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Experimental Therapeutics, Preclinical Pharmacology |
Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center and Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112
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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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Recently we developed a cytotoxic SST analogue AN-238 (9) , consisting of the SST carrier octapeptide RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) covalently linked to 2-pyrrolinodoxorubicin (AN-201), a derivative of doxorubicin (DOX), which is 500-1000 times more potent in vitro than the parent compound (10) . This analogue fully retains the cytotoxic activity of the radical and the receptor binding affinity of the peptide carrier (9) and has been previously shown to inhibit significantly the growth of various tumors that express SST receptors (11, 12, 13) .
In this study, we evaluated the antiproliferative effects of targeted cytotoxic SST analogue AN-238 in vitro and in vivo in mice bearing s.c. xenografts of human malignant glioblastoma cell line U-87 MG, which expresses high affinity binding sites for SST (14) . In addition, we studied the effect of AN-238 on the survival time of mice with U-87 MG cells inoculated orthotopically into the brain. A comparison of the antiproliferative activity of AN-238 and AN-162, a targeted SST analogue containing DOX, was also performed in nude mice bearing s.c. xenografts of U-87 MG human glioblastomas.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Cytotoxicity in Vitro.
The human malignant glioblastoma cell line U-87 MG was obtained from
American Type Culture Collection (Manassas, VA) and cultured as
described previously (14)
. Cells were passaged weekly and
routinely monitored for Mycoplasma contamination using a
detection kit (Boehringer Mannheim, Mannheim, Germany). All culture
media components were purchased from Gibco (Grand Island, NY). The
cytotoxic activity of the analogues was determined in vitro
using a colorimetric assay based on quantitation of biomass by staining
cells with crystal violet (16)
. IC50
values, defined as the drug concentration that inhibited cell growth by
50% compared with untreated control cultures, were calculated from
three independent experiments, consisting of seven replicate tests
each, after exposure of U-87 MG cells to cytotoxic agents for 116 h.
Animals.
Male athymic (NCr nu/nu) nude mice, 5–6 weeks
old, were obtained from the National Cancer Institute (Bethesda, MD).
The mice were housed in sterile cages under laminar flow hoods in a
temperature-controlled room with a 12-h light/12-h dark schedule and
were fed autoclaved chow and water ad libitum. All
experiments were performed in accord with institutional ethical
guidelines.
Experimental Protocol.
U-87 MG malignant glioblastomas growing exponentially were implanted
into five male nude mice by s.c. injection of 5 x
106 cells in the right flank. Tumors resulting
after 2 weeks in donor animals were aseptically dissected and
mechanically minced. Pieces of tumor tissue (3
mm3) were transplanted (s.c.) by trocar needle
into 100 mice under methoxyflurane (Metofane, Pittman-Moore, Mundelein,
IL) anesthesia. The take rate was 100%. In the first part of the
experiment we used tumors that had grown to approximately 400–500
mm3. Mice were divided into five groups, of five
to eight mice per group, and received the following treatment as a
single i.v. injection in the tail vein: group 1, control, vehicle
solution (5% mannitol); group 2, cytotoxic radical AN-201 at a dose of
150 nmol/kg; group 3, unconjugated mixture of the cytotoxic radical
AN-201 and the carrier RC-121 at a dose of 150 nmol/kg; group 4,
cytotoxic analogue AN-238 at a dose of 150 nmol/kg; group 5, carrier
peptide RC-121 at a dose of 150 nmol/kg. For the second part of the
experiment, we selected 19 mice bearing large tumors of approximately
900 mm3. The mice were divided into two
experimental groups of 9 and 10 animals each and received within 10
days two sequential i.v. injections of AN-201 or AN-238, respectively,
at 150 nmol/kg. In the third part of the experiment, mice bearing U-87
MG xenografts, measuring approximately 450 mm3,
were divided into two experimental groups and pretreated with RC-121
before the injection of AN-238 as follows: group 1 (n =
10 mice), RC-121 s.c. at 50 µg/day per animal for 7 days prior to the
i.v. injection of AN-238 at 150 nmol/kg; group 2 (n = 5
mice), RC-121 as a single s.c. injection of 500 µg per animal, 4 h before the administration of AN-238 at 150 nmol/kg. Three mice were
sacrificed after treatment with RC-121 for 7 days for receptor studies.
