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Research Communications |
a Modulo Progressione Neoplastica, the
b Servizio di Farmacologia e Neuroscienze, Istituto Nazionale per la Ricerca sul Cancro, the
c Sezione di Farmacologia, Dept of Oncologia, Univ. of Genova, and the
d Centro di Biotecnologie Avanzate, 16132 Genova, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: endothelial cells • monocytes • macrophages • invasion • neovascularization • somatostatin receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Somatostatin, a regulatory peptide originally described as an inhibitor of somatotropin release, has a wide range of activities (see ref 6 for review). Somatostatin and its analogs seem to be active in the inhibition of certain tumors 7, 8) . This cyclic peptide binds to seven transmembrane domain, G-protein-coupled receptors that transduce a variety of signals. At least five somatostatin receptor subtypes have been cloned and characterized (9) . All five of these receptor subtypes bind somatostatin 14 and somatostatin 28 (9) . The somatostatin receptors have been reported to affect many different intracellular pathways. For instance, somatostatin has been reported to inhibit cAMP formation, L-type calcium channel activity, and to activate K+ channels (6) . In the past few years it has been demonstrated that different somatostatin receptor subtypes are able to induce a phosphotyrosine phosphatase activity that has been proposed to be responsible for the direct antiproliferative activity of this peptide 10, 11) . Inhibitory effects of somatostatin on the growth of various normal and transformed cells have been reported 12-14) . Tumors of endocrine origin respond to both the direct antiproliferative activity of somatostatin and to the inhibitory effects through reduction of growth factor release (i.e., IGF-I) due to the endocrine activity of somatostatin (8) . An antitumor effect for nonendocrine tumors has also been reported for somatostatin analogs (15) . However, the mechanism for this apparently broad antitumor activity of somatostatin has not been elucidated.
Kaposi's sarcoma (KS)1 is a highly angiogenic lesion frequently associated with AIDS. The products of KS cells are highly angiogenic in vivo (16) and induce endothelial cell migration and invasion in vitro (17) . The HIV-Tat product also shows a potent angiogenic activity 18, 19) that is mediated by specific binding and activation of the KDR receptor for VEGF (20) . Here we report that somatostatin inhibits KS tumor cell growth in vitro and in vivo. However, the KS cells used did not express any known somatostatin receptors and did not respond to somatostatin in vitro. Instead, tumor growth inhibition was associated with a block of the angiogenesis and stimulation of endothelial cells and monocytes by the KS cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells
The human endothelial-like immortalized cell-line EAhy926,
derived from the fusion of human umbilical vein endothelial (HUVE)
cells with the A549 cell line, has been shown to have an
endothelial-like phenotype (22)
. These cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) 10% FCS h.i. (Seromed),
supplemented with glutamine (300 µg/ml), penicillin (100 U/ml), and
streptomycin (50 g/ml). Primary cultures of KS spindle cells (23)
were
grown in 50% DMEM and 50% RPMI 1640, 10% FCS supplemented with
glutamine (300 µg/ml). KS-Imm cells were isolated by our laboratory
from a kidney transplanted immunosuppressed patient and have been
described previously (24)
. HUVE cells from the ATCC (Rockville, Md.)
were cultivated on gelatin-coated plates (1,5% in PBS) in M199
containing 10% heat-inactivated fetal calf serum, 100 µg/ml heparin,
and 60 µg/ml endothelial cell growth factor (ECGS; crude extract
isolated from bovine brain) at 37°C in a 5%
CO2.
Tumor growth in vivo
KS-Imm cells (5 x 106 cells) (24)
were mixed
with liquid Matrigel to a final volume of 250 µl at 4°C and
injected subcutaneously (s.c.) in the flanks of nude (nu/nu) mice. The
animals were housed in pathogen-free conditions; mice were weighed and
tumor growth was monitored by measuring tumor size every day (25)
.
Treated animals had 50 µg of somatostatin injected s.c. twice daily.
Controls received an injection of buffer alone. Animals were killed
after 20 days and the tumors removed, photographed, and processed for
histology.
