Somatostatin analogs and chimeric somatostatin–dopamine molecules differentially regulate human growth hormone and prolactin gene expression and secretion in vitro

Molecular and Cellular Endocrinology 362 (2012) 104–109

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Somatostatin analogs and chimeric somatostatin–dopamine molecules differentially regulate human growth hormone and prolactin gene expression and secretion in vitro Anna Gruszka a,b,⇑, Michael D. Culler c, Shlomo Melmed a a b c

Division of Endocrinology, Cedars-Sinai Research Institute, University of California School of Medicine, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA Department of Endocrinology, Medical University of Lodz, 1/3 Sterling Street, 91-425 Lodz, Poland Endocrinology and Hematology Research, IPSEN, Biomeasure Inc., 27 Maple Street, Milford, MA 01757, USA

a r t i c l e

i n f o

Article history: Received 22 August 2011 Received in revised form 21 December 2011 Accepted 31 May 2012 Available online 15 June 2012 Keywords: Pituitary tumors Growth hormone Prolactin Gene expression Somatostatin analogs Dopamine agonists

a b s t r a c t We tested effects of selective somatostatin receptor 2 (SST2) agonist BIM-23120, SST5 agonist BIM-23206 and chimeric somatostatin–dopamine molecules (SRIF/DA) BIM-23A760 and BIM-23A761 on GH and PRL secretion and gene expression in human GH/PRL-secreting pituitary tumors in vitro. In ‘‘responders’’ group BIM-23120 suppressed GH levels by 26 ± 4%, BIM-23206 by 31 ± 5%, BIM23A760 by 23 ± 4%, BIM-23A761 by 39 ± 8% and D2-dopamine agonist BIM-53097 by 31 ± 5%. Using real-time PCR we demonstrated that GH inhibition was not accompanied by decreased GH mRNA levels. PRL secretion was inhibited by BIM-23A760 (29 ± 5%), BIM-23A761 (34 ± 4%), BIM-23206 (26 ± 4%) and BIM-53097 (36 ± 2%). SRIF/DA and BIM-53097 also suppressed PRL mRNA levels. Concluding, SST2 and SST5 agonists and SRIF/DA inhibit GH secretion, but do not suppress GH gene transcription. SRIF/DA and BIM-53097 inhibit both PRL secretion and PRL gene expression. SST5 agonist inhibits PRL secretion, but does not suppress PRL gene expression. D2 affinity is crucial in SRIF/DA action on PRL gene expression. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction GH secretion from the anterior pituitary cells is controlled by complex neuroendocrine signals including hypothalamic GHRH and SRIF (for review see Giustina and Veldhuis (1998) and Luque et al. (2008)). In addition, somatotroph function is modulated by ghrelin – the endogenous ligand for the GH secretagogue receptor type Ia (GHS-RIa), acting mainly at hypothalamus to induce GH secretion in synergy with GHRH (Kojima and Kangawa, 2005; Tannenbaum et al., 2003). GHRH stimulates both GH gene transcription, synthesis and release (Giustina and Veldhuis, 1998; Melmed, 2009). GHRH may also act as a coagonist for the ghrelin receptor (Casanueva et al., 2008). SRIF inhibits GH secretion, mostly as a result of acute inhibition of hormone exocytosis, and its effect on GH biosynthesis is unclear. Inhibition of GH synthesis has not been proven conclusively. Studies in pituitary cell cultures

Abbreviations: D2, D2-dopamine receptor; GH, growth hormone; GHRH, growth hormone-releasing hormone; PRL, prolactin; SRIF, somatotropin-release inhibitory factor – somatostatin; SRIF/DA, chimeric somatostatin–dopamine molecules; SST, somatostatin receptor. ⇑ Corresponding author at: Department of Endocrinology, Medical University of Lodz, 1/3 Sterling Street, 91-425 Lodz, Poland. Tel./fax: +48 42 636 54 27. E-mail address: [email protected] (A. Gruszka). 0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.05.020