Another experiment was performed in two groups of four mice each,
bearing U-87 MG xenografts measuring 500–600
mm3. The first group received an i.v. injection
of AN-238 at 150 nmol/kg, and the second group received AN-162 at 13.75
µmol/kg. Eight days after the injection, the mice treated with AN-238
were sacrificed, while the mice treated with AN-162 were anesthetized
and their tumors were partially removed. Twelve days later, when their
tumors reached a volume of approximately 500 mm3,
mice were divided into two groups of two mice each, and each group
received AN-238 at 150 nmol/kg or AN-162 at 13.75 µmol/kg.
During all experiments, tumor volume was measured twice a week using microcallipers (14) , and changes in body weights and deaths related to toxicity were recorded. At the end of the experiments, autopsy was performed and the tumor weights were measured. Samples of each tumor were either fixed in formalin for histological examination or snap-frozen at -70°C for preparation of membranes for receptor assay and for extraction of RNA for reverse transcription-PCR.
Survival Test.
U-87 MG cells were inoculated into the brains of 33 six-week old
athymic male NCr nu/nu nude mice as described previously
(14)
. Briefly, mice were anesthetized with methoxyflurane
(Metofane, Pittman-Moore, Mundelein, IL). A midline incision was made
over the anterior aspect of the cranium and the scalp retracted to the
right. A guarded 26-gauge needle was used to drill a hole 3–4 mm deep
in the skull, 3 mm to the right of the midline, just anterior to the
coronal suture. Using a syringe (Hamilton, Reno, NV) 15 µl of 0.9%
sterile sodium chloride containing 5 x 104
U-87 MG glioblastoma cells was injected and the scalp was closed with
surgical skin staples. Two days after the inoculation of the cells,
mice were randomly divided into three experimental groups of 11 mice
each and received the following treatment as a single i.v. injection:
group 1, control, vehicle solution (5% mannitol); group 2, cytotoxic
radical AN-201 at a dose of 150 nmol/kg; group 3, AN-238 at a dose of
150 nmol/kg.
Histological Methods.
Tumor samples were processed as described previously (17)
.
The extent of necrosis was evaluated with the point-counting method on
tumor slides stained with H&E. For the measurement of the number of
mitotic and apoptotic cells, 10 high power fields were considered and
the numbers of mitotic and apoptotic cells per 1000 cells (mitotic and
apoptotic indices) were calculated in H&E-stained slides.
Receptor Assay.
Binding characteristics of SSTRs on membrane fractions of U-87 MG
tumors were determined by ligand competition assays using
125I-labeled RC-160 as reported previously
(9)
. The LIGAND PC computerized curve-fitting program
(18)
was used to determine the type of receptor binding,
the dissociation constant (Kd), and
the maximal binding capacity (Bmax) of
receptors (18)
. Receptor binding affinity of cytotoxic SST
analogue AN-238 as well as its carrier peptide RC-121 to tumor
membranes was measured in displacement experiments based on competitive
inhibition of 125I-RC-160 binding using various
concentrations of AN-238 and RC-121
(10-6-10-12
M). IC50, defined as the
dose causing 50% inhibition of 125I-labeled
RC-160 binding, was calculated by a computerized curve-fitting program
(19)
.
RNA Extraction and Reverse Transcription-PCR.
Total RNA was isolated using the RNAzol B reagent (TEL-TEST, Inc.,
Friendswood, TX) following the manufacturer’s instructions. The
quantity and the quality of the RNA was assessed by spectrophotometry
at 260 and 280 nm. For the reverse transcription 1 µg of total RNA
was added in a test tube containing 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 5 mM
MgCl2, 1 mM of each
deoxyribonucleoside triphosphate, 2.5 µM random
hexamers, 1 unit of RNase inhibitor and double distilled
H2O in a final volume of 19 µl. Following
heating for 10 min at 65°C and quenching on ice, 2.5 units of Moloney
murine leukemia virus reverse transcriptase (Perkin-Elmer, Norwalk, CT)
was added and the reaction mixture was incubated for 10 min at room
temperature following an incubation at 42°C for 1 h. The
reaction was terminated by heating at 95°C for 5 min and quenching on
ice. Subsequently, cDNA was amplified by the PCR as follows: 1 µl of
the cDNA was amplified in a 50-µl solution containing 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5
mM MgCl2, 200 µM of
each deoxynucleotide triphosphate, 2.5 units of Taq DNA poly-merase
(Perkin-Elmer, Norwalk, CT) and 0.4 mM of each primer. The
primers used were 5'-TCCTCTGACTTCAACAGCGACACC-3' and
5'-TCTCTCTTCCTCTTGTGCTCTTGG-3' for human glyceraldehyde-3-phosphate
dehydrogenase (hGAPDH), 5-ATGGACATGGCGGATGAGC-CACT-3' and
5'-TACTGGTTTGGAGGTCTCCATTGA-3 for SSTR-2, and
5'-CGTCTTCATCATCTACACGG-3' and 5'-GGCCAGGTTGACGATGTTGA-3 for SST
receptor subtype-5. Samples were denatured at 94°C for 5 min and then
subjected to 40 cycles of 94°C for 1 min (denaturation), 62°C for 1
min (annealing), and 72°C for 1 min (extension) for SSTR-2; 45 cycles
of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s
for SSTR-5; or 25 cycles of 94°C for 35 s, 60°C for 40 s,
and 72°C for 40 s for hGAPDH. This was followed by a final
extension at 72°C for 5 min, using a DNA thermal cycler (Perkin-Elmer
model 2400). The number of cycles was determined in preliminary
experiments to be within the exponential range of PCR amplification.