In vivo angiogenesis
We used the Matrigel sponge model of angiogenesis introduced by
Passaniti (26)
and modified Albini (16)
. Somatostatin was mixed with
Tat, tumor necrosis factor 
(TNF–), and heparin and added to
unpolymerized liquid Matrigel at 4°C to a final volume of 600 µl.
The Matrigel suspension was slowly injected s.c. into the flank of
C57/bl6 male mice with a cold syringe. In vivo, the gel
quickly polymerizes to form a solid gel. Matrigel with buffer alone was
used as negative control. After 4 days, gels were collected and
weighed. Some of the samples were minced and diluted in water to
measure the hemoglobin content with a Drabkin reagent kit (Sigma, St.
Louis, Mo.) that was normalized to 100 mg of recovered gel and
confronted with a standard curve of mouse blood hemoglobin. Other
samples were fixed in formalin, embedded in paraffin, and sections were
stained with hematoxylin and eosin for histological analysis.
Chemotaxis and invasion assays
These assays were carried out in Boyden chambers as described by
Postlethwaite (27)
and modified by Albini (28)
. EAhy926 cells were
harvested in trypsin/EDTA solution (0.05/0.02% in PBS, Seromed),
collected by centrifugation, and resuspended in DMEM 0.1% BSA. The
lower compartment of Boyden chambers (200 µl) was filled with the
chemoattractants: KS-conditioned medium or Tat protein (10 ng/ml) with
TNF– with heparin (0.01 U/ml) diluted in DMEM with 0.1% BSA. DMEM
with 0.1% BSA alone (SFM) was used as negative control to evaluate
random migration. EAhy926 cells (1.2 x 105/400 µl
per chamber) were placed in the upper compartment along with
somatostatin at the concentrations indicated. The two compartments were
separated by a polycarbonate filter (12 µm pore size; Nucleopore,
Milan, Italy) coated with gelatin to allow for cell adhesion. Chambers
were incubated for 6 h at 37°C in humidified atmosphere
containing 5% CO2. After incubation, cells on
the upper side of the filter were removed. The cells migrated to the
lower side of the filter were fixed in 100% ethanol, stained with
toluidine blue, and 5 to 8 unit fields per filter were counted at 160x
magnification with a microscope (Zeiss). The test was run in triplicate
and repeated six times. In the chemoinvasion assay, Matrigel was added
to the filters as a barrier (28)
.
Growth assays
[3H]-Thymidine incorporation assay
DNA synthesis activity was measured by means of the
[3H]-thymidine uptake assay as previously
reported (29)
. Briefly, cells were plated at the density of 5 x
105 in 24-well plates. After 24 h, cells were serum-
and growth factor-starved for 48 h. Subsequently, cells were
treated with the test substances for 16 h, and in the last 4 h cells were pulsed with 2 µCi/ml of
[3H]-thymidine (Amersham). At the end of the
incubation time, cells were trypsinized (15 min at 37°C), extracted
in 10% trichloroacetic acid (TCA) and filtered under vacuum through
fiber glass filters (GF/A; Whatman). The filters were then washed
sequentially under vacuum with 10% and 5% TCA and 95% ethanol. The
TCA-insoluble fraction was then counted in a scintillation counter.
MTT-assay
Mitochondrial function, as an index of cell viability, was
evaluated by measuring the levels of mitochondrial dehydrogenase
activity using reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl
tetrazolium bromide (MTT) as the substrate. Its cleavage to a purple
formazan product by dehydrogenase was quantified spectrophotometrically
measuring the absorbance at 570 nm.
Isolation of monocytes
Heparinized peripheral blood from normal, HIV-negative healthy
donors was carefully layered on the top of an equal volume of Ficoll
(Seromed, Berlin, Germany) and centrifuged at 600 x g
for 40 min. After centrifugation, the mononuclear cells (peripheral
blood mononuclear cells) remaining at the sample/medium interface were
collected and purified on Percoll (Pharmacia-Biotech, Milan, Italy).
