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Endocrinology Vol. 143, No. 8 3114-3121
Copyright © 2002 by The Endocrine Society

ARTICLE

Human Chorionic Gonadotropin Inhibits Kaposi’s Sarcoma Associated Angiogenesis, Matrix Metalloprotease Activity, and Tumor Growth

Ulrich Pfeffer, Davide Bisacchi, Monica Morini, Roberto Benelli, Simona Minghelli, Angelo Vacca, Douglas M. Noonan and Adriana Albini

Laboratory of Molecular Biology (U.P., D.B., R.B., S.M., A.A.) and Tumor Progression Section (M.M., D.M.N.), National Cancer Research Institute Genova, c/o Advanced Biotechnology Center Genova, Genova 16132, Italy; and Department of Biomedical Sciences and Human Oncology (A.V.), University of Bari, Bari 70124, Italy

Address all correspondence and requests for reprints to: Adriana Albini, Laboratory of Molecular Biology, National Cancer Research Institute Genoa, Largo Rosanna Benzi 10, 16132 Genova, Italy. E-mail: . albini{at}cba.unige.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Kaposi’s sarcoma is a highly angiogenic, AIDS-associated neoplasm that is more frequent in male than in female patients. Cases of spontaneous regression during pregnancy have been reported and the pregnancy hormone human chorionic gonadotropin (hCG) has shown anti-Kaposi’s sarcoma activity in several, but not all, clinical trials. Antiproliferative and proapoptotic activities specific for Kaposi’s sarcoma (KS) cells have been shown. We report here further analyses of the anti-KS activity of the hormone and show that urinary hCG, the hCG ß-subunit, the hCG ß-core, and to a lesser extent a recombinant hCG, directly inhibit the activity of matrix metalloproteases of different origin. The hCG hormone also inhibited angiogenesis in vivo in the matrigel sponge assay as well as growth of KS cell xenografts in nude mice. The effect of the pure recombinant hormone dimer on xenograft growth was transient, indicating that the activity of intact hCG alone is not sufficient to overcome the growth potential of this tumor and suggesting that active hCG fragments or other anti-KS activities contribute to the activity of urinary hCG.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
KAPOSI’S SARCOMA IS a highly angiogenic mucocutaneous tumor that was once a rare lesion in elderly subjects (classic KS). The human herpes virus HHV-8 (Kaposi’s sarcoma associated herpes virus, KSHV) is present in all KS lesions analyzed and appears to be an important etiological factor for KS. However, HHV-8 infection is not sufficient for the formation of KS lesions, as most HHV-8 positive subjects do not develop KS. Loss of immune surveillance in renal transplant patients under immune-suppressive therapy (iatrogenic KS) or in AIDS patients (epidemic KS) is linked to the development of KS in HHV8 carriers. KS is endemic in some areas of Africa with high HHV8 infection rates (endemic KS), and in zones that overlap with the HIV epidemic, KS has become one of the most frequent tumors (1). All forms of KS show a clear sex bias even though HHV-8 sero-prevalence is equivalent in males and females (2). For example, HIV positive men are at least four times more at risk to develop the disease than HIV-positive women (for reviews see Refs. 2, 3, 4, 5, 6). It has been reported that more than 30% of HIV-positive homosexual men develop the tumor (7) although the incidence in the Western hemisphere has drastically declined with the introduction of highly active antiretroviral therapy (HAART) (8).

The observation of complete growth repression of tumorigenic KS cells in pregnant immunodeficient mice and reports of two spontaneous remissions in HIV-positive female KS patients during pregnancy led to the hypothesis that pregnancy hormones, in particular human chorionic gonadotropin (hCG), may have a protective activity against KS (9). hCG is a glycoprotein composed of two noncovalently linked subunits ({alpha} and ß) that can be found along with free forms of its subunits and a proteolytic fragment of the ß-subunit termed the "ß-core" (residues 6–40 and 55–92 linked by disulfide bridges) in the urine of women in the first trimester of gestation and in certain malignancies (10). Sera from pregnant women (9) and, later on, preparations of hCG from urine (11) were shown to act in vitro and in vivo on KS cells inducing apoptosis and inhibiting tumor growth. Subsequent clinical trials reported that more than half of the patients treated showed partial remission or stabilization of the disease (11, 12, 13). Intralesional application of the hormone yielded response rates of over 80% (11), and high remission rates even for advanced visceral lesions were reported (14). However, several parallel clinical trials with hCG did not report any significant amelioration of the disease (15, 16, 17, 18, 19), leading to speculation that there might be contaminants in the active preparations that were responsible for the observed effects in laboratory and clinical trials.

