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Experimental Therapeutics, Preclinical Pharmacology |
Institute of Molecular Pathology, University of Copenhagen, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The antiangiogenic compound TNP-470 is an inhibitor of endothelial cells (4) . The mechanism of action is not fully elucidated, but the compound is believed to act by inhibiting methionyl aminopeptidase-2, an intracellular enzyme related to protein myristoylation (5) . Accordingly, the effects of TNP-470 are mediated by the resulting inhibition of membrane proteins, such as nitric oxide synthase, which are translocated to the cell surface membrane by myristoylation (6) . TNP-470 has been shown to inhibit the growth of gliomas and other brain tumors in a number of animal studies (7, 8, 9, 10, 11) .
The combination of radiotherapy and TNP-470 has been investigated in murine mammary and lung carcinomas but not in human glioblastoma multiforme. Murata et al. (12) found that TNP-470 administered before IR did not induce tumor hypoxia, whereas TNP-470 given during fractionated radiotherapy did decrease the radiocurability of murine mammary carcinoma. In studies on murine mammary carcinoma and Lewis lung carcinoma, Teicher et al. (13 , 14) found that TNP-470 in combination with other antiangiogenic agents, given during fractionated radiotherapy, increased the inhibitory effect of radiation on tumor growth. Angiostatin, another potent antiangiogenic agent, has been used in combination with fractionated radiotherapy against human glioma on nude mice with a greater than additive effect on tumor-growth inhibition (15) .
On this basis, we evaluated the effect of combination therapy with TNP-470 and ionizing radiation on human glioblastoma xenografts in the s.c. space and intracranially in the nude mouse. In addition to the tumor response evaluation, we studied the effects on vascular morphology and on the expression pattern of a number of relevant angiogenic factors.
By this approach, the present study provides information of relevance to the introduction of antiangiogenic therapy in effective combinations with other treatment modalities.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor.
The human glioblastoma cell line U 87 MG was originally purchased from
American Type Culture Collection (Rockville, MD). Xenografts
were established by s.c. injection of cells and maintained by serial
transplantation. Prior to transplantation, the mice were anesthetized
by a s.c. injection of ketamine (10 mg/kg) and xylazine (1 mg/kg) in
0.9% NaCl solution. Through a 1-cm incision in the dorsal skin,
1-mm3 tumor blocks were s.c. implanted into both
flanks. For experiments, passage 2 and 3 were used. Intracranial tumors
were established by injection of 1–2 x 106
cells intracranially in the right hemisphere. Cells were injected 2 mm
anterior to the sulcus coronarius, 2 mm lateral to the midline at 2-mm
depth, using a stop device on the injection needle to ensure a
reproducible implantation in all of the mice.
Treatment.
Single-dose 10 Gy tumor X-radiation was given antero-posteriorly to
flank tumors and, as two opposing lateral fields including the whole
brain, to mice with brain tumors. The X-radiation was given during
anesthesia with ketamine/xylazine under aseptic conditions using a
Stabilipan (Siemens) therapeutic unit that yields 4.58 Gy/min at 300
kV.
TNP-470 (O-chloroacetyl carbamoyl fumagillol) was generously provided by Leo Pharmaceutical Products (Ballerup, Denmark). A stock solution of 70% ethanol and TNP-470, 10 mg/ml, was prepared and stored at 4°C. Immediately prior to treatment, the stock solution was diluted with 0.9% NaCl to obtain a dosing concentration of 1 mg/ml. TNP-470 was administered as a daily s.c. injection, 6.7 mg/kg/d. This treatment regimen was found in previous experiments to be efficient, with relatively low toxicity (11) .
Three parallel series of experiments were performed. In each experiment the mice were divided into four groups, given: (a) TNP-470 (6.7 mg/kg/d); (b) IR 10-Gy single dose; (c) TNP-470 + IR in the same doses; or (e) no treatment. TNP-470 was administered for 1 week immediately before IR. In all of the experiments, the IR was applied 1–2 h after the last TNP-470 dose.
In a separate experiment with s.c. tumor transplants, treatment was initiated when an average tumor diameter of 7 mm was reached, and tumors were excised 8 and 48 h after treatment. The excised tumor tissue was processed for morphology studies and expression of angiogenic factors.
Effects on tumor growth were determined in two types of experiments: (a) in s.c. tumors, in which tumor growth was measured; and (b) with intracranial tumor implants using survival as end point.
Growth Calculations.
The s.c. tumor size was measured five times per week. Two measurements
in two perpendicular dimensions (d1
and d2) were recorded using a sliding
gauge, and tumor volume [V(t)] curves were obtained
according to the following formula:
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Nonparametric tests were used for statistics because q-q plots of the growth data clearly demonstrated a non-Gaussian distribution.
