help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1762
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barbieri, F.
Right arrow Articles by Florio, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barbieri, F.
Right arrow Articles by Florio, T.
Endocrinology Vol. 149, No. 9 4736-4746
Copyright © 2008 by The Endocrine Society

Somatostatin Receptors 1, 2, and 5 Cooperate in the Somatostatin Inhibition of C6 Glioma Cell Proliferation in Vitro via a Phosphotyrosine Phosphatase-{eta}-Dependent Inhibition of Extracellularly Regulated Kinase-1/2

Federica Barbieri, Alessandra Pattarozzi, Monica Gatti, Carola Porcile, Adriana Bajetto, Angelo Ferrari, Michael D. Culler and Tullio Florio

Laboratory of Pharmacology (F.B., A.P., M.G., C.P., A.B., T.F.), Department of Oncology, Biology and Genetics, University of Genova, 16132 Genova, Italy; Istituto Zooprofilattico Sperimentale del Piemonte (A.F.), Liguria e Valle D'Aosta, National Reference Center of Veterinary and Comparative Oncology, 16132 Genova, Italy; and IPSEN (M.D.C.), Milford, Massachusetts 01757

Address all correspondence and requests for reprints to: Prof. Tullio Florio, Department of Oncology, Biology, and Genetics, University of Genova, Viale Benedetto XV, 2, 16132 Genova, Italy. E-mail: tullio.florio{at}unige.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin inhibits cell proliferation through the activation of five receptors (SSTR1–5) expressed in normal and cancer cells. We analyzed the role of individual SSTRs in the antiproliferative activity of somatostatin in C6 rat glioma cells. Somatostatin dose-dependently inhibited C6 proliferation, an effect mimicked, with different efficacy or potency, by BIM-23745, BIM-23120, BIM-23206 (agonists for SSTR1, -2, and -5) and octreotide. The activation of SSTR3 was ineffective, although all SSTRs are functionally active, as demonstrated by the inhibition of cAMP production. All SSTRs induced cytostatic effects through the activation of the phosphotyrosine phosphatase PTP{eta} and the inhibition of ERK1/2. For possible synergism between SSTR subtypes, we tested the effects of the combined treatment with two agonists (SSTR1+2 or SSTR2+5) or bifunctional compounds. The simultaneous activation of SSTR1 and SSTR2 slightly increased the efficacy of the individual compounds with an IC50 in between the single receptor activation. SSTR2+5 activation displayed a pattern of response superimposable to that of the SSTR5 agonist alone (low potency and higher efficacy, as compared with BIM-23120). The simultaneous activation of SSTR1, -2, and -5 resulted in a response similar to somatostatin. In conclusion, the cytostatic effects of somatostatin in C6 cells are mediated by the SSTR1, -2, and -5 through the same intracellular pathway: activation of PTP{eta} and inhibition of ERK1/2 activity. Somatostatin is more effective than the individual agonists. The combined activation of SSTR1 and -2 shows a partial synergism as far as antiproliferative activity, whereas SSTR2 and -5 activation results in a response resembling the SSTR5 effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOLOGICAL EFFECTS of somatostatin (SST) are mediated by a family of five G protein-coupled receptors named SST receptor (SSTR) 1–5 (1), variably expressed in both normal and cancer tissues (2, 3) and often showing the presence of multiple subtypes in the same cell. Besides its central role as an endocrine regulator of pituitary, pancreatic, and gastrointestinal hormone secretion, SST represents an endogenous regulator of proliferation for a variety of epithelial and endocrine cells (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). SST effects involve both direct and indirect mechanisms. SST indirectly controls cell growth in vivo through its inhibitory effects on the release of growth-promoting hormones (14) and through antiangiogenic mechanisms (15) involving the inhibition of proliferation and migration of endothelial cells and monocytes (16, 17) or interfering with the production of proangiogenic factors from tumor cells (18). Moreover, a direct antiproliferative activity in both normal and tumor cells has also been reported (3, 19) in which the modulation of phosphotyrosine phosphatase (PTP) activity was proposed as one of the main intracellular pathways responsible for such an effect (20, 21, 22, 23, 24, 25, 26).

In the past years, different receptor subtypes, displaying antiproliferative activity, have been reported to act via the activation of this class of enzymes. Although an increase in PTP activity was shown after LHRH, angiotensin II, and dopamine receptor activation, most of the studies have been performed on PTP activation by SSTR (27). In particular, the SST-responsive PTPs have been initially identified in the cytosolic enzymes, Src-homology 2-domain-containing tyrosine phosphatase (SHP) 1 and 2 (23, 28, 29). More recently, beside SHPs, other PTPs were also reported to be involved in the SST antiproliferative effects. In particular, we described that SST-dependent cytostatic effects in the normal thyroid cell line PC Cl3 (25), as well as in human and rat glioblastoma cells (13), were mediated by the activation of the receptor-like PTP, PTP{eta} (also called DEP-1 in humans). This is a ubiquitous PTP, mainly expressed in the brain, liver, and spleen (30). The predicted protein sequence contains a unique intracellular catalytic domain, a short transmembrane domain, and an extracellular region containing eight fibronectin type III-like repeats (30). Experimental evidence suggests that PTP{eta} activity participates in the regulation of cell growth. For example, after vascular injury, PTP{eta}/DEP-1 is down-regulated in migrating and proliferating endothelial cells (31). Moreover, the overexpression of PTP{eta}/DEP-1 in macrophages or breast cells results in a marked inhibition of cell proliferation (32, 33, 34). On the other hand, PTP{eta} expression is down-regulated by the oncogene-induced transformation of PC Cl3 thyroid cells as well as in malignant human thyroid tumors (35, 36). Reexpression of PTP{eta} into the transformed rat thyroid cells leads to a slowing down of the proliferation rate, associated with an inhibition of ERK1/2-dependent proteolysis of p27Kip1 and reacquisition of a partially differentiated phenotype (25, 37). Similarly, the transfection of PTP{eta} in glioblastoma cells that do not express it and are not sensitive to SST antiproliferative activity, leads to the loss of some neoplastic transformation markers (i.e. anchorage-dependent growth) and more importantly allows the recovery of SST sensitivity (13). More recently, we characterized, in CHO-K1 cells, the intracellular signaling pathway mediated by SSTR1 that leads to the activation of PTP{eta} and that involves the sequential activation of tyrosine kinases (Janus kinase 2 and Src) and PTPs (SHP-2) (38).

Altogether, these data indicate that PTP{eta} activity is an important transducer of SSTR-dependent antiproliferative signals.

The molecular cloning of a family of SSTRs prompted many studies devoted to the identification of specific physiological roles for each individual receptor subtype and the possible different coupling to intracellular transduction systems. However, likely due to the simultaneous expression of multiple receptors on the same cells, very little biological specificity has been observed. In particular, as far as the inhibitory effects on cell proliferation, previous studies showed that the activation of all the five SSTRs can mediate cytostatic effects. However, most of these studies have been performed in cells heterologously expressing single SSTR subtypes (21, 29, 39), and few data analyze the interaction among different SSTRs in cell lines natively expressing multiple subtypes. Moreover, it is still not established whether the SSTR subtypes able to regulate cell proliferation act through the same intracellular pathways or regulate independent signaling.