hCG, its ß-subunit and the hCG ß-core were all reported to inhibit the growth and induce apoptosis of both reactive spindle-shaped KS cells and an immortalized KS line (20, 21), with the ß-core being the most active compound (20). A ribonuclease (22) showing KS cell-specific cytotoxicity has also been identified in hCG preparations, possibly in combination with other factors. Similar apoptosis inducing activity of urinary hCG preparations has been attributed to an unknown hCG-associated factor (HAF), that was chromatographically distinct from the hCG heterodimer and eluted with or close to the ß-core fragment (23, 24).

Here we show that a urinary preparation of hCG (u-hCG) strongly blocked angiogenesis in the matrigel sponge model. Furthermore, u-hCG proved to be a novel inhibitor of matrix metalloproteases (MMP) released by various cell lines. Similar antiangiogenic and MMP inhibitory activities were observed for the hCG ß-subunit and ß-core. hCG also inhibited the growth of KS cell xenografts in nude mice. Recombinant hCG (r-hCG) tested in parallel showed similar, but not identical, activities, confirming that the hCG hormone itself has a clear anti-KS activity, although in vivo the urinary preparation had a longer lasting effect probably due to the presence of active fragments (ß-subunit or ß-core) or unidentified compounds (22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents
Six- to 7-wk-old C57BL/6 mice and 37 (CD-1)BR nude mice were obtained from Charles River (Calco, Como, Italy) and housed in specific pathogen free conditions. Urinary preparations of hCG (CG10; 10,000 IU) were obtained from Sigma (St. Louis, MO), LH, the ß-subunit, the ß-core and a recombinant form of the hormone were kindly provided by Serono (Geneva, Switzerland). Recombinant hCG and ß-subunit were obtained from transfected mouse cell lines followed by affinity chromatography purification to greater than 98% purity as analyzed by SDS-PAGE. The hCG ß-core fragment purified from urinary hCG preparations by HPLC size exclusion chromatography (25). Matrigel, an extract of the murine Engelbreth-Holm-Swarm tumor grown in C57BL/6 mice, was produced as previously described (26). Briefly, Engelbreth-Holm-Swarm tumor material was homogenized with a polytron homogenizer in a high-salt buffer (3.4 M NaCl; 50 mM Tris-Cl, pH 7.4; 4 mM EDTA with 2 mM N-ethyl-maleimide). The homogenate was separated from the supernatant by centrifugation and the procedure was repeated three times. The remaining pellet was solubilized in 2 M urea, 50 mM Tris-Cl (pH 7.4), 150 mM NaCl overnight. The supernatant was cleared by centrifugation and extensively dialyzed against Tris-saline and finally against DMEM. The resulting material, matrigel, is rich in basement membrane components (laminin, collagen IV, nidogen, and perlecan) with limited quantities of growth factors (27). Heparin was obtained from Clarisco (Schwarz Pharma S.p.A., San Grato-Lodi, Italy).

Cell culture
The KS-20 (28) and KS-26 (29) are primary spindle cell cultures derived from HIV-infected KS patients, the KS-C1 primary spindle cell culture is from an iatrogenic KS patient (29). KS-IMM cells are a spontaneously immortalized iatrogenic KS cell line (20). All KS lines were grown in Roswell Park Memorial Institute 1640 medium containing glutamine (300 µg/ml) and 10% heat-inactivated FCS. These KS cells in vitro express markers similar to KS spindle cells in vivo. KS cells may be produced as the result of a "hit and run" mechanism (30), where the initial contact with HHV8 appears to initiate a cascade of events that alter cellular phenotypes to the KS spindle cell. Primary and immortalized KS model systems, although they do not contain HHV-8, are representative of HHV-8 exposed and affected cells.