Chemotaxis assays for monocytes
Chemotaxis was performed in a 48-microwell chemotaxis chamber
(Costar-Nucleopore), using a 5 µM pore-size polyvinylpyrrolidone-free
polycarbonate filter (Costar-Nucleopore) that divides each well in two
compartments 30-32)
. The lower compartment was filled with 27 µl of
chemoattractant (Tat or KS-CM) diluted in RPMI 0.1% BSA. The
concentration of f-MLP inducing maximal migration,
10-8 M, was used as the positive control. Simple
RPMI/0.1% BSA was the negative control to evaluate background
migration. The upper compartment was filled with 50 µl of monocytes
(107 cells/ml) suspended in RPMI/0.1% BSA. The chamber was
incubated at 37°C in 5% CO2 humidified
atmosphere for 120 min. Somatostatin was added to the cell suspension
1 h before running the assay. Each point was run in sextuplicate.
The filter was then removed and the cells fixed with absolute ethanol
and stained with toluidine blue. Cells that had not migrated were
removed from the upper surface of the filter using filter paper.
Migration was assessed by scanning the filters and quantitation by
using the NIH Image program as described previously (32)
.
Polarization assay
The ability of somatostatin to inhibit monocyte polarization was
assessed following the procedure of Locati et al. (33)
, with minor
modifications (32)
. Freshly Percoll-purified monocytes (5 x
105/500 µl) were preincubated at 37°C in polypropylene
tubes for 5 min and then treated with polarizing stimuli for 10–30
min. The assay was stopped by adding an equal volume of cool 10%
formaldehyde in PBS. The percent of polarized monocytes was determined
by seeding 10 µl of cell suspension in a Bürker hemocytometer
and counting the number of polarized cells over the total number of
cells by microscopy.
Detection of somatostatin receptors
RNA isolation and reverse transcription-polymerase chain reaction
(RT-PCR)
Total RNA was isolated using the acidic phenol technique (34)
,
treated for 45 min with RNase-free Dnase, then phenol/chloroform
extracted and ethanol precipitated. cDNA was synthesized using AMV RT
(Finnzyme OY, Finland) with oligo dT (16)
primers. Ten nanograms of
cDNA was subsequently used in the PCR reaction 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°). Amplified DNA fragments were visualized by 1.7% agarose gel
electrophoresis. The primers used were SSTR1: 5'-sense primer
corresponding to amino acids 225–244 and the 3'-antisense primer
corresponding to amino acids 438–457 of the SSTR1 sequence; SSTR2: the
5'-sense primer corresponding to amino acids 71–77 and the
3'-antisense primer corresponding to amino acids 195–201 of the SSTR2
sequence; SSTR3: the 5'-sense primer corresponding to amino acids
663–682 and the 3'-antisense primer corresponding to amino acids
865–884 of the SSTR3 sequence; SSTR4: the 5'-sense primer
corresponding to amino acids 548–567 and the 3'-antisense primer
corresponding to amino acids 849–868 of the SSTR4 sequence; SSTR5: the
5'-sense primer corresponding to amino acids 598–617 and the
3'-antisense primer corresponding to amino acids 801–820 of the SSTR5
sequence; expected lengths for the amplified products were the
following: SSTR1 = 233bp, SSTR2 = 402bp, SSTR3 = 222bp;
SSTR4 = 321bp, SSTR5 = 235bp.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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The strong inhibition of KS-Imm tumor growth by somatostatin suggested
that these cells have receptors for somatostatin. However, RT-PCR
experiments demonstrated that the KS-Imm cells do not express any of
the known somatostatin receptors (Fig. 1c
). Somatostatin did
not affect KS-Imm cell growth in vitro (Fig. 1d
),
further suggesting that the response was not directly mediated by
receptors for somatostatin on KS cells. Histological examination of the
tumors showed extensive vascularization in the control, but the
somatostatin-treated tumors showed very little vascularization
(Fig. 2
a). Since angiogenesis is required for solid tumor growth, we
examined the effects of somatostatin on KS cell product-induced
angiogenesis.