Five µl of each PCR product was electrophoresed in a 8%
polyacrylamide gel and stained with silver.
Statistical Analysis.
The data are expressed as the mean ± SE. Statistical evaluation
of data were performed by Student’s t test (two-tailed).
Differences were considered statistically significant when
P 
0.05. SigmaPlot computer software (Jandel, San
Rafael, CA) was used for preparation of figures.
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RESULTS |
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. Administration of a single i.v.
injection of AN-238 at 150 nmol/kg produced a significant tumor growth
inhibition. Nineteen days after the injection, the tumor volume in
animals treated with AN-238 was significantly (P =
0.00168) reduced to 867 ± 313 mm3 as
compared with the control group, which measured 4782 ± 1012
mm3; this corresponded to a growth inhibition of
82% (Fig. 1A)
. In contrast,
tumors in mice treated with an equimolar dose of AN-201 given alone and
as an unconjugated mixture with RC-121 grew steadily and measured
3133 ± 1256 mm3 and 5200 ± 713
mm3, respectively, 19 days after the injection.
Treatment with AN-201 alone or together with RC-121 also resulted in
the death of 1 of 6 and 1 of 5 animals, respectively (Table 1)
. The
carrier peptide RC-121 was not toxic, but had no tumor inhibitory
effects after a single i.v. injection of 150 nmol/kg. Losses in body
weights occurred in all groups treated with cytotoxic agents, but the
body weights of the surviving animals returned to normal by the end of
the experiment (Fig. 2)
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and
Fig. 1B
, the final tumor volume in the mice treated twice
with AN-238 regressed to 637.5 ± 248.3 mm3
(P = 0.00046) from an initial tumor volume of
890.7 ± 378 mm3, corresponding to a
decrease of 30%. The U-87 MG tumors in animals treated with cytotoxic
radical AN-201 grew steadily even after the administration of the
second injection, reaching 3274 ± 795 mm3
19 days after the first injection of the cytotoxic radical. The tumor
volume in this group was 510% higher than that in mice treated with an
equimolar dose of AN-238. AN-201 was also more toxic than AN-238. Three
of 10 animals died in the group that received AN-201, but only 1 of 9
animals was dead after treatment with AN-238. Significant losses of
body weights were recorded in all animals, but the surviving animals
started to recuperate before the end of the experiment (Fig. 2)
.
Effect of Pretreatment with RC-121 on the Antitumor
Activity of AN-238.
In the third part of this experiment, mice bearing approximately
450-mm3 xenografts of U-87 MG glioblastomas were
pretreated s.c. with the carrier peptide SST analogue RC-121, before a
single i.v. administration of AN-238 at 150 nmol/kg. RC-121 was given
at a dose of 50 µg/day per animal for 7 days or at 500 µg per
animal 4 h before the injection with AN-238. The experiment in
which RC-121 was administered chronically at a low dose was performed
to ascertain whether pretreatment with RC-121 could produce a higher
tumor inhibition than AN-238 alone, by a possible up-regulation of the
SST receptors on the U-87 MG tumors. Acute administration of RC-121 at
a high dose was intended to produce a blockade of the SST receptors on
the tumor cells that could decrease or abolish the antitumor effect of
AN-238.