from animals (Barinaga et al., 1985; Fukata et al., 1985) have shown that SRIF regulates GH release, but has no effect on basal levels of GH synthesis or gene transcription (for review see Luque et al. (2008)). However, other studies have shown a decrease in somatotroph GH mRNA levels (Acunzo et al., 2008; Sugihara et al., 1993; Tsukamoto et al., 1994), maintaining the controversy on this issue. Interestingly, observations in primary pituitary cell cultures from pigs and baboons (Luque et al., 2006) indicate that SRIF may exert biphasic effects on GH release (i.e. GH suppression at moderate to high doses and GH stimulation at low doses). The effect of SRIF and/or its analogs on human GH gene expression has not yet been thoroughly studied. Dopamine regulates PRL secretion and gene expression acting through D2 receptors (Ben-Jonathan et al., 2008). SRIF and its analogs inhibit PRL secretion from rat PRL-secreting pituitary tumors in vivo and in vitro (Gruszka et al., 2001, 2007; Pawlikowski et al., 1997) and human PRL-secreting pituitary adenomas in vitro (Hofland et al., 2004; Jaquet et al., 1999; Shimon et al., 1997). Effects of SRIF and its analogs on human PRL gene expression have not yet been reported. Both somatostatin and dopamine receptors belong to the superfamily of G-protein-coupled receptors (GPCR). Somatostatin receptor subtypes 1, 2, 3 and 5 (SST1–SST5) predominate in normal pituitaries and pituitary adenomas (Ben-Shlomo and Melmed,

A. Gruszka et al. / Molecular and Cellular Endocrinology 362 (2012) 104–109

2010; Panetta and Patel, 1995; Pawlikowski et al., 2003; Taboada et al., 2007). D2 is the predominant dopamine receptor in normal pituitaries and pituitary adenomas (Missale et al., 1998; Neto et al., 2009; Stefaneanu et al., 2001). In most GH-secreting adenomas SST2, SST5 and D2 are co-expressed and some of somatotroph tumors also co-express SST3 and SST1, particularly mixed GH/PRL adenomas (Neto et al., 2009; Pawlikowski et al., 2008 Taboada et al., 2007). There is also evidence that human D2 receptors may form heterodimers with somatostatin receptors SSTR2 and SSTR5 with enhanced functional activity (Baragli et al., 2007; Rocheville et al., 2000). Approximately 65% of patients harboring GH-secreting pituitary adenomas treated with clinically available somatostatin receptor ligands, octreotide and lanreotide (with affinity mainly to SST2), achieve control of GH secretion (random fasting GH levels <2.5 lg/l and/or normalization of IGF1 levels). Using GH cutoff of less than 1 lg/l, approximately 33% of patients can be defined as controlled (Melmed, 2009). Novel SRIF analogs with high affinity to SST2 and SST5 represent potential future treatment option in selected patients with GH-secreting pituitary tumors. Pasireotide (SOM230), SRIF analog with high affinity to SST1, SST2, SST3 and SST5, is currently being evaluated in clinical trials in patients with GH-secreting adenomas, and short-term studies show promising results (Schmid, 2008; Petersenn et al., 2010). SRIF/DA, chimeric molecules containing structural elements of both SRIF and dopamine agonists and directed against both GPCRs were shown to be effective in controlling GH and PRL secretion in vitro in somatotroph adenomas that were partial responders to octreotide (Jaquet et al., 2005a,b; Ren et al., 2003; Saveanu et al., 2002). Previous studies on the efficacy of SRIF analogs and SRIF/DA in pituitary tumors from acromegaly patients were limited to the evaluation of GH and PRL secretion. The effect of SRIF and its analogs on human GH gene transcription is unclear, and the effects of SRIF/DA on human GH and PRL mRNA have not yet been reported. We have previously shown that inhibition of GH release is not accompanied by decreased GH mRNA levels in primary and tumorous rat pituitary cells (Gruszka et al., 2007). The aim of the present study was to simultaneously investigate the effects of selective SST2 (BIM-23120) and SST5 (BIM-23206) agonists and SRIF/DA (BIM-23A760 and BIM-23A761) on human GH and PRL secretion and gene expression in mixed GH/PRL-secreting pituitary tumors in vitro.

2. Materials and methods 2.1. Compounds Somatostatin receptor agonists: BIM-23120, BIM-23206, BIM23244, chimeric somatostatin–dopamine molecules BIM-23A760 and BIM-23A761, and D2 receptor agonist BIM-53097 were provided by Biomeasure, Inc. (Milford, MA), and their properties are shown in Table 1. Stock solutions (103 mol/l) of these substances Table 1 Human SST and D2 binding affinities of SRIF-14, SRIF analogs and D2 agonist BIM 53097 (IC50 (nmol/l)). Data are from radioligand binding assays to membranes from transfected CHO-K1 cells expressing human D2 or human SST subtypes. Values are from IPSEN, Biomeasure, Inc. as previously published (Gruszka et al., 2007).