In this study, we evaluated the antiproliferative activity of individual SSTRs in rat C6 glioma cells, comparing the effects of selective agonists for each receptor subtype with those of the native ligand as far as regulation of cell proliferation and intracellular signaling. Moreover, the modulation of the activity of each receptor subtype induced by the costimulation of other SSTRs was analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs
SST-14 was purchased from Calbiochem (Lucerne, Switzerland). The SSTR agonists (BIM compounds) were provided by Biomeasure Inc./IPSEN (Milford, MA), The selective SSTR3 agonist L-796778 (40) was kindly provided by S. Rohrer (Merck Research Laboratories, Boston, MA), and octreotide was obtained from Novartis (Basel, Switzerland). The individual binding characteristics of these compounds are listed in Table 1Go. Stock solutions (10–3 M) of these substances were prepared in 0.01 M acetic acid containing 0.1% purified human serum albumin (Sigma-Aldrich, Milano, Italy). All drugs were stored at –80 C until used. For each experiment, working solutions were made by diluting a fresh aliquot with culture medium. Vanadate, forskolin (FSK), 3-isobutyl-1-methylxanthine, and all other reagents were purchased from Sigma-Aldrich, unless otherwise specified.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Binding affinities (Kd) of SST and SST agonists used in this study

 
Antibodies
For PTP{eta} detection, antibodies raised against the intracellular region of PTP{eta}, expressed as a recombinant protein fused to glutathione S-transferase and affinity purified (36), were used in Western blot experiments at the dilution of 1:500. Anti-p44/42 and anti-phospho-p44/p42 ERK1/2 (New England Biolabs Inc., Beverly, MA) were used at the dilution of 1:1000.

Cell cultures
C6 rat glioma cells (Interlab Cell Line Collection. Genova, Italy) were grown in Ham’s F12 medium, supplemented with 10% fetal calf serum (Euroclone, Milano, Italy).

RNA isolation and RT-PCR analysis
Total RNA from cultured cells was extracted using the acidic phenol technique. Before processing, total RNA was treated with ribonuclease-free deoxyribonuclease (Roche Molecular Biochemicals, Basel, Switzerland) to remove genomic DNA contamination. RT was performed using oligo-dT(16) primer and the AMV RT (GE Healthcare, Milano, Italy), for 40 min at 42 C. Amplification profile was 94 C for 5 min, 40 cycles at 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min, followed by 7 min at 72 C using the Taq PCR Core Kit (QIAGEN, Milano, Italy). Amplified DNA fragments were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. The primers used for the amplification of rat SSTR1–5 and β-actin, as PCR efficiency control, were previously described (25, 41) and purchased from Tib MolBiol, (Genova, Italy). In addition, a negative control, in which PCR amplification was performed in the absence of the RT reaction, was included to verify the absence of genomic DNA contamination.

[3H]Thymidine incorporation assay
DNA synthesis activity was measured by means of the [3H]thymidine uptake assay, as previously reported (42). Briefly, cells were plated at the density of 5 x 104 in 24-well plates. After 24 h, cells were serum starved for 48 h and treated with the test substances for 16 h. In the last 4 h, cells were pulsed with 1 µCi/ml of [3H]thymidine (GE Healthcare). Then cells were trypsinized, extracted in 10% trichloroacetic acid (TCA), and filtered under vacuum through fiberglass filters (Millipore Co., Bedford, MA). Filters were sequentially washed, under vacuum, with 10 and 5% TCA and 95% ethanol. The TCA-insoluble fraction was measured in a scintillation counter.

Apoptosis detection
The specific enzyme immunoassay (Cell Death Detection ELISAPLUS; Roche Diagnostics, Penzberg, Germany) for the qualitative and quantitative in vitro determination of cytoplasmic histone-associated DNA fragments was used following the manufacturer’s recommendation (41). Results from ELISA are expressed as the percentage of DNA fragments relative to untreated control cultures designated as 100%.

Measurement of cAMP accumulation
C6 cells plated (5 x 105 per well) in 24-well culture plates until subconfluence were starved for 24 h in medium without fetal calf serum. Culture medium was removed from the wells, and fresh medium containing the specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (0.5 mM) was added to each well and incubated at 37 C in the absence (basal) or presence of FSK (100 nM) and SSTR agonists (1 µM for 1 h), as reported (43). Reactions were stopped by medium removal, cells were lysed, and cAMP accumulation was determined using the cAMP Biotrak EIA System according to the instructions of the manufacturer (GE Healthcare). A standard curve was generated by plotting the percent of B/Bo as function of the log cAMP concentration, where B/Bo was calculated as (standard/sample OD – nonspecific binding OD)/(zero standard OD – nonspecific binding OD). The amount of cAMP in cell supernatants was determined by interpolation of femtomoles per well of each sample and the standard curve by nonlinear regression and data points were calculated as a percentage of the FSK response (100%).

Immunoprecipitation
Total cell lysates (250 µg) were incubated with the appropriate antibody (1 µg/mg proteins) for 2 h at 4 C in RIPA buffer and then for an additional hour with IgG-coupled magnetic beads (Dynabeads; Invitrogen, Milano, Italy), because the protein A caused, per se, hydrolysis of para-nitro-phenylphosphate (pNPP) (25). After three washes with RIPA buffer, the immunocomplexes were analyzed for PTP activity and by Western blot to normalize PTP{eta} immunoprecipitation efficiency.

PTP assay
PTP activity was evaluated on immunocomplexes using the synthetic substrate pNPP in a spectrophotometric assay. pNPP is a general phosphatase substrate that in the presence of inhibitors of Ser/Thr phosphatases is specific for PTP (20). The samples were incubated at 30 C in 80 µl volume containing 20 µl of a 5x reaction buffer [250 mM HEPES (pH 7.2), 50 mM dithiothreitol, 25 mM EDTA, 500 nM microcystin-leucine-arginine (Alamone Laboratories, Jerusalem, Israel), as Ser/Thr phosphatase inhibitor], and the reaction was started by adding 20 µl 50 mM pNPP, carried out for 60 min, and stopped by adding 900 µl 0.2 N NaOH. The absorbance of the sample, directly proportional to the amount of the cleaved substrate, was measured at 410 nm (44).

Western blot
Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and the Complete protease inhibitor cocktail (Roche Diagnostics). Nuclei were removed by centrifugation (5000 rpm for 10 min at 4 C), and total protein contents were measured using the Bradford assay (Bio-Rad, Milano, Italy). Proteins (10 µg) were resuspended in 2x reducing sample buffer [2% SDS, 62.5 mM Tris (pH 6.8), 0.01% blue bromophenol, 1.43 mM 2β-mercaptoethanol, and 0.1% glycerol], electrophoresed on 10–15% SDS-polyacrylamide gels, transferred on polyvinylidene difluoride membrane (Bio-Rad), and probed with specific antibodies. The detection of immunocomplexes was performed using the ECL kit (GE Healthcare).

Statistical analysis
Experiments were performed in quadruplicate and repeated at least three times. All values are expressed as mean ± SEM. Statistical analysis was performed by means of one-way ANOVA followed by Newman-Keuls test. A P value ≤0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of individual SSTR subtypes in the antiproliferative effects of SST
To explore the individual contribution of the different SSTR subtypes in the SST-dependent inhibition of C6 cell proliferation, we first analyzed the expression of SSTRs by RT-PCR.

As shown in Fig. 1FGo, C6 cells expressed four SSTR subtypes: SSTR1, -2, -3, and -5, whereas SSTR4 mRNA was not detected.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		FIG. 1. SSTR subtype-specific inhibition of DNA synthesis. A–E, Dose-response curves of the inhibition of bFGF-stimulated DNA synthesis induced, in C6 rat glioma cultures, by SST (A), the SSTR1-preferential compound BIM-23745 (B), the SSTR2-preferential compound BIM-23120 (C), the SSTR3 agonist L-796778 (D), and the SSTR5-preferential compound BIM-23206 (E), assessed by [3H]thymidine incorporation. Results are expressed as mean percentage ± SE of basal value in the presence of bFGF stimulation of DNA synthesis. Data represent the average of three independent experiments performed in quadruplicate. *, P < 0.05; **, P < 0.01 vs. bFGF-induced DNA synthesis. IC50 values are indicated for each compound. In F, the pattern of SSTR mRNA expression in C6 rat glioma, evaluated by RT-PCR, is reported.