As shown in Table 1
and Fig. 1C
, pretreatment with RC-121
for 7 days followed by a single i.v. injection of AN-238 at 150 nmol/kg
did not produce a higher tumor inhibition than administration of AN-238
without pretreatment with RC-121. However, the necrotic area of tumors
from pretreated animals was increased by 102% as compared with the
tumors from the untreated control mice (P = 0.041),
whereas the area of necrosis in the tumors from mice that received
AN-238 alone was only 16% higher (not significant) than that of the
controls (Table 2)
. SST analogue carrier
peptide RC-121, administered s.c. at a dose of 500 µg 4 h before
the injection with AN-238 to nude mice bearing xenografts of U-87 MG
glioblastomas, blocked the tumor inhibitory effects of the cytotoxic
SST analogue AN-238 the resulting inhibition being not significant
versus control (Fig. 1C)
. The inhibition of tumor
growth produced by AN-238 alone was 82%, while administration of a
high dose of RC-121 before the injection with AN-238 reduced the tumor
inhibition to 37% (P = 0.051 versus
AN-238-treated group).This tumor inhibition was similar to that
obtained with the cytotoxic radical alone, being 32% versus
the controls (Table 1)
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shows that 8 days after the treatment with AN-162 tumors grew from
566 ± 72 mm3 to 1368 ± 439
mm3, while AN-238 produced a significantly
stronger antitumor effect (P = 0.0281), virtually
arresting tumor growth. The loss of body weights caused by AN-162 was
slightly higher than that caused by AN-238, being 13% (3.6 g)
versus 8.5% (2.5 g), respectively. This implies that
administration of a higher dose of AN-162 could have been lethal.
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, the tumors from mice treated with AN-162
continued to grow, while the tumors from mice treated with AN-238
regressed by 50% within 8 days.
Effect of AN-238 on the Survival Time of Mice Bearing
Orthotopically Implanted U-87 MG Cells.
The life span of mice inoculated orthotopically with 5 x
104 U-87 MG cells was prolonged significantly by
a single administration of cytotoxic SST analogue AN-238, as compared
to untreated mice. The mean survival times of mice in the control
group, and groups treated with AN-238 or AN-201 at 150 nmol/kg were
23.8 ± 2, 33.7 ± 3.1, and 29.4 ± 2.5 days,
respectively (Fig. 4)
. Thus, treatment
with AN-238 increased the mean survival time of mice inoculated
orthotopically with U-87 MG glioblastomas by 10 days (P = 0.00987), which represents an extension in their life span by 42%.
Treatment with cytotoxic radical AN-201 at 150 nmol/kg also increased
the survival time by 5.5 days versus the controls, but this
was not significant statistically (P = 0.0853).
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Histological Findings.
In some areas, U-87 MG tumors consisted of relatively small elongated
cells arranged in bundles. In other areas, the cells were larger and
more rounded containing pale nuclei with big nucleoli. As shown in
Table 2
, all groups that received AN-238 had a larger area of necrosis
than the controls. The most extensive necrosis was found in tumors from
the mice treated with AN-238, after 1 week of pretreatment with RC-121.
The area of necrosis in tumors from mice treated with AN-201 was
essentially equal to that of the controls. Mitotic and apoptotic
indices, as well as their ratio were not altered by the treatment with
cytotoxic SST analogue AN-238 or cytotoxic radical AN-201 (data not
shown).
Receptor Binding Studies and Reverse Transcription-PCR in
U-87 MG Tumors.
Ligand competition assay showed a single class of high affinity binding
sites for SST in membranes of U-87 MG glioblastoma xenografts with a
mean dissociation constant (Kd) of
9.77 ± 2.17 nM and a mean maximal binding
capacity (Bmax) of 835 ± 130
fmol/mg membrane protein (Fig. 5A)
. After a single or
repeated treatment with AN-238 there were no significant changes in the
binding affinities (Kd = 6.95 ±
0.95 and 7.79 ± 2.33 nM, respectively) and
the capacities (Bmax = 784 ± 153
and 715 ± 154 fmol/mg protein, respectively) of the SST
receptors. Similarly, Kd and
Bmax values were not affected when
AN-238 treatment was performed after pretreating the tumor bearing mice
with a high dose of the carrier peptide RC-121
(Kd = 10.5 ± 3.25
nM; Bmax =
930 ± 146 fmol/mg protein). In three animals treated with RC-121
alone at a dose of 50 µg/day for 7 days and then sacrificed, we found
no statistically significant changes in the binding affinities
(Kd = 9.85 ± 2.58
nM), although the concentration of receptors for
SST was slightly, but not significantly higher (1041 ± 97 fmol/mg
membrane protein) than in the control group.