SRIF-14 BIM-23A760 BIM-23A761 BIM-23120 BIM-23206 BIM-23244 BIM-53097

SST1

SST2

SST3

SST4

SST5

D2

2.3 622 462 1000 1152 >1000 >1000

0.2 0.03 0.06 0.34 166 0.3 >1000

1.4 160 52 412 1000 133 >1000

1.8 >1000 >1000 1000 1618 >1000 >1000

1.4 42.0 3.7 213.5 2.4 0.7 >1000

>1000 15 27 >1000 >1000 >1000 22

105

were prepared in 102 mol/l acetic acid containing 0.1% BSA (Sigma–Aldrich) and stored at 20 °C until used. 2.2. Human pituitary adenomas Use of human tissue was approved by the Institutional Review Board, and patients gave written consent for anonymous tissue collection. Clinical characterization of the patients is summarized in Table 2. A part of each surgically removed tumor from acromegaly patients was mechanically dispersed and enzymatically digested in DMEM (Invitrogen Corp., Grand Island, NY) containing 0.35% collagenase type IA, 0.15% hyaluronidase and 0.3% BSA (Sigma–Aldrich) at 37 °C for 40 min according to the protocol previously described (Gruszka et al., 2006). Cells were cultured in DMEM supplemented with 10% FBS for 48 h, then serum-starved for 12 h and treated with test substances. According to the results of preliminary time-course and dose-dependency experiments (data not shown), cultures were incubated with test compounds at concentrations of 108 mol/l for 24 h. Medium for hormonal assays and RNA for real-time PCR was collected. 2.3. Hormone assays Human GH in culture medium was measured using a radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA) with a sensitivity of 0.9 lg/l. Human PRL was measured by immunoradiometric assay (Coat-A-Count Prolactin IRMA, Diagnostic Products Corp., Los Angeles, CA) with a sensitivity of 0.1 lg/l. 2.4. Real-time PCR Total RNA from cultured cells was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. RNA samples were treated with DNase I (Deoxyribonuclease I, Amplification Grade, Invitrogen) to eliminate genomic DNA contamination. Total RNA was reverse transcribed into first-strand cDNA using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. For each new batch of cDNA a control sample containing no reverse transcriptase was performed (/–/RT control). Real-time PCRs were carried out in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) according to the protocol previously described (Gruszka et al., 2007), recorded and analysed using the iQ5 Optical System Software ver. 1.0 (Bio-Rad Laboratories, Inc.). Briefly, real-time PCR amplifications were carried out with 10 ll SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 5  107 mol/l forward primer, 5  107 mol/l reverse primer and 5 ll cDNA template (100 ng reverse-transcribed total RNA per well). b-Actin served as an internal reference for normalization of GH and PRL mRNA levels. Primer sequences (Invitrogen) were as follows: human GH forward: 50 -CAGGAGTGTCTTCGCCAACA-30 , human GH reverse: 50 TCCCCATCAGCGTTTGGAT-30 , human PRL forward: 50 -TCATCTGGTCACGGAAGTACGT-30 , human PRL reverse: 50 -TGCCCTCTAGAAGCCGTTTG-30 , b-actin forward: 50 -CATGTACGTTGCTATCCAGGC-30 , b-actin reverse: 50 -CTCCTTAATGTCACGCACGAT-30 . Thermal cycling profile consisted of incubation at 95 °C for 4.5 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s and elongation at 72 °C for 30 s. Samples were run in triplicate. Controls comprising either no template, or /–/RT were run in each experiment. 2.5. Statistical analysis Statistical significance of the difference between means was assessed with one-way analysis of variance (ANOVA) followed by Tu-

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Table 2 Clinical characterization of patients harboring GH/PRL-secreting pituitary tumors and hormonal secretion in vivo and in vitro. Patient

Sex

Age (yr)

Tumor size (MRI) (mm)

Hormone secretion in vivo GH

1 2 3 4 5

F M M F M

30 62 44 70 50

30  18  35 10  10 12  12 6.8  6.8 99

a

(lg/l)

