Increase in Mitotic Activity in Male Rat Anterior Pituitary Endocrinology, June 1, 2007; 148(6): 2821 - 2827. [Abstract] [Full Text] [PDF]

				E. Resmini, P. Dadati, J.-L. Ravetti, G. Zona, R. Spaziante, A. Saveanu, P. Jaquet, M. D. Culler, F. Bianchi, A. Rebora, et al. Rapid Pituitary Tumor Shrinkage with Dissociation between Antiproliferative and Antisecretory Effects of a Long-Acting Octreotide in an Acromegalic Patient J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1592 - 1599. [Abstract] [Full Text] [PDF]

				D. Ferone, A. Saveanu, M. D Culler, M. Arvigo, A. Rebora, F. Gatto, F. Minuto, and P. Jaquet Novel chimeric somatostatin analogs: facts and perspectives Eur. J. Endocrinol., April 1, 2007; 156(suppl_1): S23 - S28. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, M. R. Ambrosio, M. Bondanelli, and E. C d. Uberti Control of pituitary adenoma cell proliferation by somatostatin analogs, dopamine agonists and novel chimeric compounds Eur. J. Endocrinol., April 1, 2007; 156(suppl_1): S29 - S35. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, D. Piccin, C. Vignali, F. Tagliati, M. R. Ambrosio, M. Bondanelli, V. Cimino, A. Bianchi, H. A Schmid, M. Scanarini, et al. Pasireotide, a multiple somatostatin receptor subtypes ligand, reduces cell viability in non-functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion Endocr. Relat. Cancer, March 1, 2007; 14(1): 91 - 102. [Abstract] [Full Text] [PDF]

				K. K Y Ho Endocrinology: the next 60 years. J. Endocrinol., July 1, 2006; 190(1): 3 - 6. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, C. Vignali, A. Margutti, M. Bondanelli, G. C. Pansini, M. R. Pelizzo, et al. Selective Activation of Somatostatin Receptor Subtypes Differentially Modulates Secretion and Viability in Human Medullary Thyroid Carcinoma Primary Cultures: Potential Clinical Perspectives J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2218 - 2224. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, and E. C. degli Uberti Src Homology-2-Containing Protein Tyrosine Phosphatase-1 Restrains Cell Proliferation in Human Medullary Thyroid Carcinoma Endocrinology, June 1, 2005; 146(6): 2692 - 2698. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zatelli, M. C. Articles by degli Uberti, E. C. Search for Related Content PubMed PubMed Citation Articles by Zatelli, M. C. Articles by degli Uberti, E. C.