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Growth hormone releasing hormone plasmid supplementation, a
potential treatment for cancer cachexia, does not increase tumor
growth in nude mice
Amir S Khan,
1
Louis C Smith,
1
Ingrid W Anscombe,
1
Kathleen K Cummings,
1
Melissa A Pope,
1
and Ruxandra Draghia-Akli
1,2
1
ADViSYS, Inc., The Woodlands, Texas 77381, USA; and
2
Department of Molecular and Cellular Biology,
Baylor College of Medicine, Houston, Texas 77030, USA.
Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous
studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in
animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune
cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast
adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-
specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with
the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors
(Po.02), a 48% increase in white blood cells (Po.025) and a 300% increase in monocyte count (Po.0001), as well as an increase
in the frequency of activated CD3
þ
and CD4
þ
cells in the tumors, compared to tumors of control animals. No adverse effects were
observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the
GHRH–GH–IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia
in humans.
Cancer Gene Therapy (2005) 12, 54–60. doi:10.1038/sj.cgt.7700767
Published online 17 September 2004
Keywords: plasmid; GHRH; cachexia; electroporation; muscle
T
hree of the hormones largely responsible for postnatal
growth in animals and humans include growth
hormone releasing hormone (GHRH), which stimulates
growth hormone (GH) production and secretion from the
anterior pituitary,
1
and insulin-like growth factor-I (IGF-
I) that is responsible for many of the indirect effects of
GH.
2
The effects of these hormones on development,
growth, metabolism and regeneration have been widely
documented.
3,4
Recent studies in different animal models
and humans have also shown that GHRH has immune-
stimulatory effects, both through stimulation of the GH
axis and direct actions as an immune-modulator.
5
Conflicting data exist regarding the role of the GHRH–
GH–IGF-I axis in tumorigenesis and cancer-associated
pathology. Some studies have suggested that carcinogen-
esis is dependent upon critical plasma levels of GH and
IGF-I.
6
GH- or IGF-I-deficient animals are resistant to
chemically induced carcinogenesis.
7
Circulating IGF-I
levels play an important role in tumor development and
metastasis
8
and IGF-II mRNA levels are increased in
some tumor lines.
9
By contrast, other studies have failed
to demonstrate an effect of GH on cancer development
10
or have concluded that GH may actually improve the
efficacy of cancer chemotherapy.
11
Preclinical studies in rodents have suggested that
anabolic hormones, such as GH and IGF-I, may reverse
the catabolic state associated with cachexia, one of the
major complications of cancer and cancer therapies,
12
as
well as inhibit metastases in tumor-bearing animals.
13,14
In this study, the use of species-specific GHRH was not
necessary. Numerous studies have shown that GHRH of
different mammalian origin or GHRH analogs exert
similar effects in species such as dogs, pigs, cattle and
rodents.
15,16
In a previous study in immunocompetent
mice with implanted LL-2 adenocarcinoma cell line, we
showed that stimulation of the GH axis by intramuscular
delivery of a GHRH plasmid increased serum IGF-I
concentrations by 13% (an indicator of GHRH activity),
decreased growth of the tumor by 20% in males and 11%
in females, attenuated tumor metastases by up to 57%,
and prevented muscle atrophy. These results suggest a
role for plasmid-mediated GHRH therapy in reversing
the catabolic processes associated with cancer cachexia.
17
Received April 9, 2004.
Address correspondence and reprint requests to: Dr Ruxandra
Draghia-Akli, MD, PhD, Vice President of Research, ADViSYS,
Inc., 2700 Research Forest Drive, Suite 180, The Woodlands,
TX 77381, USA. E-mail: ruxandradraghia@advisys.net or
ada@bcm.tmc.edu
Cancer Gene Therapy (2005) 12, 54–60
r
2005 Nature Publishing Group All rights reserved 0929-1903/05 $30.00
www.nature.com
/
cgt
Similar results were obtained in dogs with spontaneous
malignancies.
18
Long-term evaluation of the cancer-
afflicted dogs that received plasmid-mediated GHRH
supplementation showed a significant improvement in
hematological parameters and quality of life.
19
Clinical
use of GHRH-expressing plasmids for cancer cachexia,
however, requires that ectopic GHRH expression does
not upregulate tumor growth or increase tumor pro-
gression.
In lieu of a constitutively active system of GHRH
delivery, regulated expression of GHRH may be required
under some circumstances. A mifepristone (MFP)-indu-
cible plasmid vector system was used in this case.
