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Endocrinology Vol. 144, No. 4 1574-1584
Copyright © 2003 by The Endocrine Society

ARTICLE

Somatostatin Inhibits Tumor Angiogenesis and Growth via Somatostatin Receptor-3-Mediated Regulation of Endothelial Nitric Oxide Synthase and Mitogen-Activated Protein Kinase Activities

T. Florio, M. Morini, V. Villa, S. Arena, A. Corsaro, S. Thellung, M. D. Culler, U. Pfeffer, D. M. Noonan, G. Schettini and A. Albini

Farmacologia e Neuroscienze (T.F., V.V., S.A., A.C., S.T., G.S.), Progressione Neoplastica (M.M., D.M.N.) e Oncologia Molecolare (U.P., A.A.), Istituto Nazionale per la Ricerca sul Cancro and Dipartimento di Biologia Oncologia e Genetica (T.F., V.V., S.A., A.C., S.T., G.S.), Sezione di Farmacologia, Università di Genova, 16132 Genova, Italy; and Biomeasure Inc. (M.D.C.), Milford, Massachusetts 01757-3650

Address all correspondence and requests for reprints to: Gennaro Schettini, Farmacologia e Neuroscienze, Istituto Nazionale per la Ricerca sul Cancro (IST) c/o Centro Biotecnologie Avanzate, Largo R. Benzi 10, 16132 Genova, Italy. E-mail: schettini{at}cba.unige.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin was reported to inhibit Kaposi’s sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASCULARIZATION SEEMS TO be a crucial process both for tumor growth and for metastatic dissemination (1), with acquisition of an angiogenic potential (the angiogenic switch) being a necessary step in tumor progression (2). The angiogenic process can be summarized as a series of distinct steps initiated through local stimulation of endothelial cells by the synthesis and secretion of a range of different cytokines and growth factors by tumor and inflammatory cells. The stimulated endothelial cells lose contact inhibition, breach their own basement membrane, and migrate toward the source of the stimulus, proliferating and finally differentiating to organize new vessels (3). In physiological angiogenesis, the balance of the production of angiogenic and antiangiogenic factors regulates the process and ensures both adequate vascularization and homeostasis. During tumor development, this balance is constantly altered in favor of an ongoing angiogenic process in the growing tumor mass (1, 3). The observations that tumor vascularization is necessary for tumor growth, progression, and metastasis and that tumor angiogenesis is a poorly regulated process involving normal host cells suggested that angiogenesis may be a primary target in therapy of numerous cancer types.

Numerous compounds with antiangiogenic properties acting at various levels in the angiogenic cascade have been reported to be effective in the inhibition of tumor development, and some have been reported to induce tumor regression (1, 4, 5, 6). Somatostatin, a broadly distributed cyclic peptide, was initially described as a powerful inhibitor of the secretion and activity of different pituitary and gastrointestinal hormones (7). More recently, somatostatin has been reported to be an effective antiproliferative agent for different epithelial and neuroendocrine tumors (8, 9). Somatostatin effects are mediated through the activation of a family of at least five G-protein-coupled receptors able to transduce a variety of intracellular signals (10). Somatostatin receptors (SSTRs) have been reported to inhibit cAMP formation and modulate Ca2+ and K+ channel activities (10). It has been demonstrated that different SSTR subtypes are able to induce phosphotyrosine phosphatase (PTP) activity, proposed to be responsible for the direct antiproliferative activity of this peptide in different normal and transformed cells (11, 12, 13, 14). It has been proposed that tumors of endocrine origin may respond to both a direct antiproliferative activity of somatostatin as well as to inhibitory effects mediated by reduced growth factor release (for example, IGF-1) caused by the endocrine activity of somatostatin (9). Because SSTR expression has also been detected, along with somatostatin antitumor activity, in nonendocrine tumors (15, 16), this peptide has been proposed as a major endogenous inhibitor of cell proliferation. However, the molecular mechanisms for the broad antitumor activity of somatostatin have not been completely elucidated.

Kaposi’s sarcoma (KS) is a highly vascularized lesion frequently associated with AIDS. The products of KS cells are strongly angiogenic in vivo (17) and induce endothelial cell migration and invasion in vitro (18). The HIV tat (transactivator) gene product also shows a potent angiogenic activity (19) mediated by specific binding and activation of the KDR receptor for vascular endothelial growth factor (VEGF) (20) and via a chemokine-like activity (21). We have previously reported that somatostatin was able to prevent growth of KS tumor xenografts in nude mice in a prevention protocol when the treatment was started the same day as tumor cell inoculation (22). The antitumor effect was induced through a PTP-dependent inhibition of the angiogenic process (22).