 
To test the specific role of each of these subtypes in the antiproliferative effects of SST, we used three peptidic agonists selective for SSTR1, -2, and -5 (BIM-23745, BIM-23120, and BIM-23206) and the peptidomimetic compound L-796778, able to exclusively bind SSTR3 (see Table 1Go). In particular, we compared, in extended dose-response experiments (0.1 pM to 10 µM), the effects of each selective agonist with those elicited by the simultaneous activation of all receptors induced by SST, evaluating both the maximal inhibition on basic fibroblast growth factor (bFGF) stimulation of DNA synthesis and the IC50 at which this effect occurred.

To observe a better SSTR modulation of C6 cell proliferation, we chose to use bFGF concentrations (30 ng/ml) able to induce a submaximal, although significant, stimulation of DNA synthesis (data not shown).

SST dose-dependently inhibited bFGF-induced DNA synthesis, being statistically significant already at the concentration of 10 pM and maximal at 10 µM. Low concentrations of SST (10 pM to 10 nM) dose-dependently abolished bFGF stimulation of DNA synthesis, whereas higher concentrations (100 nM to 10 µM) brought the [3H]thymidine incorporation significantly below the basal value. Thus, analyzing the dose-response curve, we identified a biphasic effect with a component showing IC50 value of about 7 pM and another with an IC50 of about 15 nM (Fig. 1AGo).

Using the SSTR1-selective agonist BIM-23745, a significant inhibition of bFGF-stimulated DNA synthesis was also observed, causing a reduction of [3H]thymidine incorporation to the basal level. The effect was statistically significant starting at the concentration of 1 nM and maximal at 100 nM (IC50 6.2 nM), whereas higher concentrations did not cause a further inhibition of DNA synthesis (Fig. 1BGo).

The SSTR2 agonist BIM-23120 inhibited bFGF-stimulated cell proliferation with an efficacy similar to that of BIM-23745. However, BIM-23120 showed a higher potency than the SSTR1 agonist because it caused a statistically significant effect already at the concentration of 10 pM and an IC50 of 195 pM (Table 2Go and Fig. 1CGo).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Efficacy (maximal inhibition) and potency (IC50) of SSTR1, SSTR2, and SSTR5 agonists on the inhibitory effects on bFGF-induced C6 cell DNA synthesis

 
BIM-23206, a SSTR5 agonist, caused a slight inhibition of C6 proliferation at low concentrations, whereas a more sustained effect (below the basal value) was observed only when it was used in the micromolar range. At these concentrations, a higher efficacy in the inhibition of bFGF-induced DNA synthesis, compared with the SSTR1 and SSTR2 agonists, was observed, with an IC50 of 80 nM (Fig. 1EGo).

Interestingly, the activation of SSTR3 using the SSTR3 agonist L-796778 was unable to modulate bFGF-dependent [3H]thymidine incorporation (Fig. 1DGo), thus demonstrating that SSTR3 did not contribute to SST antiproliferative effects in these cells. This observation was confirmed in experiments in which the selective SSTR3 antagonist, the tetrahydro-β-carboline derivative BN-81658, was used to reverse the SST effects. This compound binds to the human SSTR3 with an IC50 of 1.58 nM, whereas the binding to all the other SSTRs is virtually absent (45). In these experiments, the antiproliferative effects of SST were not affected by SSTR3 blockade, clearly confirming the lack of contribution of this subtype in the transduction of the SST antiproliferative activity (data not shown).

Based on these data, we conclude that the effects of SST on C6 cell proliferation are mediated by the activation of three SSTR subtypes: SSTR1, SSTR2, and SSTR5.

To evaluate whether the antiproliferative effects of the SSTR agonists are dependent on the activation of the apoptotic process, we performed specific ELISA for cytosolic mono- and oligonucleosomes.

We did not detect the presence of cytosolic oligonucleosomes in C6 cell cultures treated with SST or SSTR1, -2, -3, and -5 selective agonists (1 µM for 24 h, data not shown). Conversely, the treatment with the proapoptotic agent staurosporine, used as positive control (100 nM for 24 h) resulted in about a 4-fold increase in apoptosis, as compared with basal values (data not shown).

Effect of SST and SSTR agonist subtypes on FSK-induced intracellular cAMP accumulation
All SSTR subtypes couple in an inhibitory fashion to adenylate cyclase via G protein-linked transduction pathway. To demonstrate that all the SSTR agonists and the respective receptors are functional, we tested their ability to inhibit cAMP accumulation induced by FSK pretreatment. As reported in Fig. 2Go, the exposure to FSK (100 nM) caused a marked increase in intracellular cAMP levels (+73% vs. basal) in C6 cells, whereas in the presence of SST or the agonists selective for SSTR1, -2, -3, and -5 (BIM-23745, BIM-23120, L-796778, and BIM-23206), the FSK-dependent cAMP accumulation was significantly reduced (about –70% vs. FSK stimulation values). These results demonstrated that all SSTRs expressed in C6 cells are functionally active and all the analogs tested behave as effective SSTR agonists.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 2. Effect of SST and its analogs on intracellular cAMP levels. Effect of SST and the selective agonists for SSTR1 (BIM-23745), SSTR2 (BIM-23120), SSTR3 (L-796778), and SSTR5 (BIM-23206) on intracellular cAMP levels were tested by an immunocompetitive enzyme-immunoassay system. C6 cells were treated with 100 nM FSK in the absence or in the presence of each drug (1 µM). All compounds inhibited FSK-stimulated intracellular cAMP accumulation. Values are mean ± SE of two independent experiments performed in triplicate. *, P < 0.01 vs. FSK-stimulated cAMP accumulation.

 
Intracellular mechanisms mediating the antiproliferative activity of individual SSTRs
The same SSTR agonists were used to analyze the intracellular mechanisms regulated by each receptor subtype. We previously reported that in C6 cells, SST caused inhibition of cell proliferation via a PTP-dependent inhibition of ERK1/2 phosphorylation (13) and the consequent up-regulation of p27Kip1 (46). To evaluate the role of PTPs in the intracellular signaling of each SSTR subtype, we pretreated C6 cells with the general PTP inhibitor vanadate and measured [3H]thymidine incorporation after bFGF stimulation, in the presence or absence of SSTR agonists.

As shown in Fig. 3Go, vanadate pretreatment completely abolished the inhibitory effects of BIM-23120, BIM-23745, and BIM-23206 (SSTR1, SSTR2, and SSTR5 agonists, respectively) on C6 DNA synthesis, clearly implicating the activation of a PTP in the antiproliferative signals activated by all these receptors. Again, the SSTR3 agonist was ineffective in the presence or absence of vanadate pretreatment.


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 3. Role of PTPs in the inhibition of DNA synthesis by individual SSTRs, shown by the effect of the blockade of PTP activity, induced by vanadate treatment, on SSTR agonist (1 µM) inhibition of bFGF-stimulated C6 cell proliferation, as assessed by [3H]thymidine incorporation. Values obtained in bFGF-treated cultures were taken as baseline (100%). Results are expressed as mean ± SE percent growth inhibition vs. bFGF stimulation of DNA synthesis. Data represent the average of three independent experiments performed in quadruplicate. **, P < 0.01 vs. bFGF-induced DNA synthesis; °°, P < 0.01 vs. SSTR agonists-inhibited bFGF-induced DNA synthesis.