20
An
earlier version of this MFP-inducible GHRH system has
been shown to increase serum IGF-I, lean body mass,
body weight, and bone mineral density in SCID mice.
21
In the present study on nude mice with implanted NCI-
H358 human bronchioalveolar carcinoma
22
or MDA-
MB-468 breast adenocarcinoma cells,
23
we tested the
hypothesis that physiologic stimulation of the GHRH-
GH-IGF-I axis through a plasmid-based GHRH expres-
sion system does not stimulate tumor growth in immune-
deficient animals, and thus may prove beneficial as a
treatment for cancer cachexia.
Materials and methods
Cell culture
NCI-H358 cells (ATCC CRL-5807) and MDA-MB-468
cells (ATCC HTB-132) were obtained from ATCC
(Manassas, VA) and stored in liquid nitrogen. Cells were
rapidly thawed and plated in DMEM media (GIBCO,
Grand Island, NY) with 10% fetal bovine serum
(GIBCO, Grand Island, NY) and 1% penicillin/strepto-
mycin. Cells were grown to 80% confluence, removed by
adding 0.1% trypsin-EDTA (Gibco), centrifuged and
resuspended in 1 PBS. Cells were counted before
implantation.
DNA constructs
The plasmid pSPc5-12 contained a 360 bp SacI/BamHI
fragment of the SPc5-12 synthetic promoter.
24
To
generate pSP-hGHRH(1-40), the human GHRH cDNA
was modified by site-directed mutagenesis of human (1-
44)OH GHRH cDNA, and cloned into the BamHI/Hind
III sites of pSP-GHRH, followed by the 3
0
untranslated
region and poly(A) signal of hGH gene.
25
The GHRH-
inducible (IS) is a two-plasmid system. Plasmid pGS1633
codes for GeneSwitch regulatory protein, version 4.0 and
is controlled by a muscle-specific promoter (Valentis,
Burlingame, CA). Plasmid pGHRH(1-40) is an inducible
human GHRH plasmid. The GeneSwitch and inducible
GHRH plasmids were mixed to generate a 1:10 mol/mol
solution.
21
Control plasmid, pSP-b-gal, contained the
Escherichia. coli b-galactosidase gene under control of the
same muscle-specific promoter. Plasmids were grown in
E. coli DH5a, (GIBCO, Grand Island, NY). Endotoxin-
free plasmid (Qiagen Inc., Chatsworth, CA) preparations
were diluted to 0.8 mg/mL in sterile water and stored at
801C prior to use. Mifepristone (Sigma, St. Louis, MO)
was diluted in sesame oil and 360 mg/kg was administered
by gavage three times a week starting at Day 7.
Animals
Hsd:Athymic Nude-nu mice were obtained from Harlan
(Indianapolis, IN) or Charles River (Raleigh, NC) and
allowed to acclimate for 2 weeks. Rodents were housed
five per cage on Tek-Fresh Autoclavable Bedding
(Harlan) and given ad libitum access to food (Autocla-
vable Rodent Lab Diet 5010, Harlan) and water (reverse-
osmosis, UV-treated, sterile-filtered from municipal water
supply). No known contaminants that would interfere
with the outcome of the study were present in feed or
water. Animals were housed at 22731C, with a relative
humidity of range between 30 and 80% on a 12- hour
light/dark cycle.
Injection, electroporation, and experimental procedure
Animal groups were: Group 1 (constitutive pSP-GHRH),
Group 2 (control pSP-bgal), Group 3 (GHRH-inducible
system (GHRH-IS), no MFP), Group 4 (GHRH-IS,
MFP), Group 5 (control pSP-bgal, MFP), and Group 6
(no plasmid). All animals received a pre-experiment
physical examination by a registered veterinary technician
prior to selection for testing. At Day 7, all animals were
weighed, bled, and randomly assigned to one of six
groups (n ¼ 20/group, 10 of each sex). On Day 1,
animals were weighed, bled, and injected subcutaneously
in the flank with tumor cells in 30 mL PBS. Nude mice
received either 2 10
7
NCI-H358 cells or 1 10
7
MDA-
MB-468 cells. Mice were anesthetized on Day 0 with 0.5–
0.7mL/kg of a combination anesthetic: ketamine
(42.8 mg/mL), xylazine (8.2 mg/mL) and acepromazine
(0.7 mg/mL). A measure of 25 mL of the thawed plasmid
stock was injected into the lateral gastrocnemius muscle
using 3/10 cm
3
syringe with 26-gauge needle. At 2 minutes
after injection, the injected muscle was electroporated (3
pulses, 150 V/cm, 50 milliseconds) with a BTX ECM 830
electroporator and two-needle electrodes (BTX, San
Diego, CA), as described.