The aim of this present study is to compare the antitumor activity, in vivo, of somatostatin with that of known antitumor agents on established tumors in an intervention protocol and to analyze the role of specific SSTRs and intracellular pathways involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
KS-Imm cells isolated from a kidney transplanted immunosuppressed patient have been described previously (23). The human EAhy926 cell line derived from the fusion of human umbilical vein endothelial cells (HUVEC) with the A549 cell line has been shown to have an endothelial-like phenotype (24) that is maintained over time (25). Bovine artery endothelial cells (BAEC) were purchased from the ATCC (Rockville, MD). All cells were maintained in DMEM supplemented with 10% fetal calf serum (Invitrogen, Paisley, Scotland, UK), glutamine (300 µg/ml), penicillin (100 U/ml), and streptomycin (50 µg/ml), at 37 C in a 5%-CO2 incubator.

Tumor growth in vivo
KS-Imm cells (5 x 106 cells) were mixed with liquid matrigel, to a final vol of 250 µl at 4 C, and injected sc into the flanks of nude (nu/nu) mice. After 12 d, when tumors were clearly measurable, mice were randomized into four different groups, of nine animals each, with similar tumor masses. Group 1 received peritumor saline solution, twice a day; group 2 received a peritumor injection of 50 µg somatostatin (Calbiochem, San Diego, CA) sc, twice daily; group 3 received 1 mg/kg adriamycin (Sigma, Milano, Italy) iv in a single administration; group 4 received both adriamycin and somatostatin treatments as above. The animals were housed in pathogen-free conditions; mice were weighed and tumor growth was monitored by measurement of the tumor size every day (26). After 27 d, the animals were killed and the tumors were removed, photographed, and processed for histology.

In vivo angiogenesis
For the in vivo angiogenesis studies, we used the matrigel sponge model (27), as modified by Albini (17). Matrigel was purified from the EHS tumor as previously described (28). KS-conditioned medium (KS-CM), containing angiogenic factors (1x final concentration) and heparin (24 IU/ml), or TTH [Tat (100 ng/ml), TNF-{alpha} (2 ng/ml), and heparin (24 IU/ml)] were used as the angiogenic stimuli. Somatostatin (1 µM final concentration) and/or the other drugs used (BN81658, 100 nM; L-796778, 1 µM; nitro-L-arginine (NNA), 10 µM; sodium nitroprusside, 500 µM final concentrations) were mixed with either the KS-CM or TTH and added to unpolymerized liquid matrigel at 4 C, to a final vol of 600 µl. The matrigel suspension was slowly injected sc into the flanks of C57/bl6 male mice, using a cold syringe, where it quickly polymerizes to form a solid gel. Matrigel with buffer alone was used as negative control. After 4 d, gels were collected, weighed, and either minced and diluted in water [to measure the hemoglobin content with a Drabkin reagent kit (Sigma)], normalized to 100 mg of recovered gel and compared with a standard mouse blood hemoglobin curve [as previously reported (22)], or processed for histology.

Cell survival studies
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide]-assay. Mitochondrial function, as an index of cell viability, was evaluated by measuring the levels of mitochondrial dehydrogenase activity, using reduction of MTT as the substrate. The cleavage to a purple formazan product by dehydrogenase was quantified spectrophotometrically, measuring the absorbance at 570 nm, as previously reported (22).

Detection of apoptosis. This assay was performed using the Cell Death Detection ELISA Kit (Roche Diagnostics, Milano, Italy), as previously reported (29). Briefly, the cytosolic fraction of the cell lysates was placed into streptavidin-coated wells; a mixture of biotin-linked antihistone antibody and peroxidase-linked anti-DNA was added and incubated for 2 h. The antihistone antibody binds to the histone component of the oligonucleosomes, released from the nucleus by the apoptotic process, and fixes the immunocomplex to the well bottoms; the peroxidase linked anti-DNA antibody reacts with the DNA component of the nucleosomes. Washing steps remove the unfixed anti-DNA antibody, and the peroxidase activity is determined spectrophotometrically, with 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate) as the substrate (absorbance at 405 nm).

Cell proliferation studies
Cell proliferation was assessed using the [3H]-thymidine incorporation assay. Cells were plated at the density of 5 x 105 in 24-well plates. After 24 h, cells were serum-starved for 48 h. Subsequently, cells were treated with the test substances for 16 h and, in the last 4 h, were pulsed with 2 µCi/ml [3H]-thymidine (Amersham, Milano, Italy). DNA synthesis activity was measured as previously reported (30).

Determination of nitric oxide (NO) production
NO production was estimated by monitoring the conversion of L-arginine in L-citrulline, by measuring the production of [3H]-L-citrulline after incubation of cytosolic extracts with [3H]-L-arginine (Amersham), using a Stratagene kit according to the manufacturer’s instructions. Briefly, cells were mechanically homogenized in a buffer containing 24 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1 mM EGTA; the particulate fraction was pelleted (14,000 x g for 5 min); and the cytosolic fraction was collected and incubated (10 mg/ml) in a reaction buffer [25 mM Tris HCl (pH 7.4), 1 µM flavin adenine dinucleotide, 1 µM flavin adenine mononucleotide, 3 µM tetrahydrobiopterin] to which 1.2 mM nicotinamide adenine dinucleotide phosphate (reduced), 0.25 µCi [3H]-L-arginine, and 750 µM CaCl2 have been added. The reaction was carried out for 60 min and then stopped with 400 µl 50 mM HEPES (pH 5.5), 5 mM EDTA. The [3H]-citrulline formed, derived from NO production, was then recovered by chromatography and measured in a ß-counter.