Human fibroblasts, MRC-5 (31) and sinovial sarcoma cells were grown in DMEM, 10% fetal calf serum (FCS) supplemented with glutamine (300 µg/ml). HT1080 fibrosarcoma cells were grown in Roswell Park Memorial Institute medium, 10% FCS, glutamine (300 µg/ml) and human umbilical vascular endothelium (HUVE) cells (obtained from the ATCC, Manassas, VA) were grown in M199 medium containing 10% FCS, 10 ng/ml of acidic fibroblast growth factor and basic fibroblast growth factor, 20 ng/ml epidermal growth factor, and 160 µg/ml heparin.

In vivo angiogenesis
Matrigel (12 mg/ml) is liquid at 4 C but upon sc inoculation it rapidly solidifies to form a gel. Addition of 24–26 U/ml heparin and 12 µl of KS cell supernatants to a final volume of 600 µl results in a strong angiogenic response with formation of new vessels that grow into the matrigel, whereas no reaction is induced by matrigel with buffer alone (32). We tested the effects of hCG and derivatives by direct addition of the factors to the matrigel mixture before injection. Four days after injection, the animals were killed and the gels recovered and weighed. In some samples, the hemoglobin content was measured as an indicator of angiogenesis. The recovered gels were minced and dispersed in water, and hemoglobin released was measured using a Drabkin reagent kit 525 (Sigma), the concentration was calculated from a standard curve and normalized to 100 mg of recovered gel as previously described (32). Other samples were processed for histology by dehydration in ethanol, equilibration in Histolemon, and paraffin embedding. Five-micrometer sections were placed on poly-lysine coated slides, rehydrated, and stained with hematoxylin/eosin.

Gelatin zymography
KS-20, KS-26, KS-IMM, sinovial sarcoma, MRC-5 fibroblasts, HUVE, and HT1080 fibrosarcoma cells at 80% confluence were incubated for 24 h with serum-free medium. The supernatants were recovered and centrifuged for 5 min at 1000 rpm, diluted 1:5 with absolute ethanol and incubated at -20 C for a minimum of 2 h to precipitate protein. Precipitates were pelleted by centrifugation at 10,000 x g for 20 min at 4 C, the pellet was air-dried and dissolved in 100–200 µl 40 mM Tris, pH 7.5. The total protein content was measured by absorbance at 220 nm and by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as a protein standard.

Gelatin zymographs, used to visualize type-IV collagenases that also have a characteristic gelatinase activity (gelatinases A and B), were performed according to Heussen and Dowdle (33) with minor modifications. SDS-PAGE gels (7%) (34) containing copolymerized gelatin at a final concentration of 0.6 mg/ml were prepared. Protein samples (10 µg) were dissolved in 40 mM Tris, pH 7.5, and electrophoresed. After electrophoresis, the gel was washed 4 times (30 min each) in 2.5% Triton X-100 to remove SDS, cut into identical portions and incubated for 18 h at 37 C in a 40 mM Tris, 200 mM NaCl, 10 mM CaCl2 (pH 7.4) buffer in either the absence or the presence of recombinant hCG (20 µg/ml), ß-subunit (10 µg/ml) or ß-core (5 µg/ml). After digestion, the gel was stained with 0.1% Coomassie brilliant blue. Enzyme-digested areas were identified as white bands against a blue background.

Western blots were obtained from KS-IMM and HT1080 cell culture supernatants separated by SDS-PAGE and blotted to polyvinylidine difluoride membranes (Amersham Pharmacia Biotech, Milan, Italy) and analyzed for the presence of MMP-2 using anti-MMP-2 antibodies (AB-3; Oncogene Research Products, Cambridge, MA). Isotype-specific horseradish peroxidase conjugated secondary antibodies (DAKO Corp., Milan, Italy) were used a secondary antibodies and were revealed with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) following the manufacture’s instructions.

KS xenografts
For xenografts, KS-IMM cells were pretreated in vitro with r-hCG 16 µg/ml (Serono) or CG10 (urinary preparation from Sigma) for 1 h. Ten animals for each treatment were then injected with 5 x 106 KS-IMM cells suspended in 250 µl of 10 mg/ml matrigel. Matrigel is liquid at 4 C and rapidly solidifies at 37 C. Mice were injected im every other day either with physiological saline solution (control) or with 1 µg/g body weight r-hCG or with 1 µg/g body weight u-hCG CG10 for 26 d. Tumor dimensions were determined regularly and the differences in tumor dimensions analyzed statistically using a two-way nonparametric ANOVA test. At the end of the treatment, mice were killed and tumors were recovered for histological evaluation (see Histological evaluation of KS xenografts).