26
Animals were weighed and
bled once a week, while tumor volume was evaluated
twice a week using Promax NSK Electronic Digital
Calipers (Fred Fowler Co., Newton, MA) and Gage
Wedge for Sylvac Measuring Tools software (TAL
Technologies, Philadelphia, PA). Length, width, and
depth of the tumors were separately measured and then
used to calculate tumor volume.
Necropsy and histopathology
Animals were weighed and then euthanized by CO
2
inhalation on Days 42–43 (NCI-H358 mice) and Days 33–
34 (MDA-MB-468 mice). Organs (lungs, heart, liver,
spleen, kidneys, and the injected gastrocnemius) were
excised, weighed and checked for gross pathologies.
Gastrocnemius and any of the excised organs with
macroscopic abnormalities were fixed in 10% buffered
GHRH does not increase tumor growth in nude mice
AS Khan et al
55
Cancer Gene Therapy
formalin overnight, washed in PBS, and transferred to
70% ethanol for storage. Tumor was also excised,
weighed, fixed in 10% buffered formalin overnight, and
stored in 70% ethanol. For MDA-MB-468 males, a
complete histopathological examination was performed
on internal organs (brain, heart, lung, liver, spleen, and
kidneys), injected muscle, and tumor (IDEXX Labora-
tories, Inc., West Sacramento, CA). The tissues were
paraffin-embedded, sectioned at 4–5 mm, stained with
hematoxylin/eosin and examined microscopically by
IDEXX Laboratories. An independent licensed veterinary
pathologist read slides of each organ, including tumors of
each animal and data were recorded. Tissue from NCI-
H358 mice was paraffin embedded, sectioned at 4–5 mm,
stained with hematoxylin/eosin and examined microsco-
pically for micrometastases.
CD3 and CD4 immunohistochemistry
Tumor sections were selected from NCI-H358 male
animals from Groups 1 and 2. Sections were deparaffi-
nized and subsequently washed in PBS. Slides were
stained using goat ABC staining system (Santa Cruz
Biotechnology, Santa Cruz, CA) following the manufac-
turer’s instructions with slight modifications. Briefly, the
sections were first incubated in 0.03% hydrogen peroxide
in methanol solution to block endogenous peroxidases,
then incubated in the blocking solution (1.5% donkey
serum (Santa Cruz Biotechnology, Santa Cruz, CA) in
PBS), and finally, incubated overnight at 41C in the
primary antibody, CD3-e
0
(M-20) or CD-4 (C-18) (Santa
Cruz Biotechnology, Santa Cruz, CA), diluted 1:2000
(CD3-e
0
) and 1:1500 (CD-4) in blocking solution. After
PBS washes, the secondary antibody was applied for 30
minutes at room temperature and slides were incubated in
ABC solution for 30 minutes, as per kit instructions.
Slides were washed in PBS between each step of the
procedure. Peroxidase activity was revealed using diami-
nobenzidine (DAB) as substrate (Vector Laboratories,
Burlingame, CA) for 4 minutes. The stained sections were
visualized on an Olympus
s
BX51 microscope (Leeds
Instruments, Irving, TX) with a 20 objective, and
digital images of the sections were captured using an
Optronics MagnaFire digital color camera with the
MagnaFire 2.0 software (Optronics, Goleta, CA). The
observer was blinded to the treatment groups and counted
a random set of 9–18 fields per group. The within-animal
average was corrected for tumor volume, and then used to
calculate an average of the CD3
þ
and CD4
þ
cell counts/
tumor volume per group.
CBC and biochemistries
At necropsy, whole blood was collected in Microtainer
Brand tubes with EDTA (Becton Dickinson, Franklin
Lakes, NJ) for CBC analysis and in Microtainer Serum
Separator tubes (Becton Dickinson, Franklin Lakes, NJ)
for serum biochemistries. All tests were performed by
IDEXX Contract Research Services (West Sacramento,
CA). Parameters tested in the biochemical analysis were:
ALT (alanine aminotransferase), AST (aspartate amino-
transferase), creatinine kinase, albumin, total protein,
bilirubin, cholesterol, glucose, calcium, phosphorous,
bicarbonate, chloride, potassium, and sodium.