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). Five micrograms of protein from each sample were size-fractionated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Milano, Italy). Membranes were blocked with 25 mM Tris-HCl (pH 7.4), 140 mM NaCl, 2.5 mM KCl, 0.1% Tween 20 containing 5% nonfat milk, and probed with rabbit antibodies raised against endothelial NO synthase (eNOS) and neuronal NOS (nNOS) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the phosphorylated or total forms of ERK1/2 (New England Biolabs, Inc., Beverly, MA). The secondary antibody was a horseradish peroxidase-linked antirabbit IgG antiserum (Amersham). The antibody–reactive bands were visualized by ECL (Amersham).

RT-PCR analysis
Total RNA was isolated using the acidic phenol technique (31), treated for 45 min with ribonuclease-free deoxyribonuclease, and then phenol/chloroform was extracted and ethanol precipitated. cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (U.S. Biological, Swampscott, MA) using oligo dT (16) primers (TIB-MOLBIOL, Genova, Italy). Ten nanograms of cDNA was subsequently used in the PCR for 35 cycles (1 min at 94 C, 1 min at 60 C, and 1 min at 72 C, followed by 7 min at 72 C). The sequences of the primers used were the following: SSTR1 = sense 5'255-tat gcc aag atg aag acg gcc-2753', antisense 5'650-ttg agc ggg ctc tgg cat gag-6303' (accession no. M81829); SSTR2 = sense 5'210-tat gcc aag atg aag acc atc-2303', antisense 5'602aga ttc acc tgg cca gtt gat-5823' (accession no. M81830); SSTR3 = sense 5'560-atg agcacc agc cac atg cag tgg-5803', antisense 5'-830gtt gag cac gta gaa ggg cat-8103' (accession no. M96738); SSTR4 = sense 5'868-ctt gat gcc acc gtc aac cac-8883', antisense 5'1160-gtc ctg gtg agg ggg atg cgc-11403' (accession no. D16826); SSTR5 = sense 5'586-tgt ggg cgg acg tgc agg agg-606-3', antisense 5'861-aga agt gta gag gcc ggc gga-8413' (accession no. L14865).

Expected lengths for the amplified products were the following: SSTR1 = 395 bp, SSTR2 = 392 bp, SSTR3 = 270 bp; SSTR4 = 292 bp, SSTR5 = 235 bp.

IGF1 assay
IGF1 was measured by double-antibody RIA using immunochemicals and tracer provided by Medgenix (Fleurus, Belgium). The sensitivity of the assay was 150 pg/ml; the intra- and interassay coefficients of variation were 6% and 7.5%, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Somatostatin effects on growth of established tumors in vivo
When injected sc in nude mice, KS-Imm cells form highly angiogenic and hemorrhagic tumors with a latency of about 7 d, becoming fully developed after 10–12 d (22). To analyze the effects of somatostatin on established tumors, KS-Imm cells were injected in nude mice, and the tumors were allowed to grow for 12 d before the treatments were started with either adriamycin or somatostatin.

The tumors in the saline-treated control mice continued to grow until the end of the observational period (Fig. 1AGo). Treatment with somatostatin (50 µg, twice daily) resulted in an essentially complete blockade of tumor growth and disease stabilization, with significant differences in tumor size (P < 0.001, two-way ANOVA), as compared with controls, from d 23 to the end of the experiment (Fig. 1AGo). A single bolus of adriamycin (1 mg/kg) caused a similar pattern of tumor growth inhibition that was not significantly different from the somatostatin-induced effects (Fig. 1AGo). A combination of adriamycin and somatostatin treatments did not show differences with those of either drug alone (data not shown). No differences were noted in animal body weights, indicating limited general toxicity caused by all the treatments (data not shown). Histological examination of the tumors showed extensive vascularization in the controls, with some necrotic areas (Fig. 1BGo). In the somatostatin-treated animals, the necrosis was far more pronounced and associated with an almost complete lack of vascularization (Fig. 1BGo). Adriamycin-treated mice also showed large necrosis areas in the tumor mass, although the angiogenic process seemed to be preserved (Fig. 1BGo).