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Inhibition of angiogenesis in vivo
KS cell products induce a potent angiogenic
reaction in vivo, with an intense infiltration of new
vessels, as readily seen in macroscopic examination. Addition of
somatostatin to the Matrigel containing KS cell products clearly
blocked this angiogenic response (Fig. 2b
). Quantitation of
angiogenesis by hemoglobin content showed that somatostatin
significantly (P<0.001) reduced the angiogenic response
(Table 1
).
|
We have previously demonstrated that HIV-Tat induces angiogenesis
through the VEGF flk1/KDR receptor on endothelial cells (20)
. This
induction of angiogenesis by Tat appears to use a pathway different
than that of KS-CM, as the metalloproteinase inhibitor TIMP-2 is able
to block KS cell product-induced angiogenesis but not that of HIV-Tat
(16)
. Exposure to TNF- increases KDR levels on endothelial cells by
5- to 10-fold (35)
and enhances the response to Tat. We therefore used
Tat in combination with TNF– as a powerful angiogenic mixture
in vivo. Somatostatin strongly inhibited the angiogenesis
induced by TNF– and Tat (Fig. 2c
). Histological
examination confirmed the absence of vascularization in the
somatostatin-treated samples for both Tat/TNF–/heparin (Fig. 2d
) and KS-CM/heparin (not shown). Determination of
hemoglobin content also demonstrated a significant inhibition of
Tat/TNF–/heparin-induced angiogenesis by somatostatin (Table 1)
.
The addition of vanadate, an inhibitor of phosphotyrosine phosphatases,
counteracted the effect of somatostatin, suggesting that activation of
phosphotyrosine phosphatases is involved in the somatostatin inhibition
of Tat-induced angiogenesis. (Table 1)
Angiogenesis inhibitors can act through a number of effector targets. They can block endothelial cell growth and or their migration. In addition, angiogenesis inhibitors could also act indirectly on other effector cell types, such as monocytes, which appear to play a key role in angiogenesis. We tested the effects of somatostatin in vitro on stimulated endothelial cells and on monocytes.
Somatostatin effects on endothelial cells
Treatment with somatostatin inhibited the growth of endothelial
cells stimulated with KS cell products (Fig. 3
a). This effect was observed in primary cultures of umbilical
vein endothelial cells (HUVEC, not shown) and in the human
endothelial-like immortalized cell-line EAhy926 (Fig. 3a
).
The inhibitory effects of somatostatin on cell proliferation was
reverted by the pretreatment with vanadate (50 µM), thus confirming
the involvement of phosphotyrosine phosphatases in the somatostatin
antiproliferative activity of endothelial cells. Vanadate alone
increases cell growth due to repression of endogenous phosphatases, as
previously observed 14, 29)
.
|
During angiogenesis endothelial cells must migrate and invade through
their own basement membrane (2)
, a major barrier to cell movement (28)
.
Induction of an invasive phenotype in endothelial cells in
vitro is a hallmark of the angiogenic response in vivo,
and KS cell products induce endothelial cell invasion through a
reconstituted basement membrane in vitro in the
chemoinvasion assay (17)
. Somatostatin added to the upper chamber
dose-dependently inhibited the ability EAhy926 cells to invade in
response to KS cell products (Fig. 3b
). This inhibition was
selective for invasion, as migration in the absence of the basement
membrane barrier was not affected (not shown).
Endothelial cells express only the SSTR3 somatostatin receptor, as
assessed by RT-PCR analysis (Fig. 3c
). These data indicate
that somatostatin directly affects the endothelial response to KS cell
product stimulation. In contrast to KS cell products, somatostatin was
not able to reduce Tat/TNF–-induced endothelial cell invasion (not
shown). These data confirm that KS cell product and Tat-induced
angiogenesis occur through independent pathways. However, somatostatin
effectively blocked Tat-TNF– angiogenesis in vivo (Fig. 2c
, Table 1
), indicating that it may effect a different
target.