IGF-I radioimmunoassay
Serum was aliquoted for serum IGF-I measurement using
a mouse-specific IGF-I kit (Diagnostic Systems Labora-
tories, Inc., Webster, TX). The intra-assay variability was
6.6% for NCI-H358 males and 5.0% for NCI-H358
females.
Statistical analysis
A Microsoft Excel statistics analysis package was used.
The mean values were compared with paired t-test or
ANOVA with subsequent Student’s t-test as post hoc test.
Po.05 was taken as the level of statistical significance.
Results
Body weight
No significant differences in body weight were observed
among groups during the treatment period.
Tumor volume
As an initial observation, tumor growth rate was higher
for the NCI-H358 males (Fig 1a) than for the females
(Fig 1b). In NCI-H358 males, animals treated with the
constitutive GHRH plasmid exhibited a 40% decline in
tumor volume by Day 40 (Po.02), compared to b-
galactosidase (pSP-b-gal) plasmid controls. In NCI-H358
animals, the females treated with the constitutive GHRH
plasmid exhibited a 33% decrease in tumor volume by
Day 40 (P ¼ .14), compared to pSP-b-gal plasmid controls
and 37% decline (P ¼ .15) in final tumor weight at
necropsy. Male and female mice that received the GHRH-
IS activated at 7 days after tumor implantation, had a
23% reduction in tumor growth (Po.05 for males, and
P ¼ .15 for females, due to high variance within the
groups). Tumor volumes in MDA-MB-468 males were
not statistically significantly different (data not shown)
among groups. Female animals implanted with MDA-
MB-468 were removed from analysis due to abnormalities
in growth rate of MDA-MB-468 cells in culture and,
unlike other experiments, had inconsistent tumor devel-
opment.
CBC and serum biochemistry
In the NCI-H358 male mice treated with the constitutively
active GHRH plasmid, white blood count (WBC) was
increased 48.8% (Po.025) (Fig 2a) and percent mono-
cytes were increased 300% (Po.00005) (Fig 2b) com-
pared to pSP-b-gal plasmid controls. In MDA-MB-468
males treated with the constitutively active GHRH
plasmid, lymphocyte count was increased by 20%
(Po.045) relative to pSP-b-gal plasmid controls (Fig
2c). In the NCI-H358 female mice, with overall smaller
tumors and tumor growth rates, no biologically or
GHRH does not increase tumor growth in nude mice
AS Khan et al
56
Cancer Gene Therapy
statistically significant changes were found in the CBC or
biochemistry values.
Necropsy
Tumor weights at necropsy were 45% smaller (P ¼ .017)
in Group 1 relative to Group 2 in NCI-H358 male
animals (Fig 3a), and were 37% smaller in the NCI-H358
females, although this did not attain statistical signifi-
cance due to individual variation of tumor size (P ¼ .15)
(Fig 3b). Tumor weights were not significantly different
between Groups 1 and 2 in the MDA-MBA-468 animals.
No biologically significant changes were found in the
mean necropsy organ weights (mean organ weight/mean
body weight) of male or female mice implanted with either
cell line.
Histopathology
A complete histopathological examination was performed
on internal organs (brain, heart, lung, liver, spleen, and
kidneys), injected muscle and tumor of MDA-MBA-468
animals killed at Days 41–42. Analysis showed similar
tumors in all examined animals, with expansile and locally
invasive nodular tumors composed of cords of neoplastic
epithelial cells. There was no difference in tumor
vascularization between groups. Only one micrometasta-
sis was found, in the liver of a control animal.
Extramedullary hematopoiesis was noted in the spleen
of all animals. Two animals in the pSP-bgal plasmid
group also exhibited lymphoid hyperplasia in the spleen.
Focal areas of generally mild muscle atrophy were present
Figure 1 (a) Tumor growth as measured by tumor volume in MALE
mice implanted with NCI-H358 human bronchioalveolar carcinoma
cells. Tumors were measurable from Day 5 of the experiments, and
tumor volume measurements were taken until Day 40. (b) Tumor
growth as measured by tumor volume in Groups 1 and 2 of the
FEMALE mice implanted with NCI-H358 human bronchioalveolar
carcinoma cells. Tumors were measurable from Day 4 of the
experiments, and tumor volume measurements were taken until Day
40. *Po.02.