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  		Figure 1. A, Growth of tumors after sc injection of 5 x 106 KS-Imm cells sham treated (circles), treated twice daily with somatostatin (squares), or after a single iv injection of adriamycin at the dose of 1 mg (triangles). Twelve days after injection of the cells, the tumor mass was measured, and the experimental groups (9 mice for each group) were assigned, using mice with a super-imposable cumulative mass. Average tumor sizes ± SEM are shown. B, Hematoxylin-and-eosin-stained sections of sham-treated KS-Imm tumors (Control), tumors treated with somatostatin or with adriamycin (1 mg/kg). The large areas of vascularization (*) found in control samples are absent in the somatostatin-treated samples, that show extensive necrosis areas. In the adriamycin-treated tumors, necrosis was also observed but without reduction in the vascularization of the tumors (*). Field sizes are 760 x 570 µm. C, Matrigel pellets removed from the angiogenesis assay after 4 d. Angiogenesis in the pellet is readily observed by the color intensity. Pellets were treated with Tat (100 ng/ml), TNF-{alpha} (2 ng/ml), and heparin (24 IU/ml); somatostatin (1 µM) and BN81658 (100 nM), as indicated. D, Quantitation of the angiogenic response in the matrigel pellets through measurement of the relative hemoglobin content (values in mg/dl). KS-CM, Supernatants of KS cell cultures (conditioned medium); SST, somatostatin (1 µM); BN81658 (100 nM); NNA, 10 µM NNA; SNP, 500 µM SNP. n = 11 for TTH, TTH+SST, TTH+SST+BN81658, and KS-CM; n = 6 for KS-CM+SST, KS-CM+SST+BN81658, and KS-CM+NNA; and n = 3 for SNP and KS-CM+SST+SNP. Statistically significant differences, with respect to the positive control (TTH or KS-CM) are indicated (*, P < 0.05; **, P < 0.01).

 
Somatostatin could control in vivo tumor growth by interfering with the GH-IGF1 axis. To verify whether the prolonged sc treatment with somatostatin, caused by a leak into the systemic circulation, might cause an inhibition of GH secretion that, in turn, may affect the KS tumor growth caused by a reduction in the circulating IGF1 levels, we evaluated, by RIA, the IGF1 serum concentrations in tumor-bearing mice treated for 21 d with saline or somatostatin (50 µg, twice a day, sc, peritumoral). IGF1 serum levels were not statistically different in saline- or somatostatin-treated animals (272.3 ± 34 vs. 274.6 ± 19 ng/ml, respectively).

Effects of somatostatin on KS-Imm and endothelial cells in vitro
The different histological pattern observed in somatostatin- and adriamycin-treated tumors correlated well with the expected cellular mechanisms of the antitumor effects of these drugs. Indeed, adriamycin caused KS-Imm apoptotic cell death in vitro that was dose- and time-dependent (Fig. 2Go, A and B); whereas somatostatin, in agreement with the lack of SSTRs reported previously (22), had no effect on KS-Imm cell growth in vitro (Fig. 2AGo). In contrast, somatostatin treatment inhibited the growth of different endothelial cell lines when KS-Imm (KS-CM), which contains a wide range of angiogenic factors, was used as a proliferation stimulus as a model for the tumor-dependent angiogenesis (22). In particular, somatostatin induced a dose-dependent inhibition of DNA synthesis in endothelial cells, with the immortalized EAhy926 showing an IC50 of about 100 nM (Fig. 3AGo), BAEC with an IC50 of 5 nM (Fig. 3BGo), and HUVEC with an IC50 of 1 nM (data not shown), indicating a higher potency on primary endothelial, as opposed to immortalized cells.



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  		Figure 2. A, KS-Imm cell growth in vitro in the presence of different concentrations of adriamycin ({blacksquare}) or somatostatin ({circ}), as assessed by the MTT assay. No significant differences were observed in the somatostatin-treated cells, whereas a significant reduction of viability was induced by adriamycin. B, Effect of different concentrations of adriamycin on the activation of the apoptotic process after 24 or 48 h of treatment, measured using the cell-death ELISA kit. Values obtained in the untreated cells were taken as baseline. The reported data represent the average of three independent experiments performed in quadruplicate. *, P < 0.05; and **, P < 0.01 vs. basal value.

 


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  		Figure 3. Effects of increasing molar doses (1–1000 nM) of somatostatin (Som) on KS-Imm (KS-CM)-stimulated EAhy926 cells (A) and BAEC (B) proliferation in vitro, as assessed by [3H]-thymidine incorporation. Som dose-dependently inhibited the DNA synthesis induced by the KS-CM, in both cell lines. Similar results were observed with HUVE cells (not shown). The reported data represent the average of three independent experiments performed in quadruplicate. *, P < 0.05; **, P < 0.01 vs. basal value.