Histological evaluation of KS xenografts
Eight-micrometer sections from the formalin-fixed, paraffin-embedded tumor samples were deparaffinized by the xylene-ethanol sequence, endogenous peroxidase depleted by treatment with 0.3% H2O2 and 0.1% NaN3, followed by incubation with 0.1% trypsin (Sigma). Sections were stained using primary antibodies against factor VIII-related antigen (DAKO Corp., Glostrup, Denmark) and secondary antibodies (biotin-labeled fragments of ovine antirabbit Ig; DAKO Corp.) or horse antimouse Ig (Vector Laboratories, Inc., Burlingame, CA) according to the primary antibody, and streptavidin-peroxidase conjugate (DAKO Corp.). The color reaction was carried out in 0.05 M acetate buffer (pH 5.1), 0.02% 3-amino-9-ethylcarbazole grade II (Sigma) and 0.05% H2O2. Stained sections were washed in the same buffer, counterstained in hematoxylin, mounted in buffered glycerin, and examined under a Leitz Dialux 20 photomicroscope (Leitz, Wetzlar, Germany). Rabbit preimmune serum or an unrelated murine monoclonal IgG1 served as negative controls, according to the primary antibody. Angiogenesis was estimated by the analysis of capillaries and small venules stained with factor VIII and selected from all the stained vessels as endothelial cells, single or clustered in nests or tubes and clearly separated one from another, and either with or without a lumen. The microvessel area (mm2 x 10-2) was measured in four to six 250x fields, covering almost the whole of each of four sections per sample, within a superimposed 484-point square mesh of 12.5 mm2 x 10-2 with points of 258 µm2. The planimetric method of "point counting" with slight modifications for computer image analysis (Quantinet –500, Leica Corp.) was applied to measure the area as absolute value.

Immunofluorescence binding assay
CAOV-3, KS-IMM, HUVE, and MRC-5 cells plated in chamber slides (Nunc, Roskilde, Denmark) were fixed for 5 min with 3% paraformaldehyde in PBS and 2% sucrose. The cells were then rinsed and incubated for 1 h at room temperature with 400 U/ml of hCG labeled with biotin using the Biotin-X-NHS kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. The cells were rinsed with PBS, nonspecific binding sites saturated with 1.5% BSA in PBS, and the cells were then incubated for 50 min at room temperature with fluorescein isothiocyanate-labeled streptavidin (Sigma). Fluorescence was observed on an Olympus Corp. (Milan, Italy) BX51 microscope. Negative controls were stained identically but without biotinylated hCG.

Western blotting
Equal amounts of proteins (5 µg) were fractionated on a 6% SDS-PAGE and transferred to nitrocellulose filters (Hybond-C extra, Amersham Pharmacia Biotech) according to standard protocols. Blots were saturated in 5% dried milk in PBS with 0.05% Tween and incubated for 2 h at room temperature with an antihuman MMP-9 mouse monoclonal antibody (Chemicon, Temecula, CA) or an anti-MMP-2 mouse monoclonal antibody (Oncogene) diluted in 5% dried milk in 0.05% Tween-PBS. A horseradish peroxidase-conjugated antimouse Ig secondary antibody (Amersham Pharmacia Biotech) was used for 1 h at room temperature. Antibody binding was visualized using the electrochemiluminescence Western blotting detection system (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hCG, ß-subunit, ß-core, and recombinant hCG inhibit the angiogenic response in matrigel sponges in vivo
KS is a highly vascular tumor and as such it is sensitive to antiangiogenic agents. To establish whether hCG or its derivatives act exclusively by direct growth inhibition and stimulation of apoptosis as previously reported (20, 21), or whether antiangiogenic activities were also involved, the effects of hCG were tested in angiogenesis assays in the in vivo matrigel model as previously described (35, 36). KS cells produce several potent angiogenic factors (32), and matrigel sponges containing supernatants of KS cell cultures were rapidly colonized by new blood vessels (Fig. 1Go). The addition of hCG to the KS cell supernatant containing matrigel strongly inhibited vascularization of the sponges (Fig. 1Go). All three forms of the hormone tested showed strong, statistically significant antiangiogenic activity (ß-subunit and the highly purified recombinant hCG dimer, P < 0.01; the urinary preparation CG10, P < 0.05, ANOVA). LH, a hormone that shares the {alpha}-subunit with a structure similar to hCG but differs in the ß-subunit, did not affect angiogenesis when used at the same concentration in the same model (Fig. 1Go).