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. Cytotoxic SST
analogue AN-238 retained the high binding affinity of the peptide
hormone portion of the conjugate to membrane receptors for SST in U-87
MG tumors, displaying a similar IC50 value of
3.67 ± 0.46
.
The expression of SSTR subtype in U-87 MG glioblastomas was then
investigated. Using reverse transcription-PCR analysis, mRNA for SSTR
subtype 2, but not for SSTR subtype 5, was found in xenografts of U-87
MG glioblastomas from untreated animals (Fig. 5C)
.
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DISCUSSION |
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Our results demonstrate that AN-238 can powerfully inhibit the growth of large (approximately 500 mm3) U-87 MG tumors by some 80% without severe toxicity, while the unconjugated cytotoxic radical AN-201 has no significant effect on tumor growth and is more toxic. We also found that a single administration of AN-238 at a nontoxic dose of 150 nmol/kg arrested growth of very large (approximately 900 mm3) U-87 MG xenografts, while a second injection of AN-238 at the same dose resulted in a 30% regression. Nineteen days after the initiation of the therapy, the experiment had to be terminated because the tumor volume in the controls was very high (4782 ± 1012 mm3). Thus, although a third administration of AN-238 would have been worthy of testing, it could not be applied.
It has been observed in U-87 MG tumors that chronic treatment with SST analogue RC-160 somewhat increased the binding capacity of SST receptors (14) . To test whether such an increase could enhance the antiproliferative activity of AN-238, SST carrier analogue RC-121 was administered for 7 days at 50 µg/day per animal prior to the administration of AN-238. The resulting slight, but not significant up-regulation of SST receptors by pretreatment with RC-121 did not further enhance significantly the antitumor activity of AN-238. However, histological examination revealed that tumors from the RC-121 pretreated group had significantly (P = 0.041) higher content of necrotic cells than did the tumors from the untreated mice or tumors from mice treated only with AN-238.
When RC-121 was injected s.c. at the high dose of 500 µg per animal, 4 h before the administration of AN-238 at 150 nmol/kg, the antitumor effect of AN-238 was abolished and the resulting tumor inhibition was similar to that of cytotoxic radical AN-201. It is likely that the excess of RC-121 occupied the SST receptors on the tumors, while the unbound cytotoxic SST analogue AN-238 was hydrolyzed by carboxylesterase enzymes (EC 3.1.1.1) in blood releasing unconjugated AN-201. This finding supports strongly the concept that the antitumor effect of AN-238 is receptor-mediated.
Although AN-238 was less toxic than AN-201 in these experiments on the
basis of animal mortality, the trends in the loss of body weights in
animals treated with AN-238 or AN-201 (Fig. 2)
indicate a similar
toxicity pattern for the two compounds. This is in agreement with our
previous finding that the maximum tolerated doses of both compounds are
similar in male nude mice, about 175–200 nmol/kg for single i.v.
injections, and are determined by hematopoietic toxicity
(12)
. However, Copenhagen rats bearing R-3327-AT-1 rat
Dunning prostatic carcinoma tolerate a single i.v. injection of 300
nmol/kg without apparent toxicity, whereas 115–125 nmol/kg of AN-201
is lethal (11)
. Because in AN-238 an ester bond is used to
link AN-201 to the carrier peptide, different tolerance in these
species could be explained by variations in the carboxylesterase enzyme
activity. We found that one of our cytotoxic luteinizing
hormone-releasing hormone analogues, AN-152, in which DOX is linked to
the carrier peptide through the same ester bond, had a half-life of 10
min in nude mouse serum, 30 min in Copenhagen rat serum, and 120 min in
human serum at 37°C in vitro (12
, 20)
. The
longer half-life of the conjugate in the blood may allow a better
targeting, and apparently the DNA-intercalating antiproliferative agent
AN-201 does not cause side effects that are life-threatening after
being absorbed by noncancerous SST receptor-positive tissues. A large
difference in the carboxylesterase activities in rodents and human
beings is well known (21)
. Mice can tolerate a much higher
dose of organophosphate acetylcholinesterase (EC 3.1.1.7.) inhibitors
than primates, because these toxic agents also react with
carboxylesterases present at much higher levels in mice, but the
inhibition of these enzymes causes no toxicity (21)
. In
accordance with our hypothesis that the extremely high esterase
activity is responsible for the toxicity pattern of AN-238 in mice, we
found recently that healthy nude mice without tumors can tolerate two
consecutive i.v. injections of 400 nmol/kg AN-238 without severe
toxicity after treatment with organophosphate esterase
inhibitors.4
Because of the very low esterase activity in human beings, we can
speculate that it might be possible to give even much higher doses of
AN-238 to humans. This is important because, as we demonstrated
previously (11)
and also in this study, the effect of
AN-238 is dose-dependent.