 
A specific SSTR3 antagonist reverses somatostatin inhibition of endothelial cell growth in vitro
Previous studies indicated that SSTR3 was the predominant SSTR subtype in endothelial cells (22). To confirm a possible role of SSTR3 in the antiproliferation effects of somatostatin, we analyzed the ability of a novel nonpeptidic selective SSTR3 antagonist, BN81658, to reverse the inhibitory effects of somatostatin. This compound bound to the human SSTR3 with an IC50 of 1.58 nM, whereas the binding to all the other SSTRs was virtually absent (32). In both EAhy926 and BAE cells, the antiproliferative effects of somatostatin were abolished by the cotreatment with the SSTR3 antagonist BN81658, clearly indicating the involvement of this receptor subtype in transduction of the somatostatin effects (Fig. 4Go, A and B).



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  		Figure 4. SSTR3-selective antagonist BN81658 reverses the inhibition of somatostatin on the KS-CM-dependent cell proliferation. Effects on EAhy926 cells (A) and on BAE cells (B). Data are from measurement of the [3H]-thymidine incorporation. The reported data represent the average of three independent experiments performed in quadruplicate. **, P < 0.01 vs. basal value; °°, P < 0.01 vs. KS-CM values. In C and D, the pattern of SSTR mRNA expression in EAhy926 cells and BAEC, respectively, is shown using the RT-PCR technique.

 
Because the EAhy926 endothelial-like cells express only SSTR3, as assessed by RT-PCR analysis (Fig. 4CGo), this observation was expected. However, the role of this receptor subtype in the antiproliferative effects of somatostatin was confirmed by experiments using BAEC in which, although the BAEC express the SSTR1, SSTR3, and SSTR5 subtypes (Fig. 4DGo), the specific blockade of SSTR3 was able to revert the antiproliferative effects of somatostatin. These data confirm that the inhibition of angiogenesis through the regulation of endothelial cell proliferation may represent the main mechanism through which somatostatin controls KS growth in vivo, and suggest that somatostatin may affect the endothelial response to KS cell product stimulation through the activation of SSTR3.

SSTR3 has been reported to be the only SSTR subtype capable of transducing cytotoxic signals, causing apoptosis in SSTR3-transfected Chinese hamster ovary (CHO)-K1 cells (33). We observed that somatostatin activation of SSTR3 in endothelial cells resulted in very little (and inconstant) induction of apoptosis, as measured using a specific ELISA for cytosolic oligonucleosome production (maximum effect: +28%, after 2 d of treatment with 1 µM somatostatin, compared with +200% observed with adriamycin; see Fig. 2BGo). Thus, the somatostatin effects we observed in the endothelial cells seem to be mainly cytostatic.

Somatostatin effects on angiogenesis in vivo and reversal by BN81658
KS cell products or the HIV-associated factors TTH induce a potent angiogenic reaction in matrigel sponges implanted in vivo sc, with an intense infiltration of new vessels within 4 d, as readily seen in macroscopic examination (Fig. 1CGo). Addition of somatostatin (1 µM) to the matrigel containing either the combined treatment with TTH (Fig. 1CGo) or KS-CM almost completely blocked the angiogenic response. A quantitative estimation of the angiogenic response, by measurement of the hemoglobin content, showed that somatostatin significantly (P < 0.001) reduced the angiogenic response induced by both KS-CM and TTH stimuli (Fig. 1DGo). Coincubation with the SSTR3 antagonist BN81658 potently and significantly reversed the antiangiogenic effects of somatostatin in vivo in response to both TTH and KS-CM (Fig. 1Go, C and D). The key role of SSTR3 in the antiangiogenic activity of somatostatin in vivo was further confirmed using the selective SSTR3 agonist L-796778 (34). As reported in the Table 1Go, the compound L-796778 (1 µM) was able to inhibit the in vivo neoangiogenesis induced by KS-CM to a level comparable with that obtained with somatostatin in the same series of experiments. Moreover, the L-796778 antiangiogenic activity was reverted by coincubation with the SSTR3 antagonist BN81658, thus confirming the specificity of the effects observed.


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  		Table 1. Relative hemoglobin (Hb) content per 100 mg matrigel

 
Somatostatin inhibition of ERK1/2 activation
We then evaluated possible intracellular mechanisms that may mediate the SSTR3-dependent antiangiogenic activity of somatostatin. In particular, the effects of SSTR3 activation on two of the pathways that have been reported to be involved in the angiogenic process at endothelial cell level were investigated: ERK1/2 activation and NO production. Stimulation of EAhy926 cells with KS-CM for 10 min caused a substantial activation of ERK1/2 (Fig. 5AGo). This effect was clearly related to the proliferative activity of growth factors contained in the KS-CM, because the pretreatment with the MEK inhibitor, PD98059, completely reversed the induction of DNA synthesis induced by KS-CM (data not shown). Similarly, cotreatment with somatostatin (1 µM) also caused a parallel inhibition of proliferation (see Fig. 4AGo) and ERK1/2 phosphorylation/activation (Fig. 5AGo). The presence of the SSTR3 antagonist BN81658 again completely reversed the inhibitory activity of somatostatin also on ERK1/2 phosphorylation, resulting in a slight potentiation of ERK1/2 activity, compared with KS-CM alone (Fig. 5AGo). Similar results were obtained using purified VEGF as a stimulus. Somatostatin blocked VEGF-mediated ERK1/2 activation, an inhibition that was completely reversed by coincubation with the SSTR3 inhibitor BN81658 (Fig. 5AGo).