>200 9.7 22 37 5.7

Hormone secretion in vitro

b

IGF1 (lg/l)

GH (lg/l)

PRL (lg/l)

866 518 492 930 477

717.4 794 397 100.1 130

110 230 60 69 59

M, male; F, female. Tumors 1, 2, 3 – ‘‘responders’’, tumors 4, 5 – ‘‘non-responders’’. a Basal serum GH levels. b GH and PRL levels in control culture medium from 5  104 cells cultured for 24 h.

key’s multiple comparison test (GH and PRL secretion results) or Dunnett’s multiple comparison test (real-time PCR data). Calculations were performed with statistical software GraphPadPrism (GraphPadSoftware, Inc., San Diego, CA). p < 0.05 was considered significant. GH and PRL levels are expressed as mean ± SEM and shown as the percentage of untreated controls. GH and PRL mRNA levels were normalized to b-actin, and expressed as fold expression relative to vehicle-treated control, defined as 1.0.

by the test substances (Fig. 1F). PRL levels in this group were reduced by BIM-23A760 and BIM-23A761 (37 ± 2%, p < 0.001 and 39 ± 4%, p < 0.001 vs control, respectively), and by the D2 receptor agonist BIM-53097 (40 ± 2%, p < 0.001 vs control) but not by SST2 and/or 5 receptor agonists (Fig. 1G). Suppression of PRL secretion was accompanied by decreased PRL mRNA levels as assessed by real-time PCR (p < 0.05, Fig. 1H).

4. Discussion 3. Results 3.1. GH secretion and GH mRNA expression In three of five freshly cultured human pituitary tumors, a significant reduction of GH secretion after incubation with somatostatin analogs in vitro was found (‘‘responders’’). SST2 agonist BIM-23120 reduced GH levels by 26 ± 4% (p < 0.01), SST5 agonist BIM-23206 by 31 ± 5% (p < 0.001) and SST2 + 5 agonist BIM23244 by 41 ± 2% (p < 0.001, Fig. 1A respectively). D2 receptor agonist BIM-53097 decreased GH levels by 31 ± 5% (p < 0.001). Chimeric molecules BIM-23A760 and BIM-23A761 reduced GH levels by 23 ± 4%, p < 0.01 and 39 ± 8%, p < 0.001 vs control, respectively. The efficacy of both chimeric molecules in suppressing GH secretion was similar to that of the selective SST2 and SST5 agonists (p > 0.05). Suppression of GH secretion was not accompanied by decreased GH mRNA level as evidenced by real-time PCR results (Fig. 1B). 3.2. PRL secretion and PRL mRNA expression PRL was detected in the culture medium of all human pituitary adenomas derived from acromegaly patients and all the tumors expressed PRL mRNA as evidenced by real-time PCR. In the ‘‘responders’’ group all test compounds, except for the SST2 agonist BIM23120, reduced PRL levels (Fig. 1C). PRL suppression achieved with both chimeric molecules (29 ± 5%, p < 0.001 with BIM-23A760 and 34 ± 4%, p < 0.001 with BIM-23A761) was not different (p > 0.05) from that obtained with the selective SST5 agonist BIM-23206 (26 ± 4%, p < 0.001), SST2 + 5 agonist BIM-23244 (36 ± 3%, p < 0.001) and with D2 receptor agonist BIM-53097 (36 ± 2%, p < 0.001), respectively. Real-time PCR revealed suppression of PRL mRNA levels only in groups treated with chimeric compounds (p < 0.05) and BIM-53097 (p < 0.01, Fig. 1D). 3.3. ‘‘Non-responders’’ In two of five human pituitary tumors tested a statistically nonsignificant reduction of GH secretion (10–22%) after incubation with somatostatin analogs in vitro was found (‘‘non-responders’’, Fig. 1E). GH mRNA levels in this group were also not suppressed