The antitumor effects of AN-238 were also compared to those of AN-162, its counterpart cytotoxic SST analogue containing DOX instead of the highly potent 2-pyrrolinodoxorubicin. Under our experimental conditions, AN-162 could not inhibit the growth of large U-87 MG tumors, while treatment with AN-238 resulted in the stabilization of the tumor growth. In this experiment both AN-238 and AN-162 were used at equitoxic doses. In fact, the loss of body weights in the AN-162 treated animals was higher than that in the mice treated with AN-238, indicating that administration of a higher dose of AN-162 could be lethal. The striking difference in the antitumor activity of these two cytotoxic SST analogues could be due to differences in the mechanism of action of DOX and AN-201 and/or the limited number of receptors on tumors available for targeting. When these tumors, which have been unsuccessfully treated with AN-162, were subjected to therapy with AN-238, they regressed by 50% within 8 days, demonstrating that the 2-pyrrolinodoxorubicin-containing analogue still had full antiproliferative activity on U-87 MG tumors even after pretreatment of these tumors with the DOX-containing analogue, that could have resulted in the selection of cells resistant to chemotherapy. This is in agreement with our previous findings that AN-201 and AN-238 showed very strong antitumor effect on MX-1 human mammary carcinoma, which is resistant to DOX (13) .
The difference in the potency between these cytotoxic agents was also demonstrated by in vitro studies in which AN-201 and AN-238 exhibited approximately 150 times lower IC50 values than DOX and AN-162, respectively, in this cell line. No difference was found in the antiproliferative activity between the cytotoxic radicals and their corresponding cytotoxic SST analogues in vitro. This was probably due to a fast hydrolysis of AN-238 in vitro.
An intratumoral injection of anticancer agents has been previously suggested for the treatment of brain tumors (22 , 23) , whereas recently, the locoregional administration of the SST analogue 90Y-labeled DOTA0-D-Phe1-Tyr3-octreotide was tested successfully in glioma patients (24) . We felt that it was still worthwhile to evaluate whether systemic administration of AN-238 could extend the life span of nude mice bearing U-87 MG tumors inoculated orthotopically into the brain. Mice treated with a single nontoxic dose of 150 nmol/kg of AN-238 had their life span extended by 42% (P = 0.00987), as compared to that of the untreated mice, suggesting that AN-238 may penetrate to a certain extent the tumor blood-brain barrier when systemically administered. This is in agreement with the results of a previous study showing that somatostatin octapeptide analogue RC-160 (vapreotide, octastatin) could penetrate the weakened blood-brain tumor barrier in KHT sarcomas (25) . AN-201 did not significantly affect the survival of mice with orthotopic implants of U-87 MG tumors.
The radiolabeled ligand competition assay confirmed the high affinity binding of cytotoxic SST analogue AN-238 to the membrane fraction of U-87 MG xenografts, which is in agreement with the detection of mRNA for SST receptor subtype 2 in these tumors. Thus U-87 MG glioblastomas can be considered representative of primary brain tumors that express high levels of SSTRs and especially SSTR-2. The high affinity binding sites for SST analogues were preserved after two injections of AN-238, indicating that repeated treatment by targeted cytotoxic SST analogue could be applied.
Histological analysis showed that the antitumor activity of AN-238 was associated with an induction of necrosis, while the apoptotic index remained unaffected by the treatment. This is likely due to the high cytotoxic activity of this agent, which is at least 90 times more potent in vivo and approximately 150 times more potent in vitro than AN-162, the corresponding DOX-containing analogue, and may induce necrosis rather than apoptosis (26) in this cell line.