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  		Figure 1. Angiogenesis in matrigel sponges containing KS-IMM cell supernatants (KS CM). Matrigel sponges that contain KS CM become vascularized in vivo in C57BL/6 mice as readily observed in gels which have been removed and photographed after 4 d (controls, panels A and E). Inclusion of either CG10 hCG (panel B), recombinant hCG (panel C), or recombinant ß-subunit (panel D) in the sponges with KS CM strongly reduces the vascularization of the gels. In contrast, inclusion of LH (panel F) had no effect on the vascularization compared with controls (panel E). Quantitative estimation of the extent of vascularization obtained by measurement of the hemoglobin content indicated that addition of urinary (u-hCG) or recombinant (r-hCG) hCG as well as the ß-subunit (b-sub) significantly reduced the hemoglobin content of the gels (lower panel). Data are pooled from multiple independent experiments (n >= 6), P values of treatments vs. controls as analyzed by nonparametric statistical analysis (Kruskal-Wallis test) are *, P < 0.05; **, P < 0.01.

 
Histological evaluation of the matrigel sponges containing the KS-cell supernatants with hCG (CG10) showed substantially reduced cell infiltration into the sponges after 4 d in vivo compared with controls (Fig. 2Go). Rudimentary endothelial like structures were clearly visible in KS-cell supernatant containing sponges (Fig. 2Go, A and B), whereas similar structures were not visible in the sponges also containing hCG (Fig. 2Go, C and D).



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  		Figure 2. The histology of the matrigel sponges containing KS-IMM cell culture supernatants either alone (A and B) or with hCG (CG-10) (C and D). Gels were fixed, sectioned and stained with hematoxylin and eosin and photographed at 200 (A and C) or 400 (B and D) fold nominal magnification. Control sponges show considerable cellular infiltration and rudimentary endothelial-like structures, whereas hCG-treated sponges contained few infiltrated cells.

 
Previous studies have shown that hCG or the hCG derived ß-core fragment can inhibit KS cell growth in vitro and induce apoptosis in these cells (9, 20, 21). However, while indirect methods suggested that hCG was associated with cells in KS lesions (9), no direct binding of highly purified, 125I-labeled hCG to KS cells was observed in other studies (21). We biotinylated urinary hCG and observed strong cell surface staining of the positive control CAOV-3 cells incubated with avidin fluorescein isothiocyanate, whereas no cell surface immunofluorescence staining was observed for KS or endothelial cells (data not shown). These data suggest that hCG could affect extracellular, rather than cellular, mechanisms involved in the host-tumor interactions that are required for tumor growth and angiogenesis in vivo.

hCG and derivatives inhibit gelatinolytic activity
The absence of endothelial structures in hCG containing gels could have been due to either inhibition of invasion and/or reduced cell differentiation. MMPs are necessary for invasion of tumor cells and of activated endothelial cells during angiogenesis (37, 38, 39). MMP-2 and MMP-9, or gelatinases A and B, respectively, are MMPs essential for the degradation of basement membranes (37, 38). We analyzed whether hCG could inhibit the activity of these enzymes by gelatin zymography assays on cell supernatants in the presence or absence of hCG and its derivatives. This assay allows the direct visualization of MMP activity present in the cell supernatants after electrophoretic separation on substrate (gelatin) containing gels.

As expected, KS cells (40) and endothelial cells expressed both MMP-2 (gelatinase A, 72-kDa type-IV collagenase) and MMP-9 (gelatinase B, of 92-kDa type-IV collagenase), similar to the standard HT1080 cell line (Fig. 3AGo). Western blotting of KS-C1 supernatants stained with specific antibodies that recognize MMP-2 or MMP-9 confirmed that the bands observed in zymography corresponded to these gelatinases (Fig. 3AGo). Addition of urinary hCG to the collagenase buffer reduced the degradation of gelatin by KS-C1 and KS-IMM cell supernatants by blocking both MMP-2 and MMP-9 activity (Fig. 3AGo). MMPs from other sources, such as sinovial sarcoma, HUVE, or HT1080 cells were also inhibited (Fig. 3AGo), indicating that the antigelatinolytic effect of hCG was not cell type specific. The similar hormone LH had no effect on either MMP-2 or MMP-9 activity in these assays (data not shown). The highly purified r-hCG also inhibited gelatinolytic activity of KS cells, although to a substantially lesser extent (Fig. 3BGo). In keeping with the results of the angiogenesis assays in vivo, the recombinant ß-subunit showed strong inhibition of MMP-2 gelatinolytic activity present in KS-IMM cell supernatants (Fig. 3CGo), as did the ß-core fragment (Fig. 3CGo). The inhibition of KS and HUVE cell gelatinase activity by hCG suggests both indirect and direct inhibition of tumor angiogenesis by this hormone.