Figure 2 CBC values in tumor-bearing mice either treated with
constitutively active GHRH plasmid (pSP-GHRH) or nontreated
controls: (a) White blood cell counts (WBC) in NCI-H358 males,
*Po.025 (b) monocyte percentage in NCI-H358 males, *Po.00005
and (c) lymphocyte percentage in MDA-MB-468 male mice,
*Po.045.
GHRH does not increase tumor growth in nude mice
AS Khan et al
57
Cancer Gene Therapy
in many animals across all groups. Macrophage infiltra-
tion was also found in most animals and did not appear to
be treatment related. A survey of liver and lung sections
from NCI-H358 mice did not reveal any apparent
differences in the numbers of micrometastases.
Immunohistochemistry
Tumors collected at necropsy (Days 41–42) were exam-
ined for tumor-infiltrating T cells. CD3
þ
cell counts
corrected for tumor volume were increased 66.7%
(P ¼ .18) and CD4
þ
cell counts corrected for tumor
volume were increased 87.2% (P ¼ .15) in Group 1
animals versus Group 2 animals (Fig 4).
Serum IGF-I values
Serum IGF-I levels, while increased in the GHRH-treated
groups, were not significantly different among treatment
groups for the duration of the experiments.
Discussion
NCI-H358 human bronchioalveolar carcinoma and
MDA-MB-468 human breast adenocarcinoma cell lines
have previously been used to investigate tumor progres-
sion in mice.
27,28
This report demonstrates that plasmid-
mediated, physiologic GHRH supplementation did not
increase tumor progression in tumor-bearing nude mice.
Furthermore, male animals implanted with the NCI-H358
tumor cell line and treated with a GHRH-expressing
plasmid showed a decrease in tumor volume, increased
white blood cells and monocyte count, as well as increased
intratumoral activated CD3
þ
and CD4
þ
cells, as
compared to control animals. Finally, this work confirms
earlier work in immunocompetent animals
17
and suggests
that the mechanism responsible for tumor growth
reduction involves immune stimulation. These results
provide support for the use of GHRH-expressing
plasmids in the treatment of cancer cachexia.
The present data confirm the results of a previous study
that used LL-2 adenocarcinoma cell line in immunocom-
petent mice.
17
In both studies, pSP-GHRH-treated male
mice exhibited the highest decrease in tumor volume at
the end of the study compared to controls. In the present
study, tumor weights at necropsy were smaller in pSP-
GHRH-treated animals as compared to animals that
received GHRH-IS inducible system that was activated at
7 days after tumor implantation. Thus, during tumor-
igenesis, it is preferable to increase circulating GHRH
levels early rather than late for therapeutic success. Body
weight was not changed in any of the experiments,
confirming that at lower doses the direct effects of GHRH
on tissues may be more important than the effects
mediated through the GH axis. It is known that IGF-I
is highly dependent on metabolic state, and substantially
decreased in subjects with cancer cachexia.
29
Importantly,
the nude mice in the present experiment maintained
circulating IGF-I concentrations within physiologic lim-
its. As seen in GH-deficient patients administered
physiologic doses of GH,
30
the maintenance of normal
IGF-I levels results in improved clinical outcome, while
the adverse effects of IGF-I overstimulation are avoided.
Sexual dimorphism in the neuroendocrine regulation of
the GH axis in nude mice is largely unknown. In humans,
a gender dissociation within the GH/IGF-I axis is evident
in protracted critical illness, with men showing greater
Figure 3 Tumor weights at necropsy (Day 42 in males, Day 43 in
females) in (a) NCI-H358 male, *P ¼ .017 and in (b) NCI-H358
female mice.
Figure 4 Intratumoral CD3
þ
and CD4
þ
cell counts corrected for
tumor volume in NCI-H358 male mice treated with constitutive
GHRH plasmid, as compared to nontreated controls.
GHRH does not increase tumor growth in nude mice
AS Khan et al
58
Cancer Gene Therapy
loss of pulsatility and regularity within the GH secretory
pattern than women (despite indistinguishable total GH
output) and concomitantly lower IGF-I levels;
31
as a
clinical consequence, females appear to be protected
against, at least in part, adverse outcome from prolonged
critical illness. In the present study, we found that certain
tumors develop at a slower rate in female compared to
male mice, remarkably consistent with our previously
published study.