In conclusion, we have shown that cytotoxic SST analogue AN-238, consisting of 2-pyrrolinodoxorubicin, a superactive derivative of DOX, linked to the SST octapeptide analogue RC-121, could be targeted to malignant glioblastomas that express receptors for SST, producing regression of even very large tumors, while 2-pyrrolinodoxorubicin itself is ineffective and more toxic. Additional experiments, involving additional models of glioblastomas and various dosage regimens, are required to establish the full spectrum of antitumor effects of AN-238 and to predict more accurately the subset of primary glioblastomas that might respond to such therapy. Our results suggest that targeted chemotherapy with cytotoxic SST analogues such as AN-238 could be a promising new modality for the management of malignant brain tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The work described in this paper was supported
by the Medical Research Service of the Veterans Affairs Department and
a grant from ASTA Medica (Frankfurt am Main, Germany) to Tulane
University (all to A. V. S.). 
2 To whom requests for reprints should be
addressed, at VA Medical Center, 1601 Perdido Str., New Orleans, LA
70112-1262. Phone: 504-589-5230; Fax: 504-566-1625.
3 The abbreviations used are: SST,
somatostatin; SSTR, SST-receptor; SSTR-2, subtype 2; DOX, doxorubicin;
AN-201, 2-pyrrolinodoxorubicin.
4 A. Nagy, A. Plonowski, and A. V. Schally.
Stability of cytotoxic luteinizing hormone-releasing hormone conjugate
(AN-152) containing doxorubicin 14-O-hemiglutarate in mouse
and human serum in vitro: implications for the design of
preclinical trials. Proc. Natl. Acad. Sci. USA, 97:829–834, 2000.
Received 9/ 1/99; revised 11/ 8/99; accepted 11/12/99.
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 P. J. Loehrer Sr, W. Wang, D. H. Johnson, and D. S. Ettinger Octreotide Alone or With Prednisone in Patients With Advanced Thymoma and Thymic Carcinoma: An Eastern Cooperative Oncology Group Phase II Trial J. Clin. Oncol., January 15, 2004; 22(2): 293 - 299. [Abstract] [Full Text] [PDF]
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 L. J. Hofland and S. W. J. Lamberts The Pathophysiological Consequences of Somatostatin Receptor Internalization and Resistance Endocr. Rev., February 1, 2003; 24(1): 28 - 47. [Abstract] [Full Text] [PDF]
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K. Szepeshazi, A. V. Schally, G. Halmos, B. Sun, F. Hebert, B. Csernus, and A. Nagy Targeting of Cytotoxic Somatostatin Analog AN-238 to Somatostatin Receptor Subtypes 5 and/or 3 in Experimental Pancreatic Cancers Clin. Cancer Res., September 1, 2001; 7(9): 2854 - 2861. [Abstract] [Full Text] [PDF]
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 N. Benali, P. Cordelier, D. Calise, P. Pages, P. Rochaix, A. Nagy, J.-P. Esteve, P. M. Pour, A. V. Schally, N. Vaysse, et al. Inhibition of growth and metastatic progression of pancreatic carcinoma in hamster after somatostatin receptor subtype 2 (sst2) gene expression and administration of cytotoxic somatostatin analog AN-238 PNAS, August 1, 2000; 97(16): 9180 - 9185. [Abstract] [Full Text] [PDF]
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, On day
15, the tumor volume of 3 of 4 animals exceeded 3,500 mm3
and the animals were sacrificed. Thus, the tumor volume on day 15 is
shown. B, changes in the tumor volume in nude mice
treated with 1 or 2 i.v. injections of AN-238 or AN-201 at 150
nmol/kg. *, P < 0.05 versus
control; **, P < 0.01 versus
control. C, effect of pretreatment with RC-121
administered s.c. at 50 µg/day per animal for 7 days or at 500
µg/animal 4 h before the administration of AN-238 at 150
nmol/kg. Vertical bars, SEM. *, P =
0.051 versus the AN-238-treated group and not
significant versus control.
). 100% specific binding is defined as the
difference between binding in the absence and in the presence of
10-5 M RC-160. Each point represents the mean
of triplicate determinations. C, polyacrylamide gel
electrophoresis of reverse-transcribed and subsequently PCR-amplified
mRNAs for SSTR-2 and hGAPDH in three representative xenografts of U-87
MG human glioblastomas from untreated nude mice (lanes
1–3); M, molecular weight DNA marker;
N, negative control; P, positive control
(NCI-H-69 human small cell lung carcinoma).