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  		Figure 3. MMP inhibition by hCG as determined by gelatin zymography. Proteins from cell culture supernatants were separated by SDS-PAGE in gelatin containing gels. After incubation in collagenase buffer and staining, gelatinolytic activity becomes evident as negatively stained bands that correspond to areas of digestion due to MMP-2 or MMP-9 activity, as indicated. A, Identical parallel gels containing supernatants of KS-C1 (lane 1), KS-IMM cells (lane 2), sinovial sarcoma (lane 3), or HT1080 fibrosarcoma (lane 4) or HUVE cells (lane 5) were incubated in either buffer alone (control) or in buffer containing 200 U/ml (CG10) u-hCG. The reduction in gelatinase digestion in the gel incubated with u-hCG is evident. Western blotting (wb) of KS-C1 supernatants with antibodies to either MMP-9 or MMP-2 confirmed that the bands observed in zymography correspond to these molecules. The hormone LH had no activity. B, Identical parallel gels containing supernatants of KS-20 (lane 1), KS-26 cells (lane 2) or sinovial sarcoma (lane 3) cells were incubated in either buffer alone (left panel) or in buffer containing 20 µg/ml recombinant hCG (right panel). The reduction in gelatinase digestion in the gel incubated with r-hCG is less evident. Gels containing supernatants of KS-IMM cells, incubated either in buffer alone (controls) or in buffer containing 10 µg/ml ß-subunit (ß-subunit) or 5ug/ml ß-core (ß-core). Both the ß-subunit and the ß-core strongly inhibited MMP-2 gelatinase activity.

 
hCG inhibits KS tumor growth
To assess the activity of hCG on the growth of KS cells in vivo, KS-IMM cells were resuspended in matrigel and inoculated sc in nude mice. The cells had been pretreated for 1 h with either a urinary preparation of hCG (u-hCG, 16 µg/ml) or the recombinant form of the hormone (r-hCG, 16 µg/ml) or buffer alone. In each experiment, 10 mice for each treatment were injected im every other day for 26 d with 1 µg of the hormone preparations for each gram of body weight or with carrier alone, and tumor size was measured each day. The tumors of the control group injected with carrier alone grew rapidly after inoculation and reached a large volume after approximately 17–20 d (Fig. 4Go). Over the first 15 d, the growth of both u-hCG and r-hCG treated tumors was significantly lower than that of controls. Analysis with two-way ANOVA indicated that the difference in tumor growth was statistically significant at d 12–15 for hCG (P < 0.01 at d 12 and 15; P < 0.001 at d 13 and 14) and on d 13–14 for u-hCG (P < 0.05) compared with controls (Fig. 4Go, inset). After d 15, r-hCG tumors grew as fast as those of control animals, reaching the same mean dimensions as the tumors of control animals at the end of the treatment. Over the full time course, the tumor sizes in animals treated with urinary hCG were significantly reduced with respect to controls from d 19 on (P < 0.001, d 19–26) (Fig. 4Go). This growth retardation led to a mean reduction of tumor dimension of approximately 75% in u-hCG treated mice at the end of the interval of observation (26 d). During the first 2 wk of tumor development recombinant hCG showed slightly stronger effects than that observed for the urinary preparations (Fig. 4Go, inset), but only the urinary preparations showed sustained inhibitory activity. This indicates that the effect of the recombinant form of the hormone is limited to the first phase of tumor growth, after which the growth inhibitory effect is overridden by the tumor.