Immunohistochemistry.
Tissue was frozen in cooled isopenthane, and frozen sections were fixed
in formalin and postfixed in ethanol:acetic acid (2:1). CD31
immunostaining was performed on sections from tumors in each group.
Sections were washed in PBS and TBS and incubated with 10%
normal rabbit serum for 30 min. They were then incubated with a mixture
of two monoclonal rat antimouse CD31 antibodies at a dilution of 20
µg/ml overnight at 4°C. The antibodies used were clone 390 (Serotec
Ltd.) and MEC 13.3 (PharMingen). Rat IgG2a (Serotec Ltd.) was used as a
negative control. Sections were incubated with biotin-conjugated rabbit
antirat immunoglobulin (DAKO), at a dilution of 1:600 (2.3 µg/ml) for
30 min, washed, and incubated with alkaline phosphatase-conjugated
streptavidin (DAKO) at a dilution of 1:200 (1.5 µg/ml) for 30 min. As
substrate for the alkaline phosphatase reaction, we used freshly
prepared Fast Red Substrate System (DAKO), followed by a 10-min wash in
tap water. After this, sections were counterstained with hematoxylin
and mounted with aqueous mounting media.
Vessel Density.
Vessel density was recorded as the number of point counts of
CD31-positive vessels per field, at x200, viewed through an ocular
Chalkley Point Array (Graticules Limited, Tonbridge, United
Kingdom). Ten fields per section, randomly selected from nonnecrotic
areas of tumors from control and TNP-470 groups, were examined with a
Leica DMRB microscope.
Preparation for Electron Microscopy.
After a brief fixation in 70% Karnovsky’s fixative (3%
paraformaldehyde and 3.5% glutaraldehyde in 0.1 M
cacodylate buffer) each tumor was cut into tissue slices of 760 µm
with an EMS Automatic Oscillating Tissue Slicer (Electron
Microscopy Sciences, Fort Washington, PA). The slices were
immersed in Karnovsky’s fixative for 1 h and subsequently washed
and stored in 0.1 M cacodylate buffer. Six to ten small
tissue blocks were randomly sampled from tumor slices from each group
using a biopsy needle with a diameter of 1 mm. The tissue blocks were
further fixed in 1% osmium tetraoxide in 0.1 M cacodylate
buffer, dehydrated in ethanol followed by 1,2-propylenoxide, and
embedded in Epon.
Ultrathin (70–80 nm) sections were cut on a RMC, MT 6000-XL ultramicrotome. Sections were mounted on copper grids and contrasted with 4% uranyl-acetate (30 min) and Reynolds lead-citrate (2.5 min). Ultrathin sections were examined in a JEOL electron microscope.
Northern Blotting.
Tumor tissue blocks of approximately 0.25 g were frozen in liquid
nitrogen. Polyadenylated RNA was extracted and isolated using the
guanidinium thiocyanate-based QuickPrep mRNA Purification Kit (Amersham
Pharmacia Biotech). The mRNA concentration was measured by
spectrophotometry. Three µg of denatured mRNA were separated on 1%
agarose gels containing 6.6% formaldehyde and were subsequently
transferred by capillary electrophoresis in 10x SSC buffer to a nylon
membrane (GeneScreenPlus, NEN Research Products). The mRNA was
cross-linked to the membranes with 1200 J UV light in a UV stratalinker
1800 (Stratagene). Membranes were prehybridized for 3 h in 5%
dextran sulfate, 50% formamide, 1% SDS, 1 M NaCl, and 100
µg/ml salmon sperm DNA at 42°C and hybridized with
[32P]-labeled cDNA probes overnight at
42°C. The mRNA expression was visualized on a phosphorimager (STORM
840, Molecular Dynamics Inc.), quantified by the software program Image
Quant version 5.0 (Molecular Dynamics Inc.), and the VEGF, bFGF,
angiopoietin-1, and angiopoietin-2 expression was adjusted to
the GAPDH expression. To compare data from different membranes, mRNA
from U87 cells in vitro was loaded on all of the gels as a
common standard. We used cDNA probes encoding VEGF, bFGF,
angiopoietin-1 and angiopoietin-2, and GAPDH (Clontech).
Immunoblotting.
Tissue blocks were thawed on ice and homogenized in lysis buffer with
four short bursts on a Vibra Cell 50 (Sonics & Materials Inc.). The
protein concentration was determined with a BCA Protein Assay
(Pierce Inc.) and 20/40 µg of protein from each tumor were separated
on 1.0-mm NuPage 10% Bis-Tris gels (NOVEX) and transferred onto
PVDF membranes (NOVEX) by semi-dry blotting.