Monocytes/macrophages also appear to be major mediators of angiogenesis (36) . Monocytes infiltrate early in response to angiogenic stimuli and subsequently produce factors that stimulate endothelial cells to form new vessels. Inhibition of monocyte recruitment could also inhibit tumor-induced angiogenesis. Since Tat potently induces monocyte migration and invasion in vitro and in vivo 16, 18, 32, 37) , we tested whether somatostatin was able to affect monocytes.
Somatostatin effects on monocytes
Treatment of monocytes with 50 µM somatostatin reduced KS cell
product-induced migration by ~50% (Fig. 4
a). HIV-Tat is a potent inducer of monocyte migration. Again,
somatostatin inhibited the response of monocytes to Tat (Fig. 4b
). Polarization of monocytes is an effective indicator of
the activation of these cells toward a migratory stimulus. Monocytes
treated with f-MLP or KS-CM show significantly increased polarization
as compared with untreated controls. Simultaneous treatment with
somatostatin inhibited polarization in response to KS-CM (Fig. 4c
). Treatment of monocytes with 50 µM somatostatin
inhibited monocyte migration to f-MLP by ~35%.
|
Monocytes appear to respond directly to somatostatin, suggesting these
cells have somatostatin receptors. In fact, RT-PCR demonstrated the
presence of the SSTR2, SSTR3, and SSTR5 somatostatin receptors on these
cells (Fig. 4d
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Somatostatin has been proposed to be a potential antitumor agent. Although somatostatin is very effective in inhibiting tumor cell proliferation in vitro, in vivo treatments have been much less successful (see ref 38 ). Endocrine tumors that express high levels of somatostatin receptors are the oncotypes most responsive to somatostatin 7, 8) . Recently, the antiproliferative activity of somatostatin in endocrine tumors has been proposed to be dependent on the SSTR-mediated activation of phosphotyrosine phosphatase activity (10) . Although occasional somatostatin receptors have been detected in nonendocrine tumors (39) , the mechanisms of the antiproliferative activity of somatostatin in many tumors has remained obscure.
Limited preliminary data suggest that somatostatin may inhibit angiogenesis in the chick CAM 40, 41) , and somatostatin receptors have been reported to be expressed at high density on endothelial cells near tumors (42) . However, there are no reports on the effects of somatostatin on tumor-induced angiogenesis or on endothelial cells stimulated by tumor-derived products.
Kaposi's sarcoma represents a prototype of tumor angiogenesis. Here we have provided evidence that somatostatin can inhibit the growth in vivo of Kaposi sarcoma tumors as well as angiogenesis in Matrigel pellets in vivo. Somatostatin also inhibited KS cell product-induced endothelial cell invasion of a reconstituted basement membrane (Matrigel) in vitro; however, migration in the absence of the barrier was not inhibited. To elucidate whether the in vivo effect was direct (on endothelial cells) or indirect (on monocyte/macrophage mediators), we studied the effects of somatostatin on endothelial cell growth, migration, and invasion of basement membranes. Cells of endothelial origin (EAhy926) were stimulated to grow by KS sarcoma products. Somatostatin dose-dependently inhibited KS-CM-induced growth. This effect was likely mediated by the somatostatin receptor subtype SSTR3, since it was the only somatostatin receptor to be identified in these cells by RT-PCR analysis. Moreover, the phosphotyrosine inhibitor vanadate (43) reverted this effect. These data suggest that activation of phosphotyrosine phosphatases by SSTR3, whose coupling with this transduction system has been reported (44) , is responsible for the inhibitory effects of somatostatin on EAhy926 cell proliferation. These data imply that somatostatin interferes specifically with the tyrosine kinase signaling pathways in endothelial cells closely associated with angiogenesis and endothelial cell invasion.
In contrast, somatostatin was not able to reduce Tat/TNF––induced
endothelial cell invasion in vitro, indicating that
somatostatin might have different targets in the block of
Tat/TNF––induced angiogenesis. However, the activation of
phosphotyrosine phosphatase activity appears in this case also to
participate in the somatostatin effect, since coincubation with
vanadate completely reverted the antiangiogenic activity of
somatostatin.