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  		Figure 4. Growth of KS-IMM xenografts in nude mice. Animals received 5 x 106 KS-IMM cells suspended in matrigel by injection under the skin and were injected im every other day with either physiological saline solution (Control) or 1 µg/g body weight r-hCG or 1 µg/g body weight CG10 u-hCG for 26 d. Inset, Growth curves of the tumors over the first 15 d of the experiment. Both the u-hCG and r-hCG inhibited tumor growth within the first 15 d; however, only u-hCG inhibited tumor growth over the full time course of the experiment.

 
Tumor histology
The tumor sections were stained with antibodies against factor VIII as a marker for blood vessels, counterstained with hematoxylin and histologically analyzed to determine the extent of xenograft vascularization (Fig. 5Go). Tumors in animals treated with hCG showed a reduced vascularization, which was particularly evident and statistically significant (P < 0.01) for the urinary preparation (Fig. 5Go). The treatment of the tumors with the recombinant form yielded some reduction in vessel formation, which, however, did not reach statistical significance. This is consistent with the limited effect of r-hCG on tumor development observed at later time points.



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  		Figure 5. Histology of KS-IMM xenografts after 26 d. Histology of xenografts of KS-IMM cells in nude mice treated with vehicle (A) or urinary hCG (B) stained with antibodies specific for factor VIII related antigen. Lower panel, KS-IMM xenografts treated with vehicle alone (control), recombinant hCG (r-hCG) or u-hCG were analyzed considering endothelial cells, either single or clustered in nests or tubes and clearly separated from one another. Microvessel area (mm2 x 10-2) was measured in four to six fields, covering almost the whole of each of four sections per sample. The data are given as the mean ± SD, P < 0.01 for u-hCG compared with controls using Duncan’s paired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The variability of hCG effectiveness in both experimental systems and in the clinic led to the hypothesis that the KS suppressive activity was due to contaminants present in the active preparations. The only defined anti-KS moiety that has been purified from urinary hCG preparations is a ribonuclease (22). Kachra and co-workers (41) identified low molecular weight anti-KS compounds but were not able to identify its molecular nature. Chromatographic purification of anti-KS activities from urinary hCG preparations (23) yielded a fraction with anti- KS activity that overlapped with a major urinary derivative of hCG, the ß-core, which we had shown to induce apoptosis in KS cells (20). The authors excluded the ß-core as being the active component based on the observation that an affinity purified ß-core preparation did not show activity. However, affinity purification of the ß-core from urinary hCG preparations comprised harsh treatments that may have affected activity (25, 42); thus, the possibility that the ß-core has significant anti-KS activity cannot be excluded.

Other antineoplastic factors have been obtained from urine, including inhibin, activin A, endostatin, and angiostatin (43, 44). The anticancer activity of many of these agents is associated with apoptosis of tumor-associated endothelial cells (see Ref. 45 for review). Among urinary hCG-associated molecules, antineoplastic urinary protein is, at present, the only described molecule reported to have antiangiogenic effects (46). We observed a strong antiangiogenic activity for the ß-core, the ß-subunit, and recombinant hCG as quantitatively analyzed in the matrigel sponge model in vivo. The histology of matrigel sponges containing KS cells supernatants showed cellular infiltration and vascular/lacunar-like structures. In the presence of urinary hCG, a reduced cellularity and lack of vascular development was observed. The reduction of host cell infiltration and the lack of effects on HUVE cell proliferation in vitro led us to hypothesize that hCG and its derivatives could act on other targets of the angiogenic process. Tumor invasion and angiogenesis are linked to metalloprotease activity, as both tumor and endothelial cells use these enzymes, in particular MMP-2 and 9, to extravasate and invade the extracellular matrix (36). Here we made the novel observation that hCG and hCG derivatives are able to inhibit MMP activity in gel zymography. The inhibition of endothelial-secreted MMP-2 and MMP-9 by urinary hCG could explain the antiangiogenic effects observed in vivo, an activity associated with MMP inhibitors (see Ref. 47). The reduced angiogenesis and host cell infiltration into the matrigel sponge in hCG treated samples is most likely linked to MMP inhibition (32). The direct effect of hCG on MMP activity could be explained by the potential of the cysteine-rich hormone to chelate the bivalent metal ions needed for the activity of these metalloproteases (48, 49). The highly purified r-hCG showed less inhibition of MMP activity in vitro than either the urinary preparation or the ß-subunit or ß-core, which may reflect the limited tumor suppressive activity observed in vivo associated with a suboptimal inhibition of tumor vascularization. It is possible that the major MMP inhibitory activity of hCG resides in the ß-subunit or its fragments, as the ß-subunit showed strong inhibition of angiogenesis in vivo and MMP activity in vitro. This is supported by the lack of activity of LH in inhibition of both MMP activity and angiogenesis in vivo, which suggests that the {alpha}-subunit common to both hCG and LH does not harbor these activities. It is interesting to note that the antiangiogenic activity of urinary hCG fragments reflects the observation that fragments of other proteolytically processed proteins, such as angiostatin and endostatin, have antiangiogenic activity (43, 44).