17
Additional studies are required to
elucidate the mechanism of sexual dimorphism of the
GHRH axis on tumor progression.
Enhancement of immune function is also one of the
possible mechanisms of the decline in tumor growth
observed in the present study. A substantial body of
research exists to support the production of GHRH, GH
and IGF-I by cells of the immune system.
32
In the present
study, tumor-bearing male mice treated with GHRH
plasmid demonstrated significant increases in WBC,
monocytes, and lymphocytes, confirming a similar
observation in dogs with late-stage malignancy.
18
Furthermore, the numbers of intratumoral CD3
þ
and
CD4
þ
cells were higher in male NCI-H358 tumor-bearing
animals treated with GHRH plasmid compared to
controls, which could reflect an immune response against
the tumor itself.
33
The data support the presence of an antitumor immune
function in athymic nude mice. Since nude mice lack a
normal thymus, they are characterized by low numbers of
mature T cells.
34
These animals have measurable numbers
of Thy-1
þ
and CD8
þ
and small numbers of CD4
þ
T
cells can be found. In addition, a second T-cell receptor
(TCR), gamma delta-TCR, is expressed in the spleens of
nude mice. Lake et al
35
demonstrate the quantitative
measurement of TCR expression by T cells that mature in
athymic nude mice, and they suggest that the extrathymic
environment, although inefficient, is nevertheless permis-
sive for the maturation of alpha/beta and gamma/delta
TCR-expressing T cells. Kennedy et al
36
provide further
evidence of extrathymic T-cell maturation and that nude
mice accrue increasing numbers of lymphocytes bearing
Thy-1, CD3, CD4, and CD8 with age. Radzikowski et al
37
have reported that splenocytes from young immunodefi-
cient mice retain their cytotoxic immune response to a
challenge. This suggests a possible mechanism for the
antitumor response demonstrated by the mice injected
with GHRH-expressing plasmid in this study.
Schally and colleagues have introduced evidence that
GHRH antagonists may inhibit tumor growth,
38
through
specific splice-variant GHRH receptors on tumor cells.
39
Synthetic GHRH antagonists do not inhibit the bioactiv-
ity of endogenous GHRH, but act primarily through the
inhibition of autocrine production of growth factors in
tumor cells.
40,41
Thus, an endocrine stimulation of the
GHRH axis that results in augmentation of immune
function will likely benefit affected patients, while not
stimulating malignant cell growth.
GHRH expression does not increase tumor growth in
immunodeficient, tumor-bearing mice. The benefits of
plasmid-mediated GHRH treatment may be related to
increased immune activity against the tumor in addition
to anabolic effects on the tumor-bearing animal. An
improved metabolic status of a patient in advanced stages
of cancer may increase the likelihood of the patient
surviving the radiotherapy and/or chemotherapy cancer
treatment. The findings in the present study suggest that
physiological stimulation of the GHRH-GH-IGF-I axis
in patients with cancer will likely enhance the quality of
life and may increase survival time.
Acknowledgments
We particularly thank Dr Malcolm Brenner and the
Center for Cell and Gene Therapy for continuous support
and useful discussions. We also thank Dr Jeff Nordstrom
and Ms Catherine Tone for the editorial correction of this
manuscript. We acknowledge support for this study from
ADViSYS, Inc. (The Woodlands, TX).
References
1. Muller EE, Locatelli V, Cocchi D. Neuroendocrine control
of growth hormone secretion. Physiol Rev. 1999;79:511–607.
2. Laron Z. Insulin-like growth factor 1 (IGF-1): a growth
hormone. Mol Pathol. 2001;54:311–316.
3. Thorner MO, Chapman IM, Gaylinn BD, Pezzoli SS,
Hartman ML. Growth hormone-releasing hormone and
growth hormone-releasing peptide as therapeutic agents to
enhance growth hormone secretion in disease and aging.
Recent Prog Horm Res. 1997;52:215–244 discussion 244-246.
4. Piwien-Pilipuk G, Huo JS, Schwartz J. Growth hormone
signal transduction. J Pediatr Endocrinol Metab. 2002;
15:771–786.
5. Khorram O, Garthwaite M, Golos T. The influence of aging
and sex hormones on expression of growth hormone-
releasing hormone in the human immune system. J Clin
Endocrinol Metab. 2001;86:3157–3161.