 
Thus, we directly evaluated the capability of each SSTR subtype to activate PTP{eta}, the main effector of SST antiproliferative activity in C6 cells (13). For this purpose, we measured PTP{eta} specific activity in a spectrophotometric assay (20), after immunoprecipitation of this PTP from control (untreated) or SST agonist-treated cells (Fig. 4AGo). Equal PTP{eta} protein content in the immunoprecipitates was demonstrated in Western blot experiments, as reported (25) (Fig. 4BGo).


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 4. Regulation of PTP{eta} activity by individual SSTR activation. A, PTP{eta} activity in control (basal) or SSTR agonist-treated cells as assessed. PTP{eta} specific activity was evaluated after immunoprecipitation of control or SSTR agonist-treated C6 cells using the synthetic substrate pNPP in a spectrophotometric analysis. **, P < 0.01 vs. basal value. SST treatment (100 nM, 30 min) increased PTP{eta} activity about four times. The SSTR1 agonist BIM-23745 had a similar effect, whereas the SSTR2- and SSTR5-selective compounds BIM-23120 and BIM-23206 showed a slightly lower effect (+302 and +150% over basal activity). Activation of SSTR3 by L-796778 or the inhibition of SSTR3 by BN-81658 (a nonpeptidic selective SSTR3 antagonist), in the presence of SST stimulation, were ineffective. B, Western blot (Wb) experiments were performed to demonstrate equal PTP{eta} protein content in aliquots of proteins immunoprecipitated (Ipt) to measure PTP activity.

 
SST treatment (100 nM, 30 min) increased PTP{eta} activity about four times. The SSTR1 agonist BIM-23745 had a similar effect (+407%), whereas the SSTR2 selective compound BIM-23120 showed a slight lower effect (+302% over basal activity) (Fig. 4AGo). The SSTR5 agonist BIM-23206 was also able to induce PTP{eta} activity but to a significantly lower level (+150%), in agreement with the lower efficacy in the inhibition of cell proliferation observed at the concentration of 100 nM (see Fig. 1EGo). The lack of effects of SSTR3 on cell proliferation was paralleled by its absence of effects on PTP{eta} activity measured either treating the cells with the selective agonist L-796778 (+30% over basal) or cotreating the cells with SST and the selective SSTR3 antagonist BN-81658 (–13% of SST-stimulated PTP activity, Fig. 4AGo). Taken together, these findings demonstrate the direct regulation of PTP{eta} activity by SST and selective SSTR1, -2, and -5 agonists.

We previously demonstrated that ERK1/2 represents one of the major molecular targets of PTP{eta} to induce cell growth arrest. Thus, we evaluated the effects of SST and SSTR agonists on ERK1/2 activation induced by bFGF. Our data show that SST (Fig. 5AGo, left panel) and BIM-23745, BIM-23120, and BIM-23206 (Fig. 5AGo, right panel) significantly reduced bFGF-dependent ERK1/2 phosphorylation. As expected from the previous experiments, L-796778 did not inhibit the phosphorylation/activation of ERK1/2 induced by bFGF (Fig. 5AGo, right panel). All these effects were quantified by densitometric analysis, as reported in Fig. 5BGo.


Figure 5
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 5. SSTR agonists induce inhibition of ERK1/2 activity in C6 cells. A, Representative experiments showing the effects of SSTR agonists on bFGF-induced ERK1/2 activation. ERK1/2 phosphorylation in C6 glioma cells was measured in Western blot experiments using phosphospecific antibodies (upper panels). Equal loading of proteins was demonstrated by using a total ERK1/2 antibody in a parallel gel using the same cell lysates (lower panels). SST (left panel) and the selective agonists of SSTR1 (BIM-23745), SSTR2 (BIM-23120), and SSTR5 (BIM-23206) significantly inhibited ERK1/2 phosphorylation, whereas the activation of SSTR3 (L-796778) was ineffective. Similar results were obtained in three independent experiments. B, Quantification of the effects of SSTR agonists on bFGF-induced ERK1 and ERK2 activation by densitometric analysis of three independent Western blot determinations.

 
Thus, we suggest that SSTR1, -2, and -5 are able to inhibit C6 cell proliferation, via the activation of PTP{eta} and the subsequent dephosphorylation of ERK1/2.

Synergism among the different SSTR subtypes in the regulation of C6 cell proliferation
In both cell proliferation and biochemical experiments, SST constantly showed a higher efficacy than the receptor selective agonists. Thus, in consideration of the simultaneous activation of all the SSTR subtypes induced by the natural ligand and the reported functional interaction (47, 48) and heterodimerization processes between the different SSTR subtypes (49, 50), we tested whether bifunctional agonists, or the cotreatment with two selective compounds, could better mimic SST effects. All these results, obtained after the calculation of both the IC50 (drug potency) and the maximal inhibition of bFGF stimulation of DNA synthesis (drug efficacy), are summarized in Table 2Go.

In a first series of dose-response experiments (0.1 pM to 10 µM), we compared the antiproliferative effects of the combined treatment with BIM-23745 and BIM-23120 with those of the two individual agonists and with the effects of BIM-23704, a compound able to activate both SSTR1 and SSTR2 (see Table 1Go). In fact, these subtypes are often coexpressed in many normal and tumor cell types, including gliomas (13, 51).

The combined activation of these two receptors caused an increase in the efficacy of the antiproliferative effects when compared with SSTR1 and -2 agonists alone, displaying an IC50 intermediate between that observed with the selective agonists alone (BIM-23704 at 1.5 nM and BIM-23745+BIM-23120 at 2.3 nM). Both potency and efficacy in inhibiting DNA synthesis were slightly higher for the biselective agonist BIM-23704 than using a combined treatment with SSTR1- and SSTR2-specific compounds (Table 2Go).

Similar experiments were performed activating simultaneously SSTR2 and SSTR5. These experiments were particularly relevant because the SST agonists most widely used in humans, octreotide (OCT) and lanreotide, display high affinity for these two SSTR subtypes (1). Interestingly, also in these experiments, the combined treatment with the two agonists (BIM-23120+BIM-23206), with the biselective compound BIM-23190, or with the clinically used drug OCT showed a similar response and, more importantly, the data obtained showed a complete dominance of the SSTR5-like effect (high efficacy and low potency) over the SSTR2 response (high potency and low efficacy). In fact, all the treatments (BIM-23190, BIM-23120+BIM-23206, and OCT) caused a dramatic right-bound shift of the dose-response curve and lowered the IC50, with values close to those induced by the pure SSTR5 agonist BIM-23206. On the other hand, we observed an increase in the maximal inhibition at the highest concentrations tested, compared with the SSTR2-selective compound, and thus resembling those induced by the SSTR5 agonist alone (Table 2Go).

Finally, to further characterize the role of each receptor subtype in SST activity, we used the bispecific SSTR1/SSTR2 agonist BIM-23704 in combination with the SSTR5-preferential compound BIM-23206. The combined activation of the three receptors induced a marked antiproliferative effect characterized by an IC50 (0.1 nM) similar to that observed with the SST. Indeed, both potency and efficacy were only slightly lower for the combined treatment with SSTR1+2- and SSTR5-specific compounds compared with SST treatment (Table 2Go).

Then we verified whether the effects of the SST analog OCT, which interacts with SSTR2 and -5 (and with low affinity to SSTR3, see Table 1Go) can be ascribed to the same intracellular signaling depicted with the other selective agonists. OCT (1 µM) exhibited a suppression of cAMP levels induced by FSK (100 nM) at levels similar to those observed with the preferential SSTR2, -3, and -5 agonists (data not shown).