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  		Figure 5. A, Inhibition by somatostatin (1 µM) treatment of KS-CM and VEGF (100 nM) stimulated ERK1/2 activation in EAhy926 cells, as measured by Western blot using phospho-specific antibodies. The somatostatin effect was reversed by pretreatment with the SSTR3 antagonist, BN81658 (60 nM). B, Inhibition by somatostatin (1 µM) treatment of KS-CM stimulated ERK1/2 activation in BAEC, as measured by Western blot using phospho-specific antibodies. The somatostatin effect was reversed by pretreatment with the SSTR3 antagonist, BN81658 (60 nM). Parallel blots performed using proteins from the same cell lysates were hybridized with antibodies recognizing total ERK1/2, showing that the same amount of proteins was loaded in the gels (right panels). CTRL, Control.

 
BAE cells that express the SSTR 1, 3, and 5 subtypes (Fig. 4DGo) also showed the same pattern of results: somatostatin abolished KS-CM-induced ERK1/2 activation (Fig. 5BGo), which was almost completely reversed by the BN81658 compound. However, in these cells, the rebound in KS-CM-dependent ERK1/2 activation observed in EAhy926 cells in the presence of BN81658 did not occur.

Somatostatin inhibition of eNOS activation
NO, a second-messenger molecule that plays a pivotal role in angiogenesis, is produced by a family of NOS enzymes. The Ca2+/calmodulin-dependent enzymes nNOS and eNOS are constitutively expressed in muscle cells, fibroblasts, and various epithelial cells, in addition to neuronal and endothelial cells. Inducible NOS is an isoform of NO synthase whose activity is mainly regulated transcriptionally (35, 36). As expected, both EAhy926 and BAEC expressed high levels of eNOS, both at the mRNA level as detected by RT-PCR (data not shown) and at the protein level as detected by Western blot, whereas nNOS was not detected in either cell line (Fig. 6Go, A and B). In addition, inducible NOS was not detected in EAhy926 or BAEC cells, either under basal conditions or after stimulation with KS-CM (data not shown). Thus, eNOS activity seems to account for the majority of NO produced in both EAhy926 and BAE cells, as expected for endothelial cell lines. Given the role of NO in angiogenesis, we therefore examined the effects of somatostatin on eNOS activity.



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  		Figure 6. Western blot analysis of the expression of the constitutive NOS isoforms in EAhy926 (A) and BAE (B) cells. In both cell lines, eNOS (e), but not nNOS (n), was detected. Effect of somatostatin treatment on KS-CM stimulated eNOS activity and reversal by coincubation with the SSTR3 antagonist BN81658 (60 nM) in EAhy926 cells (C) and BAEC (D). In EAhy926 cells, the pretreatment with the MEK inhibitor PD98059 (10 µM) modified neither the eNOS activation induced by the KS-CM nor the inhibition by somatostatin (A). The reported data represent the average of three independent experiments performed in triplicate. **, P < 0.01 vs. basal value; °°, P < 0.01 vs. KS-CM values.

 
Treatment of EAhy926 cells with KS-CM significantly increased eNOS activity. Coadministration of somatostatin inhibited eNOS activity induced by KS-CM (-60%) (Fig. 6CGo). Blocking SSTR3 with BN81658 totally abolished somatostatin inhibition of NO production (Fig. 6CGo), whereas the MEK inhibitor, PD98059, did not affect the activation of eNOS by KC-CM or the somatostatin inhibition of eNOS activation (Fig. 6CGo). These data suggested that both MAPK and NO production seem to be involved in the angiogenic activity of the KS-CM and are both targeted by the intracellular signaling pathways of somatostatin in endothelial cells; however, they seem to be independent. This was further verified by evaluating the effects of somatostatin on KS-CM-dependent activation of MAPK in the presence of a high concentration of sodium nitroprusside (SNP, 500 µM), a NO donor that maintains an elevated NO concentration. Somatostatin inhibited the KS-CM activation of ERK 1/2, even in the presence of high NO concentrations, and SNP treatment per se did not activate MAPK (data not shown).

Treatment of BAEC with KS-CM also induced eNOS activation; similarly, this effect was totally blocked by somatostatin (Fig. 6DGo), and the somatostatin block was significantly reversed (about 80%) by BN81658 (Fig. 6DGo), again indicating a key role for SSTR3.

Role of eNOS inhibition in the antiangiogenic activity of somatostatin
The role of somatostatin inhibition of eNOS was also analyzed in the in vivo angiogenesis assay. Blocking eNOS by inclusion of NNA (10 µM), a powerful NOS inhibitor, in the matrigel inhibited the angiogenic activity of KS-CM to the same extent or greater than somatostatin (Fig. 1DGo). These data indicate that the production of NO is a necessary step for the generation of new vessels in vivo.