Membranes were blocked in Tris-buffered saline (pH 7.4), 10% skim milk
powder, and 0.1% Tween 20, and washed in Tris-buffered saline (pH
7.4). Incubation with primary and secondary antibodies for 1–16 h was
followed by washing in Tris-buffered saline (pH 7.4) and 0.1% Tween
20. The protein expression was visualized with ECL+ Plus (Amersham) and
quantified by chemifluorescence scanning on a STORM 840 (Molecular
Dynamics Inc.). Expression of VEGF protein was adjusted to the
-tubulin expression. The following antibodies and concentrations
were used: (a) monoclonal mouse-antihuman VEGF (1 µg/ml,
PharMingen); and (b) polyclonal goat-antihuman bFGF (0.2
µg/ml; R&D Systems). Monoclonal mouse-anti--tubulin
(1:10,000) was obtained from Sigma Chemical Co.
Horseradish peroxidase-conjugated polyclonal
goat-antimouse-immunoglobulin (1:3000 and 1:5000) and horseradish
peroxidase-conjugated polyclonal rabbit-antigoat-immunoglobulin
(1:2000) was obtained from DAKO.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tables 1
and 2
).
All of the tumors eventually regrew, but some tumors in the combination
group started growing very late after treatment. The time for tumors in
the combination group to reach a volume of 600
mm3 (T600, Table 1
) was significant longer than in all of the other tumor groups.
Intracranially implanted U87 tumors were treated with TNP-470, IR, or
combination therapy in the same doses. In these mice, TNP-470 treatment
did not affect survival (Fig. 2)
, whereas
the expected effect of single-dose IR was observed.
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).
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). At the ultramorphological
level, untreated tumor vessel endothelium in nonnecrotic areas was
generally found to be continuous with tight junctions between
endothelial cells (Fig. 4B
). With light microscopy, an
increased thickness of vessel walls was observed in TNP-470-treated
tumors (Fig. 3C
) and, correspondingly, by electron
microscopy, an approximately 400–700-nm thick layer of electron-dense
material was ubiquitously observed along the endothelial basement
membrane (Fig. 4C
). This material was found in
TNP-470-treated tumors with and without additional radiotherapy but not
in untreated controls nor in tumors treated with ionizing radiation
only.
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), despite a pronounced growth-retarding
effect of TNP-470. The mean number of vessel counts (Chalkley point
counts) per field in TNP-470-treated tumors was 5.0 (4.5- 5.5) compared
with 4.6 (4.2–5.0) in control tumors (parentheses indicate 95%
confidence intervals). A t test for equality of means
yielded no significant difference (P = 0.13).
Effect of Treatment on Expression of Angiogenic Factors.
The expression of VEGF, bFGF, angiopoietin-1, and angiopoietin-2 at
mRNA level in different treatment groups was examined using Northern
blots. Four membranes, each comprising tumor-tissue mRNA from one of
the mice from each treatment group, were analyzed. We found no
significant difference in GAPDH-adjusted expression of VEGF,
angiopoietin-2, and bFGF between groups (Table 3
and Fig. 5
), whereas a significantly greater
expression of angiopoietin-1 was observed at 48 h after withdrawal
of the 1-week TNP-470 treatment (Table 3)
.
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The mRNA expression of VEGF, bFGF, angiopoietin-1, and angiopoietin-2
per total amount of mRNA was, without exception, greater in cultured
U87 cells in vitro than in the corresponding U87 tumors
grown in vivo (Fig. 5)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In contrast, similar treatment schedules with TNP-470 had no effect on the survival of mice with intracranial tumors, given alone or in combination with IR. This discrepancy may reflect differences in tumor vessels due to differences in tumor microenvironment. In the present study, the TNP-470 treatment of intracranial tumors was applied while tumors were relatively small, i.e., from day 7 to 14 after the injection of tumor cells. Recently, Holash et al. (16) proposed a preangiogenic phase, in which small tumor implants are predominantly supported by cooption of existing brain vessels. They observed that intracranially injected glioma cells grew around existing vessels in the first 2 weeks, and that de novo angiogenesis did not start until later. This could explain the presumable lack of effect on the intracranial formation of tumor vessels in the present study because a much smaller tumor volume is lethal in the brain. To what extent the blood-brain barrier also plays a role for the delivery of TNP-470 to endothelial cells in brain tumors is uncertain. The microenvironment of the brain stimulates formation of a blood-brain barrier, and the U87 tumor environment may disrupt the vascular barriers through release of VEGF and other cytokines. According to the competitive environment hypothesis, the outcome of the competition between these environmental factors determines the intracranial tumor vessel permeability (17) . Previous reports on TNP-470 treatment of intracranial gliomas are equivocal. Takamiya et al. (18) treated mice with U87 tumors with TNP-470 from day 5 after implantation until death and found prolonged survival compared with controls. Wilson and Penar (19) found no effect of TNP-470 on survival in mice with intracranial gliosarcomas.