KS-IMM tumor growth in vivo was inhibited by the urinary preparation of the hormone, an effect that was reproduced by the recombinant form in the initial growth phase of the tumors. Tumor histology showed decreased formation of microvessels for recombinant and urinary hCG-treated samples by 24% and 43%, respectively, indicating that the antitumor effect was due, at least in part, to reduced angiogenesis. This is a demonstration of an antitumor activity for a pure, recombinant form of hCG, although this activity was only transient. It is not clear why the recombinant effect was transient, the microvessel counts suggested that the antiangiogenic effect of r-hCG was insufficient to block tumor growth, again possibly related to a lower level of MMP inhibitory activity. The suppression of tumor growth by urinary hCG over the entire observation period could be due to either an enhanced activity or stability of the u-hCG compared with the recombinant form, or to the presence of additional antitumor activities in urinary hormone preparations. These factors could include hCG fragments with strong MMP inhibitory activity, such as the ß-subunit or ß-core, or other uncharacterized HAFs. Because both the ß-subunit and r-hCG were produced in similar recombinant systems, the lower activity of r-hCG does not seem to be due to production artifacts. It may be possible that the {alpha}ß dimer in the highly purified recombinant material does not have the same activity as the free ß-subunit or its derivatives.

The sex bias of KS indicates that this tumor is clearly influenced by sex hormones, suggesting that female steroids could exert a protective function or male steroids could promote KS growth. This hormone sensitivity appears to be conserved in the KS-IMM cell line, as this line forms smaller, slower growing tumors in female mice than in male mice (50). Because hCG induces the synthesis of steroid hormones by the gonads, the effects observed in the xenograft model could also reflect the induction of steroidogenesis in the recipient mice. We therefore analyzed steroid hormone receptor expression in the KS-IMM cell line by RT-PCR. In these cells, {alpha} and ß estrogen receptors and progesterone receptors were not detected. However, these cells showed clear androgen receptor mRNA expression (data not shown). These data indicate that indirect effects of hCG mediated by female sex steroids can be excluded. While hCG induces testosterone production, it would be unlikely that this is related to the KS tumor growth suppression since the tumor preferentially grows in males.

In conclusion, we show that hCG and the hCG ß-subunit inhibit angiogenesis in vivo that is associated with an inhibition of the enzymatic activity of MMPs and reduction of KS tumor growth. This novel mechanism could explain several controversies surrounding the activity of this hormone on KS and suggest that hCG could also inhibit growth of other angiogenic tumors.


   Acknowledgments
 
We thank Dr. Silvia Donini (Serono) for reagents, Dr. Isabella Paglieri, Dr. Maria Grazia Aluigi, Dr. Giorgia Orengo, Sebastiano Carlone and Luciana Masiello for preliminary experiments, and Dr. Anna Rapetti and Monica Barabino (National Cancer Research Institute Genoa) for data searches and assistance.


   Footnotes
 
This work was supported by Ministero della Sanitá, Italy, Progetto AIDS and Progetto Finalizzato, the Associazione Italiana per la Ricerca Sul Cancro (AIRC), and the Fondazione San Paolo. D.B. is a fellow of the Fondazione Italiana per la Ricerca sul Cancro (FIRC).

Abbreviations: FCS, Fetal calf serum; HAART, highly active antiretroviral therapy; HAF, hcg-associated factor; hCG, human chroionic gonadotropin; HHV-8, human herpes virus 8; HUVE, human umbelical vascular endothelium; KS, Kaposi’s sarcoma; KSHV, Kaposi’s sarcoma associated herpes virus; MMP, matrix metalloproteases; r-hCG, recombinant hCG; u-hCG, urinary hCG.

Received December 18, 2001.

Accepted for publication April 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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