Interestingly, although no angiogenic effects were observed in vivo by the presence of the NO donor SNP alone, even at high concentrations (Fig. 1DGo), this compound, when added to matrigel containing KSCM and somatostatin, completely reverted somatostatin inhibition of angiogenesis in vivo (Fig. 1DGo). These data clearly indicate that although NO production alone is not sufficient for angiogenesis, suppression of KS-CM-induced NO production is a prerequisite for the antiangiogenic effects of somatostatin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Somatostatin has been proposed to be one of the most important endogenous regulators of cell proliferation (7, 10). Indeed, multiple mechanisms have been described for its antiproliferative activity, both in normal and tumor cells, mostly through induction of cytostatic effects. Somatostatin acts through the activation of a family of five G-protein-coupled seven-transmembrane receptors (10). However, we recently demonstrated that somatostatin may also interfere with SSTR-negative tumor formation through a pure antiangiogenic mechanism. Indeed, treatment with somatostatin prevented the growth of sc injected KS-Imm cells in mice, an experimental model of KS, although this human tumor-derived cell line does not express any subtype of SSTRs (22). Somatostatin effects were solely dependent on the inhibition of intratumoral endothelial cell proliferation and invasion and monocyte activation and migration (22).

In this paper, we extend these studies by evaluating the capability of the antiangiogenic properties of somatostatin to inhibit the growth of established KS tumors. Similar to that previously observed with treatment from the day of tumor cell injection (prevention), somatostatin markedly inhibited the growth of the established KS tumors (intervention), with an initial reduction of the tumor mass and subsequent disease stabilization for the duration of the observation period. The effects of somatostatin were equivalent to treatment with adriamycin, a powerful apoptotic in vitro and in vivo agent for Kaposi’s cells, which produced a similar pattern of tumor growth inhibition. In this study, we also excluded that the in vivo somatostatin antiproliferative activity may be mediated by an endocrine effect on the GH-IGF1 axis, because IGF1 levels were not changed after somatostatin treatment. Although the two compounds acted through different cell targets (KS cells for adriamycin and endothelial cells for somatostatin) and intracellular mechanisms (i.e. direct apoptotic effects for adriamycin and a pure antiangiogenic activity for somatostatin), combined treatment with somatostatin and adriamycin did not show additive effects. In addition, a higher-dose schedule for adriamycin (7.5 mg/kg) showed effects similar to somatostatin treatment (data not shown). These data indicate that treatment schedules used here produced a maximal growth inhibition in this model that could not be further enhanced by cytotoxic agents.

Different SSTR subtypes may play different roles in the physiological activity of somatostatin (7). The endothelial cell lines analyzed (EAhy926 and BAEC, see Fig. 4Go and HUVEC, data not shown) all express mRNA for SSTR3, either as the only expressed receptor subtype or in combination with other SSTRs, and are growth-arrested after somatostatin treatment, in vitro. It was reported that multiple SSTRs are able to inhibit in vitro cell proliferation, mainly acting synergistically. It is interesting to note, in this respect, that BAEC that express multiple SSTRs are much more sensitive to the somatostatin inhibition of cell proliferation than the EAhy926 cells that express only SSTR3. This observation suggests that also at endothelial cell level, a synergism between SSTRs may occur on the inhibition of cell proliferation. Addition of the SSTR3 antagonist, BN81658, to the matrigel pellets significantly reversed the antiangiogenic effects of somatostatin under both KS-CM- and TTH-stimulated conditions. Further, the pretreatment with BN81658 reversed the antiproliferative activity of somatostatin in vitro, even in cell lines that express multiple SSTR subtypes (i.e. BAEC). Although the possible involvement of other SSTR subtypes in the observed somatostatin effects cannot be excluded, these data clearly indicate that the activation of SSTR3 is necessary for the antiangiogenic actions of somatostatin.

Two main intracellular pathways seemed to be involved of the KS-CM-dependent endothelial cell proliferation that were both inhibited by somatostatin treatment: the activation of ERK1/2 and the production of NO. ERK1/2 is generally considered to play a fundamental role in proliferation in most normal or transformed cell systems, and NOS activation represents an important mediator of angiogenesis, in vivo (37). The cytostatic effects of somatostatin have been reported to either inhibit (14, 38) or activate (39, 40) ERK1/2, depending on the cell type, resulting in increased expression of CDK inhibitors (p27kip1 and p21cip1, respectively) and growth arrest. Our data indicate that somatostatin blocks proliferation through a SSTR3-dependent inhibition of ERK1/2. This is the first observation that the activation of endogenous SSTR3 is able to suppress phosphorylation/activation of ERK1/2. Previous in vitro studies demonstrated that somatostatin inactivated ERK1/2 through activation of both receptor-like and cytosolic PTPs (14, 41). The involvement of PTPs (and consequent ERK1/2 inactivation) in somatostatin action is consistent with previous in vivo data showing that the antiangiogenic activity of somatostatin is blocked by the PTP inhibitor vanadate (22). This activity is also consistent with the role of SSTR3 in somatostatin action in vitro, because a direct activation of the cytosolic PTP, SHP-2, by SSTR3 (42) and subsequent inactivation of the MAPK Kinase Kinase, Raf-1 (43), have also been demonstrated.