We observed specific morphological effects of IR on tumor endothelium that apparently were avoided by pretreatment with TNP-470. Single-dose radiation in the same doses as used here has been shown to induce an immediate increase in vascular permeability (20) . On this basis, we suggest that TNP-470 inhibits the acute endothelial cell membrane damage and edema that are normally seen after irradiation. We, therefore, propose a potential clinical synergy of the combination of TNP-470 and radiotherapy because TNP-470 apparently prevents an acute side effect of ionizing IR while adding to its beneficial effects.
The extensive accumulation of basement membrane material in vessels of tumors treated with TNP-470 is, to our knowledge, a new observation. Perivascular amorphous matrices not organized as a conventional basal lamina have been observed after gentamicin and cisplatin treatment in normal brain and in some malignant tumors and benign lesions4 (21) . The composition and nature of the basement membrane-related material found in TNP-470-treated tumors are not defined at present. Angiopoietin-1 is considered a regulator of vascular remodeling subsequent to the formation of proliferating vascular sprouts by interaction with pericytes through pathways distinct from those of VEGF (22 , 23) and also in concerted action with VEGF (16) . The increased expression of this proangiogenic factor after termination of TNP-470 treatment may be related to the morphological changes discussed above. This potential relationship deserves further experimental validation.
The tumor vessel endothelium in the nonirradiated tumors in this study was generally continuous with tight junctions between endothelial cells. Fenestrations were rarely seen. Others have found that U87 tumors on nude mice have fenestrations in 37% of vessels, with 1–44 fenestrations per vessel profile (24) . The reason for this difference is not clear. One explanation may be the passage number used because they (24) used U87 tumors passed more than 7 times on nude mice to select for aggressive, faster growing, and more angiogenic tumors. We used passage 3 for our morphological studies.
There is a theoretical problem with combining radiotherapy and antiangiogenesis because the biological effect of ionizing irradiation is sensitive to hypoxia. Antiangiogenic agents, by their inhibition of vessel formation, are sometimes believed to decrease vessel density, and, as a consequence, tumor hypoxia would increase. Assuming that angiogenesis is a growth-limiting step in tumor growth—which is why antiangiogenic therapy works—the ratio of tumor cells to vessels, i.e., vessel density, must remain relatively constant in an individual tumor unless its nutritive demands are changed. Concordantly, we observed no significant change in Chalkley counts of tumor vessels after 1 week of TNP-470 treatment despite a significant growth inhibition. Also, with a 6-week TNP-470 schedule in human glioblastoma xenografts, no changes in vascular density were reported (7) , whereas others have actually found a decrease in vessel density after TNP-470 treatment of brain tumors (8, 9, 10) .
TNP-470 in combination with another antiangiogenic compound, minocycline, has been shown to actually decrease tumor hypoxia (25) . We found no change in the VEGF expression, and, because VEGF is known to be up-regulated by hypoxia (26, 27, 28) , this further supports our notion that TNP-470 does not induce hypoxia.
In summary, pretreatment with TNP-470 significantly enhanced the growth-retarding effect of ionizing IR, while preventing radiation-induced microvascular damage. A broadening of the endothelial basement membrane and an increased expression of angiopoietin-1 seem to be involved in this protective mechanism. The vessel density was found unaltered by the antiangiogenic treatment, which further adds to the evidence that vessel counts are not reliable parameters for the evaluation of antiangiogenic effect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by The Danish Cancer Research
Foundation, The Danish Medical Research Council (Grant 9700929), and
Danish Cancer Society (Grant 9810034). 
2 To whom requests for reprints should be
addressed, at Molecular Pathology, University of Copenhagen, Frederik
V’s Vej 11, DK-2100 Copenhagen, Denmark. Phone: 45-3532-6006; Fax:
45-3532-6081; E-mail: paulk{at}pai.ku.dk
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; bFGF, basic fibroblast growth factor; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
4 B. Eyden. Perivascular amorphous matrices
containing laminin and type IV collagen not organized as a conventional
basal lamina: identification by electron microscopy and implications
for the control of cell biological processes, submitted for
publication.
Received 9/30/99; revised 12/20/99; accepted 12/20/99.
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