Next, we investigated whether OCT was able to induce a PTP-dependent inhibition of ERK1/2 phosphorylation (Fig. 6Go). Thus, we evaluated the effects of OCT treatment on bFGF-induced ERK1/2 activation in the presence and in absence of the PTP inhibitor vanadate. Our results show that OCT significantly inhibited bFGF-dependent ERK1/2 phosphorylation at the concentrations of 1 and 10 µM (Fig. 6AGo). Moreover, pretreatment with vanadate (30 µM) significantly reversed the inhibitory activity of OCT (Fig. 6AGo). These effects were quantified by densitometric analysis and reported in Fig. 6BGo.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 6. OCT induces inhibition of ERK1/2 activity in C6 cells. A, Representative experiments showing the effects of OCT (1 and 10 µM) on bFGF-induced ERK1/2 activation in the absence and in presence of the inhibition of PTP activity, induced by vanadate pretreatment. ERK1/2 phosphorylation in C6 glioma cells was evaluated in Western blot experiments using phosphospecific antibodies (upper panel). Equal loading of proteins was demonstrated by using a total ERK1/2 antibody in a parallel gel using the same cell lysates (lower panel). Similar results were obtained in three independent experiments. B, Quantification of the effects of OCT on bFGF-induced ERK1 and ERK2 activation by densitometric analysis of three independent Western blot determinations.

 
These data confirmed that the activity of PTPs is required to induce the antiproliferative effects of OCT and to block the ERK1/2 phosphorylation induced by the proliferative stimulus of bFGF, as previously demonstrated for SST and its analogs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the cloning of the SSTR subfamily, a number of studies addressed the issue of the identification of a specific intracellular signaling for each receptor subtype and how these biochemical events may correlate with specific biological functions. Although the results obtained to date demonstrated that the specific response resides more in the cell types analyzed than in different responses of individual SSTR subtypes, it is important to consider that most studies were performed using transfected receptors. In fact, this experimental setting, due to the expression (and in most cases overexpression) in heterologous cells may have a nonnatural coupling, not necessarily reflecting the transduction signaling occurring in cells natively expressing these receptors.

We took advantage of the characterization of the antiproliferative effects and the biochemical correlates induced by SST that we performed in the C6 rat glioma line (13). We demonstrated that in these cells, SST dose-dependently reduced C6 cell proliferation via an inhibition of ERK1/2 phosphorylation directly operated by the tyrosine phosphatase PTP{eta}. Thus, we aimed to evaluate the specific contribution of each of the SSTRs expressed in these cells (namely SSTR1, -2, -3, and -5) in the cytostatic effects of SST, the identification of specific intracellular pathways responsible of such an effect, and the possible interaction among the different SSTR subtypes.

The first result we got from our study is that three of four SSTRs natively expressed in C6 cells, when selectively activated, are able to induce a statistically significant inhibition of cell proliferation in vitro. The involvement of multiple SSTRs in the antiproliferative activity of SST was also reported in neuroblastoma cells, in which BIM-23745 and BIM-23120 inhibited cell proliferation and MAPK activity, whereas BIM-23206 was less active (52).

In our study, only the selective activation of SSTR3 was devoid of antiproliferative effects. SSTR3 activation was previously reported to induce apoptosis in different cell types (39); however, it was also shown that in cells coexpressing SSTR3 and SSTR2, heterodimers are generated between these two receptors in which only SSTR2 is active (49). Thus, we cannot exclude that in C6 cells, SSTR3 is constitutively dimerized with SSTR2 and thus inactivated. However, SSTR3, as all the other receptor subtypes natively expressed in C6 cells, are functional because they respond to the exposure to analogs by inhibiting FSK-stimulated cAMP accumulation. The observation that the SSTR3-specific agonist L-796778 decreased cAMP accumulation in vitro indicates also that this compound is a functional agonist and that its inability to decrease cell proliferation in C6 cells is dependent only on the intracellular coupling of SSTR3.

Apoptosis was reported to be induced by SST in different cell lines (MCF-7 and AtT20) (53, 54) as well as in human somatotroph adenoma cells (12). In this process, the SSTRs involved are SSTR2 and SSTR3; SSTR2 acts in the absence of the activation of proapoptotic genes (i.e. p53 and Bcl2/Bax) although requiring PTPs at least in some of the cellular systems studied (12, 55), whereas the apoptotic mechanism activated by SSTR3 is PTP and p53/Bax dependent (39). However, the induction of apoptosis by SSTRs is still under discussion and discrepant reported data seem to be mainly dependent on the cell type studied (17, 56).

To identify a possible proapoptotic activity of SST, SSTR-selective agonist effects on DNA fragmentation was evaluated.

In our experimental conditions, C6 cells did not undergo apoptosis after treatment with SST or all the SSTR-selective agonists tested, including the SSTR2/5 bispecific drug OCT, thus supporting the hypothesis that the antiproliferative effects mediated by natively expressed SSTRs did not involve apoptotic cell death.

A number of previous studies reported that the heterologous expression of individual SSTRs in SSTR-negative cells may induce different intracellular signaling to regulate cell functioning (57, 58, 59, 60). Interestingly, when we analyzed the signaling of natively expressed SSTR in the same cellular background, all the effective receptors appeared to act through the same intracellular mechanisms as we previously detailed in these cells for SST (13). Indeed, the inhibition of DNA synthesis induced by agonists for SSTR1, -2, and -5 was completely reversed by preincubating the cells with the PTP inhibitor vanadate, and the activation of each of these subtypes increased PTP{eta} activity and inhibited ERK1/2 phosphorylation. Although we previously showed the capability of SSTR1 to activate PTP{eta} (38), this is the first report showing that this PTP represents also an intracellular effector for SSTR2 and SSTR5, at least in this cell type. Moreover, although the involvement of other specific PTPs (namely SHP-1) (22) was suggested for SSTR2, as far as SSTR5, contradictory results were obtained using SSTR5-transfected CHO cells (58, 61), and thus, this is one of the first studies in which a specific PTP was shown to be coupled to native SSTR5. In C6 cells, as well as in CHO-K1 cells transfected with SSTR1, the treatment with a SSTR1-selective agonist resulted in a complex interplay of PTPs (SHP-2) and tyrosine kinases (Janus kinase 2 and src) leading to PTP{eta} activation (38). Further studies will be required to verify whether also the SSTR2- and SSTR5-mediated activation of PTP{eta} requires the same intracellular pathway.

In our experimental model, the analysis of the effects of the single receptor in the same cell populations allowed us to identify a common intracellular pathway (the activation of PTP{eta} and the subsequent inhibition of ERK1/2 phosphorylation) responsible for the same biological effect (inhibition of DNA synthesis). Importantly, the observation that all these receptors induce the same antiproliferative response may justify the higher efficacy of SST (agonist of all the SSTRs) than the SSTR-selective compounds tested. In fact, only when we used a combined treatment with compounds able to activate SSTR1, -2, and -5 could we reproduce an effect comparable with that of the natural ligand.

As far as for the effects induced by the selective agonists for the single subtypes, different potencies and efficacies were observed on cell proliferation, although the same intracellular pathways were activated. This apparent discrepancy may reflect either the number of active receptors or their coupling efficiency or the intrinsic activity of the compounds. In fact, although the SSTR5 agonist BIM-23206 showed the highest efficacy (maximal inhibition), the SSTR2 agonist BIM-23120 displayed the highest potency (with an IC50 rank as follows: BIM-23120 >> BIM-23745 > BIM-23206).