Upon ligand activation, CHO-K1 cells transfected with SSTR3 have been reported to respond by induction of apoptosis through a complex mechanism involving the wild-type tumor suppressor protein p53, the proapoptotic protein Bax (33), intracellular acidification, and the induction of an acidic endonuclease (44). We did not observe any significant induction of apoptosis after somatostatin treatment of the SSTR3-expressing EAhy926 cells, indicating that, in endothelial cells, somatostatin acts mainly through a cytostatic pathway, as reported also for other cell types (13, 45).

Interestingly, although the modulation of ERK1/2 by SSTR has been reported in many cell types, there are very few reports on the somatostatin effects on NO production. One report showed a direct activation of nNOS by somatostatin in transfected CHO cells through the modulation of SSTR2 (46). Although NO is now widely recognized as pivotal mediator of the angiogenic response, and a role for somatostatin as an antiangiogenic drug has been proposed (22, 47), we demonstrate here, for the first time, a direct inhibitory role of somatostatin in regulating eNOS activity, through an SSTR3-mediated mechanism.

The regulation of NO production and ERK1/2 activity by somatostatin seem to be independent processes. Somatostatin inhibited ERK1/2 activation even in the presence of the NO donor, SNP. Similarly, a MEK inhibitor did not modify the regulation of NO synthesis, indicating that these two metabolic pathways, activated by somatostatin, do not converge. In vivo, however, the antiangiogenic activity of somatostatin was completely abolished in the presence of SNP, indicating that reduction of NO levels is a necessary step for inhibition of angiogenesis. This observation is in keeping with previous data showing that NO is necessary for tumor angiogenesis (37). Our data suggest that, although NO production alone is not sufficient to induce new blood vessel formation in vivo, NOS inhibition completely blocks growth factor-induced angiogenesis. We observed here that NO alone is not sufficient to induce the activation of proliferation pathways such as ERK1/2. Further, most angiogenic factors, such as VEGF and basic fibroblast growth factor, in addition to inducing NO production, also activate ERK1/2 (48, 49). Our observations support the Garcia-Cardena and Folkman hypothesis (50) that, in the presence of growth factor stimulation, the vasodilating properties of NO are required for endothelial cell proliferation. Elongation and spreading of endothelia seem to represent a permissive event for endothelial cell mitosis and migration (51), processes likely requiring ERK1/2 activation. Somatostatin interference with both eNOS and ERK1/2 activities at the endothelial cell level indicates that it is a compound with a potent antiangiogenic potential.

In conclusion, our data show that the antiangiogenic properties of somatostatin are a potentially significant anticancer tool, even for SSTR-negative tumors such as KS. Moreover, these effects require the activation of SSTR3, which, in turn, transduces a signal that results in the inhibition of eNOS and ERK1/2, two key pathways of angiogenesis.


   Acknowledgments
 
We gratefully thank Prof. A. Barreca and Dr. Sidoti (University of Genova) for helping us in serum IGF1 measurements. We thank S. Carlone and L. Masiello for expert technical assistance in the initial in vivo studies. The compound L-796778 was kindly provided by Merck Research Laboratories.


   Footnotes
 
This work was supported by grants from the EU Contract QlG3-CT-1999-00908 (to G.S.), the Istituto Superiore di Sanità, Progetto AIDS (to G.S., A.A., and D.M.N.) and Italy-USA sulla Terapia dei Tumori (to A.A. and D.M.N.), the Italian Association for Cancer Research (to A.A., G.S., and D.M.N.), and the Minstero della Sanità, Progetto Finalizzato (to G.S., A.A., and D.M.N.). V. Villa is the recipient of a fellowship from the Italian Foundation for Cancer Research.

Abbreviations: BAEC, Bovine artery endothelial cells; CHO, Chinese hamster ovary; eNOS, endothelial nitric oxide synthase; HUVEC, human umbilical vein endothelial cells; KS, Kaposi’s sarcoma; KS-CM, KS-conditioned medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide; NNA, nitro-L-arginine; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, NO synthase; PTP, phosphotyrosine phosphatase; SNP, sodium nitroprusside; SSTR, somatostatin receptor; TTH, Tat (100 ng/ml), TNF-{alpha} (2 ng/ml), and heparin (24 IU/ml); VEGF, vascular endothelial growth factor.

Received September 9, 2002.

Accepted for publication December 19, 2002.


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 Results
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