6. Shim M, Cohen P. IGFs and human cancer: implications
regarding the risk of growth hormone therapy. Horm Res.
1999;51(Suppl 3):42–51.
7. Ramsey MM, Ingram RL, Cashion AB, et al. Growth
hormone-deficient dwarf animals are resistant to dimethyl-
benzanthracine (DMBA)-induced mammary carcinogenesis.
Endocrinology. 2002;143:4139–4142.
8. Wu Y, Yakar S, Zhao L, Hennighausen L, LeRoith D.
Circulating insulin-like growth factor-I levels regulate colon
cancer growth and metastasis. Cancer Res. 2002;62:1030–
1035.
9. Guo N, Ye JJ, Liang SJ, et al. The role of insulin-like
growth factor-II in cancer growth and progression evi-
denced by the use of ribozymes and prostate cancer
progression models. Growth Horm IGF Res. 2003;13:44–53.
10. Beentjes JA, van Gorkom BA, Sluiter WJ, de Vries EG,
Kleibeuker JH, Dullaart RP. One year growth hormone
replacement therapy does not alter colonic epithelial cell
proliferation in growth hormone deficient adults. Clin
Endocrinol (Oxf). 2000;52:457–462.
11. Cherbonnier C, Deas O, Vassal G, et al. Human growth
hormone gene transfer into tumor cells may improve cancer
chemotherapy. Cancer Gene Ther. 2002;9:497–504.
12. Kotler DP. Cachexia. Ann Intern Med. 2000;133:622–634.
GHRH does not increase tumor growth in nude mice
AS Khan et al
59
Cancer Gene Therapy
13. Torosian MH. Growth hormone and prostate cancer
growth and metastasis in tumor-bearing animals. J Pediatr
Endocrinol. 1993;6:93–97.
14. Wang W, Iresjo BM, Karlsson L, Svanberg E. Provision of
rhIGF-I/IGFBP-3 complex attenuated development of
cancer cachexia in an experimental tumor model. Clin Nutr.
2000;19:127–132.
15. Hashizume T, Ohtsuki K, Sasaki K, et al. Effects of growth
hormone-releasing hormone (GRF) analogs, bovine and rat
GRF on growth hormone secretion in cattle in vivo. Endocr
J. 1997;44:811–817.
16. Campbell RM, Stricker P, Miller R, et al. Enhanced stability
and potency of novel growth hormone-releasing factor
(GRF) analogues derived from rodent and human GRF
sequences. Peptides. 1994;15:489–495.
17. Khan AS, Anscombe IW, Cummings KK, Pope MA, Smith
LC, Draghia-Akli R. Effects of plasmid-mediated growth
hormone releasing hormone supplementation on LL-2
adenocarcinoma in mice. Mol Ther. 2003;8:459–466.
18. Draghia-Akli R, Hahn KA, King GK, Cummings K,
Carpenter RH. Effects of plasmid mediated growth hor-
mone releasing hormone in severely debilitated dogs with
cancer. Mol Ther. 2002;6:830–836.
19. Tone CM, Cardoza DM, Carpenter RH, Draghia-Akli R.
Long-term effects of plasmid-mediated growth hormone
releasing hormone in dogs. Cancer Gene Ther. 2004;11:389–
396.
20. Abruzzese RV, Godin D, Mehta V, et al. Ligand-dependent
regulation of vascular endothelial growth factor and
erythropoietin expression by a plasmid-based autoinducible
GeneSwitch system. Mol Ther. 2000;2:276–287.
21. Draghia-Akli R, Malone PB, Hill LA, Ellis KM, Schwartz
RJ, Nordstrom JL. Enhanced animal growth via ligand-
regulated GHRH myogenic-injectable vectors. FASEB J.
2002;16:426–428.
22. McLemore TL, Eggleston JC, Shoemaker RH, et al.
Comparison of intrapulmonary, percutaneous intrathoracic,
and subcutaneous models for the propagation of human
pulmonary and nonpulmonary cancer cell lines in athymic
nude mice. Cancer Res. 1988;48:2880–2886.
23. Cailleau R, Olive M, Cruciger QV. Long-term human breast
carcinoma cell lines of metastatic origin: preliminary
characterization. In vitro. 1978;14:911–915.
24. Li X, Eastman EM, Schwartz RJ, Draghia-Akli R.
Synthetic muscle promoters: activities exceeding naturally
occurring regulatory sequences. Nat Biotechnol. 1999;17:
241–245.
25. Draghia-Akli R, Fiorotto ML, Hill LA, Malone PB, Deaver
DR, Schwartz RJ. Myogenic expression of an injectable
protease-resistant growth hormone-releasing hormone aug-
ments long-term growth in pigs. Nat Biotechnol. 1999;17:
1179–1183.
26. Khan AS, Fiorotto ML, Hill LA, et al. Nonhereditary
enhancement of progeny growth. Endocrinology. 2002;143:
3561–3567.
27. Zhang L, Akbulut H, Tang Y, et al. Adenoviral vectors with
E1A regulated by tumor-specific promoters are selectively
cytolytic for breast cancer and melanoma. Mol Ther. 2002;
6:386–393.
28. Fernandez Y, Gu B, Martinez A, Torregrosa A, Sierra A.
Inhibition of apoptosis in human breast cancer cells: role in
tumor progression to the metastatic state. Int J Cancer.
2002;101:317–326.
29. Simons JP, Schols AM, Buurman WA, Wouters EF. Weight
loss and low body cell mass in males with lung cancer:
relationship with systemic inflammation, acute-phase re-
sponse, resting energy expenditure, and catabolic and
anabolic hormones. Clin Sci (Lond). 1999;97:215–223.
30. Pincelli AI, Bragato R, Scacchi M, et al. Three weekly
injections (TWI) of low-dose growth hormone (GH) restore
low normal circulating IGF-I concentrations and reverse
cardiac abnormalities associated with adult onset GH
deficiency (GHD). J Endocrinol Invest. 2003;26:420–428.
31. Van den Berghe G, Baxter RC, Weekers F, Wouters P,
Bowers CY, Veldhuis JD. A paradoxical gender dissociation
within the growth hormone/insulin-like growth factor I axis
during protracted critical illness. J Clin Endocrinol Metab.
2000;85:183–192.
32. Burgess W, Liu Q, Zhou J, et al. The immune-endocrine
loop during aging: role of growth hormone and insulin-like
growth factor-I. Neuroimmunomodulation. 1999;6:56–68.
33. Wakabayashi O, Yamazaki K, Oizumi S, et al. CD4(+) T
cells in cancer stroma, not CD8(+) T cells in cancer cell
nests, are associated with favorable prognosis in human
non-small cell lung cancers. Cancer Sci. 2003;94:1003–1009.
34. Ferrick DA, Sambhara SR, Chadwick BS, Miller RG, Mak
TW. The T-cell receptor repertoire is strikingly similar in
older nude mice compared to normal adult mice. Thymus.
1989;13:103–111.
35. Lake JP, Pierce CW, Kennedy JD. CD8
+
alpha/beta or
gamma/delta T cell receptor-bearing T cells from athymic
nude mice are cytolytically active in vivo. J Immunol.
1991;147:1121–1126.
36. Kennedy JD, Pierce CW, Lake JP. Extrathymic T cell
maturation. Phenotypic analysis of T cell subsets in
nude mice as a function of age. J Immunol. 1992;148:
1620–1629.
37. Radzikowski C, Rygaard J, Budzynski W, et al. Strain- and
age-dependent natural and activated in vitro cytotoxicity in
athymic nude mice. APMIS. 1994;102:481–488.
38. Kiaris H, Koutsilieris M, Kalofoutis A, Schally AV. Growth
hormone-releasing hormone and extra-pituitary tumorigen-
esis: therapeutic and diagnostic applications of growth
hormone-releasing hormone antagonists. Expert Opin In-
vestig Drugs. 2003;12:1385–1394.
39. Kineman RD. Antitumorigenic actions of growth hormone-
releasing hormone antagonists. Proc Natl Acad Sci USA.
2000;97:532–534.
40. Chatzistamou I, Schally AV, Varga JL, et al. Inhibition of
growth and reduction in tumorigenicity of UCI-107 ovarian
cancer by antagonists of growth hormone-releasing hor-
mone and vasoactive intestinal peptide. J Cancer Res Clin
Oncol. 2001;127:645–652.
41. Chatzistamou I, Schally AV, Varga JL, et al. Inhibition of
growth and metastases of MDA-MB-435 human estrogen-
independent breast cancers by an antagonist of growth
hormone-releasing hormone. Anticancer Drugs. 2001;12:
761–768.
GHRH does not increase tumor growth in nude mice
AS Khan et al
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Cancer Gene Therapy