Interestingly, comparing the dose-response curves of the selective agonists with that of SST, we identified, in the natural ligand effects, two distinct components as far as antiproliferative activity: a first one showing an IC50 of about 0.068 nM, resembling the effect of BIM-23120, and a second one with an IC50 of about 15 nM, which was much closer to that of BIM-23745 and BIM-23206. This observation suggests that the response induced in vitro by SST, showing a stronger effect than that dependent on the individual receptor subtypes, may be due to the cumulative activation of all the SSTRs expressed by C6 cells. To address this hypothesis we compared the dose-response curves of SST and BIM-23745, BIM-23120, and BIM-23206 with those obtained by the cotreatment with SSTR1+SSTR2 and SSTR2+SSTR5 agonists and with other compounds able to activate the two combinations of receptor subtypes (BIM-23704 or BIM-23190 and OCT). We chose to evaluate these SSTR subtype costimulation combinations because on one hand, SSTR1 and SSTR2 are the most frequently SSTRs detected in different tumor histotypes, including brain tumors (2, 4, 13, 51), and on the other, SSTR2 and SSTR5 represent the selective target of the clinically approved SST agonists lanreotide and OCT (1). Interestingly, the effect of the cotreatment with the two combinations of selective agonists gave a response similar to that induced by the respective biselective compounds, although showing a slighter lower efficacy and higher IC50. This observation represents important evidence that the results obtained are dependent more on the result from the simultaneous activation of the two receptors than from intrinsic properties of the compounds (i.e. partial vs. full agonists).

A partially different scenario was observed according to the different combination of SSTRs activated. The simultaneous activation of SSTR1 and SSTR2 was additive because the final biological response showed an increase in efficacy in comparison with that obtained after the single receptor activation, although showing an IC50 intermediate between that of BIM-23120 and BIM-23745. Conversely, the concomitant stimulation of SSTR2 and SSTR5 resulted in an almost complete SSTR5-dominant response that was characterized by maximal inhibition of cell proliferation and IC50 resembling those of BIM-23206 (high inhibition level of DNA synthesis but only at high concentrations of drugs).

In recent years, many studies addressed the functional interactions between SSTR subtypes and in particular between SSTR2 and SSTR5, because the combined activation of these receptors is responsible for the effects of the clinically approved SST analogs OCT and lanreotide. In particular, it was reported that there was a synergism between SSTR2 and SSTR5 on hormone secretion in which the inhibition of SSTR2 activity completely reversed also the SSTR5-dependent synergistic reduction of GH release but not the SSTR5 effects when this receptor was selectively activated (47). However, an inhibition of SSTR5 on SSTR2-mediated biological effects similar to that we observe here was more recently reported in AtT20 cells on intracellular Ca2+ oscillations (48). In that study, the attenuating regulation induced by SSTR5 on SSTR2 was proposed to be dependent on a retaining of SSTR2 in the membrane preventing its internalization (48). Although we did not address directly this issue, we cannot exclude such a mechanism also in our cells, considering that we did not observe a difference in the transduction systems activated by the SSTR subtypes analyzed. Our results are partially different from those reported by Feindt et al. (62) in which the antiproliferative activity of OCT was detected only for short incubation times. Indeed, we observed a significant inhibition at 16 h of treatment. However, in our experiments, we induced inhibition of DNA synthesis at higher concentrations than those used in that paper. In fact, in agreement with the previous report, also in our experiments, 1–10 nM concentrations of octreotide were ineffective after 16 h of treatment. Importantly, in that study the authors did not consider the role of SSTR5 that we believe may be important to reduce cell proliferation at higher agonist concentrations.

Another important issue, always observed in the in vitro studies, that still needs to find a definitive explanation, is the consistent higher efficacy of the native SST compared with the synthetic analogs. Although a definitive answer cannot be provided from our data, we cannot exclude a role for SSTR3 in such a response. Indeed, although the activation of this subtype per se did not influence C6 cell proliferation, SSTR3 may modulate the response of the other subtypes when costimulated by SST, due to its capability to heterodimerize with other SSTRs (49). Thus, at least on this parameter, the activation of all the SSTR subtypes expressed may be required to obtain the maximal inhibitory activity of SST.

In conclusion, we report that different SSTR subtypes (1 , 2 , and 5) are able to affect cell proliferation of C6 glioma cells in vitro through the activation of the same intracellular pathway (PTP{eta}-dependent inhibition of ERK1/2 phosphorylation) and that the simultaneous activation of all the subtypes seems to be required to obtain maximal effects.


   Acknowledgments
 
We gratefully thank Susan Rorher for providing us with L-796778 and Dr. Alessandro Massa for his invaluable technical and scientific support.


   Footnotes
 
Disclosure Statement: F.B. A.P., M.G., C.P. A.B., A.F., and T.F. have nothing to declare. M.D.C. is an employee of IPSEN.

First Published Online June 19, 2008

Abbreviations: FSK, Forskolin; OCT, octreotide; pNPP, para-nitro-phenylphosphate; PTP, phosphotyrosine phosphatase; SHP, Src-homology 2-domain-containing tyrosine phosphatase; SST, somatostatin; SSTR, SST receptor; TCA, trichloroacetic acid.

Received December 19, 2007.

Accepted for publication June 6, 2008.