A major mediator of tumor angiogenesis in vivo are monocytes. Infiltrated monocytes are able to produce survival factors, which, in turn, activate endothelial cells and favor vascularization of tumors. We therefore analyzed whether somatostatin is capable of affecting monocyte chemotaxis. We found that somatostatin was indeed able to reduce monocyte migration. This is in agreement with a previous study showing somatostatin inhibition of growth-hormone-induced monocyte chemotaxis (45) . To explain the biological effects of somatostatin on monocytes, we investigated the expression of its receptors. RT-PCR analysis demonstrated that monocytes express three subclasses of somatostatin receptors: SSTR2, 3, and 5. The KS-Imm cells express neither somatostatin receptors nor growth inhibition by somatostatin; therefore, the antitumor effect is clearly indirect, through the inhibition of vascular supply. As is thought to occur for angiostatin and endostatin 4, 5) , somatostatin behaves in this case as a `pure' angiogenesis inhibitor. In addition, somatostatin demonstrated a multiple antiangiogenic activity, inhibiting endothelial cells both directly and indirectly through monocyte inhibition. This permitted the blockade of two different types of potent angiogenic stimulation and suggests that successful antiangiogenic therapies will need to affect multiple cell types.
In summary, our data show that Kaposi's sarcoma tumor growth is inhibited by somatostatin and that angiogenesis is the target of inhibition. Somatostatin clinical trials in oncology so far have shown no clear benefit to the patient for nonendocrine tumors (38) . Our demonstration that somatostatin can act as a `pure' antiangiogenic drug, affecting tumor cells that do not have somatostatin receptors, suggests that clinical trials of somatostatin in oncology should have a different design from those previously reported (see ref 38 ). Antiangiogenic therapy should in fact be continuous, since in most cases antiangiogenic drugs are `angiostatic' rather than cytotoxic (1) . It might be that somastostatin used as an adjuvant antiangiogenic treatment, administrated after or during conventional therapy (surgical, radiological and chemical), may result in benefit to the patient.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Abbreviations: BSA, bovine serum albumin; DMEM,
Dulbecco's modified Eagle's medium; KS, Kaposi's sarcoma; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide; PBS,
phosphate-buffered saline; RT-PCR, reverse transcription-polymerase
chain reaction; s.c., subcutaneously; TCA, trichloroacetic acid; TNF,
tumor necrosis factor.
Received for publication September 14, 1998.
Revision received November 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. Sodhi, S. Montaner, V. Patel, M. Zohar, C. Bais, E. A. Mesri, and J. S. Gutkind The Kaposi's Sarcoma-associated Herpes Virus G Protein-coupled Receptor Up-Regulates Vascular Endothelial Growth Factor Expression and Secretion through Mitogen-activated Protein Kinase and p38 Pathways Acting on Hypoxia-inducible Factor 1{{alpha}} Cancer Res., September 1, 2000; 60(17): 4873 - 4880. [Abstract] [Full Text]
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A. Albini, C. Marchisone, F. Del Grosso, R. Benelli, L. Masiello, C. Tacchetti, M. Bono, M. Ferrantini, C. Rozera, M. Truini, et al. Inhibition of Angiogenesis and Vascular Tumor Growth by Interferon-Producing Cells : A Gene Therapy Approach Am. J. Pathol., April 1, 2000; 156(4): 1381 - 1393. [Abstract] [Full Text] [PDF]
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) or treated twice daily
with somatostatin (
). Average tumor size is shown, differences in
tumor sizes of the two groups were statistically significant from 14
days on to the end of the experiment (P<0.03).
b) Average mouse weight (± SD) of animals
treated with or without somatostatin, no differences were observed.
c) RT-PCR of somatostatin receptors from mRNA of KS-Imm
cells. None of the primers for SSTR 1-5 amplified a product, whereas
the control ß-actin primers amplified the expected product,
confirming sample integrity. d) KS-Imm cell growth
in vitro with (
) or without (
) somatostatin, as
assessed by the MTT assay; no significant differences were observed.