4.1. GH secretion and GH mRNA expression We have shown that inhibition of GH release in human GH/PRLsecreting adenomas by SST2 and SST5 receptor agonists and SRIF/ DA in vitro is not accompanied by decreased GH mRNA levels. Previously, we obtained similar results in primary rat pituitary cells and in GH3 rat pituitary tumor cells (Gruszka et al., 2007). The effect of SRIF and its analogs on human GH gene expression has not yet been thoroughly studied. Previous studies with SRIF analogs and SRIF/DA in human GH and/or PRL secreting pituitary tumors were usually confined to the evaluation of GH and PRL secretion, but not GH and PRL synthesis or gene transcription. Only two studies reported GH mRNA levels in human GH-secreting pituitary adenomas after octreotide treatment. In both, GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. Hofland et al. (1992) did not observe effects of 10 nM octreotide on GH mRNA levels after 24 h incubation of three adenomas. After 96 h incubation, GH mRNA levels increased in two, and were slightly decreased in one of three adenomas tested. Tsukamoto et al. (1994) showed that GH mRNA levels were significantly lower in GH-secreting pituitary adenoma tissue obtained from seven patients treated preoperatively with octreotide than in adenomas derived from 18 non-treated patients. Our results support the hypothesis that although SRIF is a potent inhibitor of GH release, it does not directly suppress GH mRNA levels in human pituitary cells in vitro, as evidenced by real-time PCR. However, SRIF may be indirectly involved in the regulation of human GH gene expression. Morishita et al. (2003) tested effects of GHRH and SRIF on GH gene 50 -promoter activity in MtT/S cells and found that SRIF modestly but significantly suppressed GHRH-induced GH gene transcription, although SRIF alone did not influence basal promoter activity. Further studies are required to elucidate the effect of SRIF and its analogs on GHRH-induced human GH gene transcription. In our study in ‘‘responders’’ group (Fig. 1A) BIM-23A760 was similarly effective in suppressing GH secretion to BIM-23120, and BIM-23A761 was similarly effective to BIM-23244. We did not observe any synergistic effects of chimeric SRIF/DA molecules on GH suppression. Previously Jaquet et al. (2005b) reported that in six of 13 tumors from acromegaly patients chimeric molecules BIM 23A387, BIM 23A760 and BIM 23A761 achieved greater maximal

107

0

% PRL change

-10 -20 -30 -40

*

97

O6

20

60

61

C

97

44

O6

61

20

1.4 1.2

-40 -50

# #

0.4

X 0.2

97

O6

1.2

20

1.4

1.0 0.8 0.6 0.4 0.2 0.0

97

O6

0.0

F GH mRNA - normalized expression

97

O6

20

61

60

C

H

0.6

20

97

O6

61

60

C

0.0

0.8

61

0.2

1.0

60

0.4

1.2

-30

-60

C

0.6

*

* *

1.4

PRL mRNA - normalized expression

0.8

D

PRL mRNA – normalized expression

97

O6

20

61

60

C

GH mRNA - normalized expression

1.0

*

*

-50

1.4

-50

*

*

-40

*

*

-50

-30

60

*

*

-40

-20

C

X

0

0

-20 % GH change

% PRL change

X

20

% GH change

-30

G

E

-10

-20

1.2

0

-10

-10

B

60

C

C

97

44

O6

20

61

A

60

C

A. Gruszka et al. / Molecular and Cellular Endocrinology 362 (2012) 104–109

1.0 0.8 0.6 #

0.4 0.2

#

#

0.0

Fig. 1. Effects of chimeric somatostatin–dopamine molecules (SST2, SST5 and D2 agonists) BIM-23A760 [60] and BIM-23A761 [61], D2 agonist BIM-53097 [97] and somatostatin receptors agonists: BIM-23120 [20] (SST2 agonist), BIM-23206 [06] (SST5 agonist) and BIM-23244 [44] (SST2 and SST5 agonist) on GH and PRL secretion and mRNA expression in human pituitary tumors from acromegalic patients after 24 h incubation (all test substances at concentrations of 108 mol/l). Results are expressed as mean ± SEM percentage GH or PRL secretion change vs vehicle-treated control cells. GH and PRL mRNA levels were analyzed by real-time PCR. Values (mean ± SEM) are expressed as fold change from vehicle-treated control, defined as 1.0. GH and PRL mRNA levels were normalized to b-actin mRNA. ⁄p < 0.001; p < 0.01 and #p < 0.05 vs control (C) A–D – ‘‘responders’’, E–H – ‘‘non-responders’’.

suppression of GH secretion than octreotide. In five of 13 tumors a similar GH suppression with BIM 23A387, BIM-23244 and octreotide was obtained (Jaquet et al., 2005b). Clearly, high binding affinity for the given somatostatin or dopamine receptor does not ensure a good cell response to the compound, and the receptor binding affinity is not always directly proportional to the potency of the compound in the specific cell. Thus, not only the binding affinity of the compound, but the cell type, receptor profile, possible interactions between receptors and defects downstream of the receptors are responsible for the final effect of the given ligand. Best results of treatment with SRIF/DA are likely achieved in cells co-expressing both somatostatin (SST2 and/or SST5) and D2

receptors. According to the previous studies, SST2, SST5 and D2 receptors, in variable amounts, are co-expressed in most human GH-secreting adenomas (Ferone et al., 2008; Pawlikowski et al., 2008; Taboada et al., 2007). In GH-secreting adenomas only SST2, but not SST5 expression, was positively correlated with the suppression of GH secretion by octreotide in vitro and in vivo and with IGF1 suppression by SRIF analogs in vivo (Pawlikowski et al., 2008; Taboada et al., 2007). D2 expression was positively correlated with GH and PRL suppression by D2 agonist quinagolide in vitro (Ferone et al., 2008). However, there is growing evidence that the presence of somatostatin and dopamine receptors does not always ensure a good response to the given ligand. In nonfunctioning pituitary adenomas (NFPAs) the level of expression of somatostatin receptors

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was not correlated with the pharmacological responses to SRIF analogs or SRIF/DA in vitro (Gruszka et al., 2006; Florio et al., 2008), and D2 mRNA level in non-responsive NFPAs was even higher than in the responsive ones (Florio et al., 2008). In another study (Fusco et al., 2008) no correlation was found between SST5 mRNA levels and SST5 agonist BIM-23206-mediated PRL suppression in human prolactinomas. It seems that other mechanisms, like defects downstream of the receptors, can be involved in SRIF analogs and SRIF/DA resistance. It has been previously shown that somatostatin and D2-dopamine receptors can form heterodimers. Heterodimerization may induce modification of ligand binding and synergy in receptor activation as demonstrated for SST5 and D2 in transfected heterologous cells (Rocheville et al., 2000). The D2-SST5 dimer had a greater affinity for both D2 and SST5 agonists, and presented enhanced coupling with adenylyl cyclase (Rocheville et al., 2000). Baragli et al. (2007) have shown that D2 and SST2 heterodimerization is associated with reciprocal modification of ligand binding, improved EC50 in cAMP inhibition and increased internalization for SST2. However, these results were obtained in non-pituitary cell lines (CHO or HEK293) stably expressing exogenous receptors, and there is so far no evidence for receptor dimerization in pituitary cells. Recently, somatostatin and D2 receptors interaction has been demonstrated in prostate cancer cell line, LNCaP, and in non-small cell lung cancer line, Calu-6 (Arvigo et al., 2010). Postreceptor mechanisms involved in the enhanced potency following receptor dimer activation have not yet been characterized. It also remains to be established whether only one or both receptors of the dimer account for its activity. The mechanism of SRIF/DA action has not been fully clarified and it could be different in various cell types (for review see Saveanu et al. (2008)). It has been hypothesized (Jaquet et al., 2005b), that: (a) dopastatins can induce heterodimerization of somatostatin and D2 receptors resulting in receptor conformation with enhanced inhibitory activity, (b) dopastatins may trigger transduction pathways different from those triggered by specific SST2, SST5 and D2 agonists. In our limited group of human mixed GH/PRL-secreting tumors dopastatins were similarly effective in inhibiting GH secretion as selective SST2 and SST5 agonists, and similarly effective in inhibiting PRL secretion as D2 agonist, and no synergistic effects on GH and PRL suppression were observed. Unexpectedly, we observed that D2 agonist BIM-53097 was very effective in suppressing GH secretion from human GH/PRL-secreting pituitary tumors in the ‘‘responders’’ group. It is well known that in vivo D2 agonists inhibit GH secretion in approximately 20% of acromegaly patients. According to the recent meta-analysis of 227 patients (Sandret et al., 2011) cabergoline single-agent therapy normalizes IGF1 levels in one third of patients with acromegaly. Our results show that D2 agonist BIM-53097 may represent a potential future treatment option in selected patients with GH and/ or PRL-secreting tumors, however long term clinical studies are required to evaluate the effectiveness of this compound.