   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. Reubi JC, Waser B, Schaer JC, Laissue JA 2001 Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. Eur J Nucl Med 28:836–846[CrossRef][Medline]
  3. Moller LN, Stidsen CE, Hartmann B, Holst JJ 2003 Somatostatin receptors. Biochim Biophys Acta 1616:1–84[Medline]
  4. Arena S, Barbieri F, Thellung S, Pirani P, Corsaro A, Villa V, Dadati P, Dorcaratto A, Lapertosa G, Ravetti JL, Spaziante R, Schettini G, Florio T 2004 Expression of somatostatin receptor mRNA in human meningiomas and their implication in in vitro antiproliferative activity. J Neurooncol 66:155–166[CrossRef][Medline]
  5. Siriwardana G, Bradford A, Coy D, Zeitler P 2006 Autocrine/paracrine regulation of breast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol Endocrinol 20:2010–2019[Abstract/Free Full Text]
  6. Hubina E, Nanzer AM, Hanson MR, Ciccarelli E, Losa M, Gaia D, Papotti M, Terreni MR, Khalaf S, Jordan S, Czirjak S, Hanzely Z, Nagy GM, Goth MI, Grossman AB, Korbonits M 2006 Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours. Eur J Endocrinol 155:371–379[Abstract/Free Full Text]
  7. Florio T, Thellung S, Corsaro A, Bocca L, Arena S, Pattarozzi A, Villa V, Massa A, Diana F, Schettini D, Barbieri F, Ravetti JL, Spaziante R, Giusti M, Schettini G 2003 Characterization of the intracellular mechanisms mediating somatostatin and lanreotide inhibition of DNA synthesis and growth hormone release from dispersed human GH-secreting pituitary adenoma cells in vitro. Clin Endocrinol (Oxf) 59:115–128[CrossRef][Medline]
  8. Florio T, Scorziello A, Fattore M, D'Alto V, Salzano S, Rossi G, Berlingieri MT, Fusco A, Schettini G 1996 Somatostatin inhibits PC Cl3 thyroid cell proliferation through the modulation of phosphotyrosine activity. Impairment of the somatostatinergic effects by stable expression of E1A viral oncogene. J Biol Chem 271:6129–6136[Abstract/Free Full Text]
  9. Tagliati F, Zatelli MC, Bottoni A, Piccin D, Luchin A, Culler MD, Degli Uberti EC 2006 Role of complex cyclin d1/cdk4 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro. Endocrinology 147:3530–3538[Abstract/Free Full Text]
  10. Guillermet-Guibert J, Saint-Laurent N, Davenne L, Rochaix P, Cuvillier O, Culler MD, Pradayrol L, Buscail L, Susini C, Bousquet C 2007 Novel synergistic mechanism for sst2 somatostatin and TNF{alpha} receptors to induce apoptosis: crosstalk between NF-{kappa}B and JNK pathways. Cell Death Differ 14:197–208[CrossRef][Medline]
  11. Cattaneo MG, Amoroso D, Gussoni G, Sanguini AM, Vicentini LM 1996 A somatostatin analogue inhibits MAP kinase activation and cell proliferation in human neuroblastoma and in human small cell lung carcinoma cell lines. FEBS Lett 397:164–168[CrossRef][Medline]
  12. Ferrante E, Pellegrini C, Bondioni S, Peverelli E, Locatelli M, Gelmini P, Luciani P, Peri A, Mantovani G, Bosari S, Beck-Peccoz P, Spada A, Lania A 2006 Octreotide promotes apoptosis in human somatotroph tumor cells by activating somatostatin receptor type 2. Endocr Relat Cancer 13:955–962[Abstract/Free Full Text]
  13. Massa A, Barbieri F, Aiello C, Arena S, Pattarozzi A, Pirani P, Corsaro A, Iuliano R, Fusco A, Zona G, Spaziante R, Florio T, Schettini G 2004 The expression of the phosphotyrosine phosphatase DEP-1/PTP{eta} dictates the responsivity of glioma cells to somatostatin inhibition of cell proliferation. J Biol Chem 279:29004–29012[Abstract/Free Full Text]
  14. Susini C, Buscail L 2006 Rationale for the use of somatostatin analogs as antitumor agents. Ann Oncol 17:1733–1742[Abstract/Free Full Text]
  15. Danesi R, Agen C, Benelli U, Paolo AD, Nardini D, Bocci G, Basolo F, Campagni A, Tacca MD 1997 Inhibition of experimental angiogenesis by the somatostatin analogue octreotide acetate (SMS 201-995). Clin Cancer Res 3:265–272[Abstract]
  16. Albini A, Florio T, Giunciuglio D, Masiello L, Carlone S, Corsaro A, Thellung S, Cai T, Noonan DM, Schettini G 1999 Somatostatin controls Kaposi’s sarcoma tumor growth through inhibition of angiogenesis. FASEB J 13:647–655[Abstract/Free Full Text]
  17. Florio T, Morini M, Villa V, Arena S, Corsaro A, Thellung S, Culler MD, Pfeffer U, Noonan DM, Schettini G, Albini A 2003 Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities. Endocrinology 144:1574–1584[Abstract/Free Full Text]
  18. Mentlein R, Eichler O, Forstreuter F, Held-Feindt J 2001 Somatostatin inhibits the production of vascular endothelial growth factor in human glioma cells. Int J Cancer 92:545–550[CrossRef][Medline]
  19. Florio T 2008 Molecular mechanisms of the antiproliferative activity of somatostatin receptors (SSTRs) in neuroendocrine tumors. Front Biosci 13:822–840[CrossRef][Medline]
  20. Pan MG, Florio T, Stork PJ 1992 G protein activation of a hormone-stimulated phosphatase in human tumor cells. Science 256:1215–1217[Abstract/Free Full Text]
  21. Buscail L, Delesque N, Esteve JP, Saint-Laurent N, Prats H, Clerc P, Robberecht P, Bell GI, Liebow C, Schally AV, et al 1994 Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2. Proc Natl Acad Sci USA 91:2315–2319[Abstract/Free Full Text]
  22. Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent N, Theveniau M, Nahmias C, Vaysse N, Susini C 1997 The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling. J Biol Chem 272:24448–24454[Abstract/Free Full Text]
  23. Reardon DB, Dent P, Wood SL, Kong T, Sturgill TW 1997 Activation in vitro of somatostatin receptor subtypes 2, 3, or 4 stimulates protein tyrosine phosphatase activity in membranes from transfected Ras-transformed NIH 3T3 cells: coexpression with catalytically inactive SHP-2 blocks responsiveness. Mol Endocrinol 11:1062–1069[Abstract/Free Full Text]
  24. Florio T, Rim C, Hershberger RE, Loda M, Stork PJ 1994 The somatostatin receptor SSTR1 is coupled to phosphotyrosine phosphatase activity in CHO-K1 cells. Mol Endocrinol 8:1289–1297[Abstract/Free Full Text]
  25. Florio T, Arena S, Thellung S, Iuliano R, Corsaro A, Massa A, Pattarozzi A, Bajetto A, Trapasso F, Fusco A, Schettini G 2001 The activation of the phosphotyrosine phosphatase {eta} (r-PTP{eta}) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation. Mol Endocrinol 15:1838–1852[Abstract/Free Full Text]
  26. Ferjoux G, Lopez F, Esteve JP, Ferrand A, Vivier E, Vely F, Saint-Laurent N, Pradayrol L, Buscail L, Susini C 2003 Critical role of Src and SHP-2 in sst2 somatostatin receptor-mediated activation of SHP-1 and inhibition of cell proliferation. Mol Biol Cell 14:3911–3928[Abstract/Free Full Text]
  27. Florio T 2008 Somatostatin/somatostatin receptor signalling: phosphotyrosine phosphatases. Mol Cell Endocrinol 286:40–48[CrossRef][Medline]
  28. Bousquet C, Delesque N, Lopez F, Saint-Laurent N, Esteve JP, Bedecs K, Buscail L, Vaysse N, Susini C 1998 sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1. J Biol Chem 273:7099–7106[Abstract/Free Full Text]
  29. Florio T, Yao H, Carey KD, Dillon TJ, Stork PJ 1999 Somatostatin activation of mitogen-activated protein kinase via somatostatin receptor 1 (SSTR1). Mol Endocrinol 13:24–37[Abstract/Free Full Text]
  30. Zhang L, Martelli ML, Battaglia C, Trapasso F, Tramontano D, Viglietto G, Porcellini A, Santoro M, Fusco A 1997 Thyroid cell transformation inhibits the expression of a novel rat protein tyrosine phosphatase. Exp Cell Res 235:62–70[CrossRef][Medline]
  31. Borges LG, Seifert RA, Grant FJ, Hart CE, Disteche CM, Edelhoff S, Solca FF, Lieberman MA, Lindner V, Fischer EH, Lok S, Bowen-Pope DF 1996 Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells. Circ Res 79:570–580[Abstract/Free Full Text]
  32. Keane MM, Lowrey GA, Ettenberg SA, Dayton MA, Lipkowitz S 1996 The protein tyrosine phosphatase DEP-1 is induced during differentiation and inhibits growth of breast cancer cells. Cancer Res 56:4236–4243[Abstract/Free Full Text]