4.2. PRL secretion and PRL mRNA expression Dopamine is the major regulator of prolactin secretion and gene expression (Ben-Jonathan et al., 2008). SRIF and its analogs inhibit PRL secretion in rat and human PRL-secreting pituitary tumors (Gruszka et al., 2001; Hofland et al., 2004; Shimon et al., 1997). Interestingly, in human prolactinomas inhibition of PRL secretion was observed only in vitro (Jaquet et al., 1999; Shimon et al., 1997), but not in vivo. We recently tested effects of SST2 and SST5 receptor agonists and chimeric somatostatin–dopamine molecules on PRL secretion and gene expression in primary rat pituitary cells and rat pituitary

tumor cell lines (Gruszka et al., 2007). Effects of SRIF and its analogs on human PRL gene expression have not yet been reported. Here, PRL was detected in the culture medium of all human pituitary adenomas derived from acromegaly patients (Table 2) and all the tumors expressed PRL mRNA as evidenced by real-time PCR. In all tumors both PRL secretion and PRL gene expression were suppressed by somatostatin–dopamine chimeric compounds as well as the D2 agonist BIM-53097. The chimeric molecules at concentrations of 108 mol/l were similarly effective as the D2 receptor agonist BIM-53097 in suppressing PRL secretion or PRL mRNA levels. Despite their affinity for SST5, both chimeric molecules acted like a D2 agonist in terms of inhibiting PRL secretion, and no synergistic effects were found. In the ’’responders’’ group PRL secretion, but not gene expression, was also decreased by the SST5 receptor agonist. SST5 agonist BIM-23206 was slightly less potent in inhibiting PRL secretion than BIM-23244 showing a higher affinity for SST5 (Fig. 1C). Previously it was reported that SST5 agonist BIM-23268 combined with D2 agonist quinagolide produced a partial additive effect on PRL suppression in human prolactinomas (Jaquet et al., 1999). In six of 13 GH/PRL-secreting pituitary tumors chimeric molecules BIM-23A758, BIM-23A760 and BIM-23A761 produced maximal PRL suppression greater than octreotide (Jaquet et al., 2005b). Recently Fusco et al. (2008) reported that SST5 receptor agonist BIM-23206 inhibited PRL release from dopamine agonist sensitive prolactinomas similarly to cabergoline, but was not effective in suppressing PRL secretion from dopamine agonist resistant tumors in vitro. Similarly to our study, Fusco et al. (2008) did not observe any additive effects of the chimeric molecule BIM23A760 on PRL suppression in human prolactinomas as compared with the D2 receptor agonist cabergoline. The SST5 receptor has previously been shown to exclusively regulate PRL secretion from human prolactinoma cells (Shimon et al., 1997). Our results indicate that the SST5 receptor agonist inhibits PRL secretion from human mixed GH/PRL-secreting pituitary adenomas in vitro, but does not suppress PRL gene expression. Thus, the action of the SRIF/DA on PRL gene expression in human pituitary tumors appears to depend primarily on their D2 receptor affinity. 5. Conclusions We conclude that in mixed GH/PRL human pituitary adenomas derived from acromegaly patients suppression of GH secretion in vitro achieved with SST2 and SST5 receptor agonists and chimeric somatostatin–dopamine molecules is not accompanied by decreased GH mRNA level under basal conditions. The SST5 receptor agonist inhibits PRL secretion from mixed GH/PRL human pituitary adenomas in vitro, but does not suppress PRL gene expression. SRIF/DA and the D2 agonist BIM-53097 inhibit both PRL secretion and PRL gene expression. D2 receptor affinity has a crucial role in the action of chimeric somatostatin–dopamine molecules on PRL gene expression in human pituitary tumors. Our results give further insight into mechanisms for SRIF analogs and SRIF/DA action. Long term in vivo studies are needed to evaluate the effectiveness of SRIF/DA and new SST and D2 agonists in the treatment of patients with GH and/or PRL-secreting tumors. Declaration of interest A.G. has nothing to declare. M.D.C. is employed by IPSEN. S.M. receives grant support (current and annual) from IPSEN. Funding This work was supported by a Grant from IPSEN.

A. Gruszka et al. / Molecular and Cellular Endocrinology 362 (2012) 104–109

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