  33. Osborne JM, den Elzen N, Lichanska AM, Costelloe EO, Yamada T, Cassady AI, Hume DA 1998 Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS. J Leukoc Biol 64:692–701[Abstract]
  34. Kellie S, Craggs G, Bird IN, Jones GE 2004 The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation. J Cell Sci 117:609–618[Abstract/Free Full Text]
  35. Florio T, Scorziello A, Thellung S, Salzano S, Berlingieri MT, Fusco A, Schettini G 1997 Oncogene transformation of PC Cl3 clonal thyroid cell line induces an autonomous pattern of proliferation that correlates with a loss of basal and stimulated phosphotyrosine phosphatase activity. Endocrinology 138:3756–3763[Abstract/Free Full Text]
  36. Trapasso F, Iuliano R, Boccia A, Stella A, Visconti R, Bruni P, Baldassarre G, Santoro M, Viglietto G, Fusco A 2000 Rat protein tyrosine phosphatase {eta} suppresses the neoplastic phenotype of retrovirally transformed thyroid cells through the stabilization of p27(Kip1). Mol Cell Biol 20:9236–9246[Abstract/Free Full Text]
  37. Iuliano R, Trapasso F, Le Pera I, Schepis F, Sama I, Clodomiro A, Dumon KR, Santoro M, Chiariotti L, Viglietto G, Fusco A 2003 An adenovirus carrying the rat protein tyrosine phosphatase {eta} suppresses the growth of human thyroid carcinoma cell lines in vitro and in vivo. Cancer Res 63:882–886[Abstract/Free Full Text]
  38. Arena S, Pattarozzi A, Massa A, Esteve JP, Iuliano R, Fusco A, Susini C, Florio T 2007 An intracellular multi-effector complex mediates somatostatin receptor 1 activation of phospho-tyrosine phosphatase {eta}. Mol Endocrinol 21:229–246[Abstract/Free Full Text]
  39. Sharma K, Patel YC, Srikant CB 1996 Subtype-selective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3. Mol Endocrinol 10:1688–1696[Abstract/Free Full Text]
  40. Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor F, Mitra SW, Degrado SJ, Shu M, Klopp JM, Cai SJ, Blake A, Chan WW, Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM 1998 Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 282:737–740[Abstract/Free Full Text]
  41. Porcile C, Stanzione S, Piccioli P, Bajetto A, Barbero S, Bisaglia M, Bonavia R, Florio T, Schettini G 2003 Pyrrolidinedithiocarbamate induces apoptosis in cerebellar granule cells: involvement of AP-1 and MAP kinases. Neurochem Int 43:31–38[CrossRef][Medline]
  42. Florio T, Pan MG, Newman B, Hershberger RE, Civelli O, Stork PJ 1992 Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity. J Biol Chem 267:24169–24172[Abstract/Free Full Text]
  43. Grimaldi M, Florio T, Schettini G 1997 Somatostatin inhibits interleukin 6 release from rat cortical type I astrocytes via the inhibition of adenylyl cyclase. Biochem Biophys Res Commun 235:242–248[CrossRef][Medline]
  44. Dunphy WG, Kumagai A 1991 The cdc25 protein contains an intrinsic phosphatase activity. Cell 67:189–196[CrossRef][Medline]
  45. Poitout L, Roubert P, Contour-Galcera MO, Moinet C, Lannoy J, Pommier J, Plas P, Bigg D, Thurieau C 2001 Identification of potent non-peptide somatostatin antagonists with sst3 selectivity. J Med Chem 44:2990–3000[CrossRef][Medline]
  46. Massa A, Barbieri F, Aiello C, Iuliano R, Arena S, Pattarozzi A, Corsaro A, Villa V, Fusco A, Zona G, Spaziante R, Schettini G, Florio T 2004 The phosphotyrosine phosphatase {eta} mediates somatostatin inhibition of glioma proliferation via the dephosphorylation of ERK1/2. Ann NY Acad Sci 1030:264–274[CrossRef][Medline]
  47. Ren SG, Taylor J, 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]
  48. Ben-Shlomo A, Wawrowsky KA, Proekt I, Wolkenfeld NM, Ren SG, Taylor J, Culler MD, Melmed S 2005 Somatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion. J Biol Chem 280:24011–24021[Abstract/Free Full Text]
  49. Pfeiffer M, Koch T, Schroder H, Klutzny M, Kirscht S, Kreienkamp HJ, Hollt V, Schulz S 2001 Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst3 receptor function by heterodimerization with sst2A. J Biol Chem 276:14027–14036[Abstract/Free Full Text]
  50. Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC 2000 Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288:154–157[Abstract/Free Full Text]
  51. Feindt J, Becker I, Blomer U, Hugo HH, Mehdorn HM, Krisch B, Mentlein R 1995 Expression of somatostatin receptor subtypes in cultured astrocytes and gliomas. J Neurochem 65:1997–2005[Medline]
  52. Cattaneo MG, Taylor JE, Culler MD, Nisoli E, Vicentini LM 2000 Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells. FEBS Lett 481:271–276[CrossRef][Medline]
  53. Srikant CB 1995 Cell cycle dependent induction of apoptosis by somatostatin analog SMS 201-995 in AtT-20 mouse pituitary cells. Biochem Biophys Res Commun 209:400–406[CrossRef][Medline]
  54. Sharma K, Srikant CB 1998 G protein coupled receptor signaled apoptosis is associated with activation of a cation insensitive acidic endonuclease and intracellular acidification. Biochem Biophys Res Commun 242:134–140[CrossRef][Medline]
  55. Guillermet J, Saint-Laurent N, Rochaix P, Cuvillier O, Levade T, Schally AV, Pradayrol L, Buscail L, Susini C, Bousquet C 2003 Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis. Proc Natl Acad Sci USA 100:155–160[Abstract/Free Full Text]
  56. Ferone D, Pivonello R, Van Hagen PM, Dalm VA, Lichtenauer-Kaligis EG, Waaijers M, Van Koetsveld PM, Mooy DM, Colao A, Minuto F, Lamberts SW, Hofland LJ 2002 Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes. Am J Physiol Endocrinol Metab 283:E1056–E1066
  57. Hou C, Gilbert RL, Barber DL 1994 Subtype-specific signaling mechanisms of somatostatin receptors SSTR1 and SSTR2. J Biol Chem 269:10357–10362[Abstract/Free Full Text]
  58. Buscail L, Esteve JP, Saint-Laurent N, Bertrand V, Reisine T, O'Carroll AM, Bell GI, Schally AV, Vaysse N, Susini C 1995 Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms. Proc Natl Acad Sci USA 92:1580–1584[Abstract/Free Full Text]
  59. Chen L, Tashjian Jr AH 1999 Identification of distinct signalling pathways for somatostatin receptors SSTR1 and SSTR2 as revealed by microphysiometry. Cell Signal 11:499–505[CrossRef][Medline]
  60. Arena S, Pattarozzi A, Corsaro A, Schettini G, Florio T 2005 Somatostatin receptor subtype-dependent regulation of nitric oxide release: involvement of different intracellular pathways. Mol Endocrinol 19:255–267[Abstract/Free Full Text]
  61. Sharma K, Patel YC, Srikant CB 1999 C-terminal region of human somatostatin receptor 5 is required for induction of Rb and G1 cell cycle arrest. Mol Endocrinol 13:82–90[Abstract/Free Full Text]
  62. Feindt J, Mentlein R, Krisch B 1997 Time-dependent influence of the somatostatin analogue octreotide on the proliferation of rat astrocytes and glioma cells. Brain Res 746:309–313[CrossRef][Medline]
  63. Jaquet P, Gunz G, Saveanu A, Dufour H, Taylor J, Dong J, Kim S, Moreau JP, Enjalbert A, Culler MD 2005 Efficacy of chimeric molecules directed toward multiple somatostatin and dopamine receptors on inhibition of GH and prolactin secretion from GH-secreting pituitary adenomas classified as partially responsive to somatostatin analog therapy. Eur J Endocrinol 153:135–141[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
F. Barbieri, A. Pattarozzi, M. Gatti, C. Aiello, A. Quintero, G. Lunardi, A. Bajetto, A. Ferrari, M. D. Culler, and T. Florio
Differential efficacy of SSTR1, -2, and -5 agonists in the inhibition of C6 glioma growth in nude mice
Am J Physiol Endocrinol Metab, November 1, 2009; 297(5): E1078 - E1088.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barbieri, F.
Right arrow Articles by Florio, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barbieri, F.
Right arrow Articles by Florio, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals