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Proc. Natl. Acad. Sci . USA
Vol. 96, pp. 3098–3103, March 1999
Medical Sciences
Inhibition of growth, production of insulin-like growth factor-II
(IGF-II), and expression of IGF-II mRNA of human cancer cell
lines by antagonistic analogs of growth hormone-releasing
hormone in vitro
VALE
´RJ. CSERNUS*†‡,ANDREW V. SCHALLY*†§,HIPPOKRATIS KIARIS*†,AND PATRICIA ARMATIS*
*Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center, New Orleans, LA 70112-1262; and †Department of Medicine, Tulane University
School of Medicine, New Orleans, LA 70112-2699
Contributed by Andrew V. Schally, January 14, 1999
ABSTR ACT Antagonistic analogs of growth hormone-
releasing hormone (GHRH) suppress growth of various tu-
mors in vivo. This effect is exerted in part through inhibition
of the GHRH–GH–insulin-like growth factor (IGF)-I ax is.
Nevertheless, because autocrineyparacrine control of prolif-
eration by IGF-II also is a major factor in many tumors, the
interference with this growth-stimulating pathway would offer
another approach to tumor control. We thus investigated
whether GHRH antagonists MZ-4-71 and MZ-5-156 also act
on the tumor cells directly by blocking the production of
IGF-II. An increase in the IGF-II concentration in the media
during culture was found in 13 of 26 human cancer cell lines
tested. Reverse transcription–PCR studies on 8 of these cell
lines showed that they also expressed IGF-II mRNA. Antag-
onists of GHRH significantly inhibited the rate of prolifera-
tion of mammary (MDA-MB-468 and ZR-75–1), prostatic
(PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990,
and Capan-2) cancer cell lines as shown by colorimetric and
[
3
H]thymidine incorporation tests and reduced the expression
of IGF-II mRNA in the cells and the concentration of IGF-II
secreted into the culture medium. Growth and IGF-II pro-
duction of lung (H-23 and H-69) and ovarian (OV-1063)
cancer cells that express mRNA for IGF-II and excrete large
quantities of IGF-II also was marginally suppressed by the
antagonists. These findings suggest that antagonistic analogs
of GHRH can inhibit growth of certain tumors not only by
inhibiting the GHRH–GH–IGF-I axis, but also by reducing the
IGF-II production and by interfering with the autocrine
regulatory pathway.
Insulin-like growth factors-I and -II (IGF-I and -II) are
involved in the proliferation of various human cancers (1–4).
Much evidence supports the view that growth hormone-
releasing hormone–growth hormone (GHRH–GH)–IGF-I
axis greatly inf luences the biologic behavior of many common
neoplasms (2, 5–7). The tumor-promoting effect of IGFs has
been shown in osteosarcomas and prostate cancers (8). Some
tumor cells contain abundant IGF-I receptors and respond
mitogenically to IGFs present in their microenvironment (6,
7). Activation of IGF receptors promotes growth of neuroec-
todermal tumors, neuroblastomas, choriocarcinomas, breast
cancers, myelomas, and liver cancer (4–7, 9). mAb IR-3, which
blocks IGF-I receptor function, inhibited growth of small-cell
lung cancer, neuroblastoma, breast cancer, and rhabdomyo-
sarcoma cells cultured in serum-free medium (10, 11). Local
growth and metastatic behavior of IGF-I-responsive sarcomas
are reduced by hypophysectomy and restored by administra-
tion of growth hormone (12, 13). It has been shown that growth
of breast cancer was significantly reduced in mice with a
genetically suppressed GH–IGF-I axis (14).
These investigations (1–14) and others support the hypoth-
esis that aggressive behavior of many neoplasms may be
reduced by interfering with the GHRH–GH–IGF-I axis. One
of the pharmacological approaches aimed at achieving an
inhibition could be based on utilization of antagonistic analogs
of GHRH. GHRH antagonists developed by us (2, 15, 16)
inhibit the growth of human renal adenocarcinoma, prostate
cancers and small-cell and non-small-cell lung carcinomas,
osteosarcomas, and other tumors xenografted into nude mice
(17–21). It was suggested that suppressive effects of antago-
nistic analogs of GHRH on tumor growth in vivo could be
caused in part by a reduction in pituitary GH release and the
subsequent decrease in production of IGF-I in the liver (2, 21).
However, the reduction in serum IGF-I level did not always
parallel tumor suppression and tumor levels of IGF-I and
IGF-II in renal cancers, lung cancers, and prostate cancers
were greatly inhibited after therapy with GHRH antagonists
(17–20). This observation suggests that, in addition to hepatic
IGF-I, the inhibitory effect of antagonistic analogs of GHRH
on tumor growth may be mediated by regulation of tumor
levels of IGF-I and -II.
The serum level of IGF-II, unlike that of IGF-I, is indepen-
dent of the functional state of GHRH–GH axis. Unlike IGF-I,
which is mostly produced in the liver, IGF-II is synthesized by
a wide variety of tissues. IGF-II is considered one of the key
cell-survival factors (22), and its secretion is controlled pri-
marily by the local environment of the cells. The observation
that certain tumor cells proliferate in the absence of serum-
derived growth factors gave rise to the idea that such cells are
capable of secreting their own growth factors. Various studies
demonstrated IGF-II production and expression of IGF-II
mRNA in several tumor-cell lines especially in diverse sarco-
mas and neural tumors (1, 23, 24). The presence of receptors
for both IGF-I and IGF-II also was shown in several tumor
cells (1, 2, 10, 23, 25). These studies provide evidence that IGFs
produced by these cells may play a fundamental role in their
proliferation.
Autocrineyparacrine regulatory mechanisms involving
IGF-II are implicated in proliferation of normal tissues as liver,
colon, lung, or bone and also participate in nerve regeneration
and wound healing (1, 4, 26). IGF-II also affects growth of
various tumors like neuroblastomas, chondrosarcomas, Wilms
tumor, mesothelial tumors, and cancers of breast, colon,
prostate, endometrium, and liver in autocrineyparacrine fash-
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: GH, growth hormone; GH-RH, GH-releasing hor-
mone; IGF-I, insulin-like growth factor-I; IGF-II, insulin-like growth
factor-II; RT, reverse transcription.
‡On leave from the Department of Anatomy, University Medical
School of Pe´cs, H-7643 Pe´cs, Hungary.
§To whom reprint requests should be addressed. at: Veterans Affairs
Medical Center, 1601 Perdido Street, New Orleans, LA 70112-1262.
3098
ion (1, 2, 4, 27–33). Interrupting the autocrine regulatory circle
of IGF-II could provide an efficacious approach to inhibiting
various cancers. In addition to blocking the function of the IGF
receptors on the surface of tumor cells, this goal also can be
achieved by reducing IGF-II production of the cells. The
mechanism of the control of IGF-II production in tumor cells,
however, has not been elucidated so far.
Besides the hypothalamus, GHRH also is produced in
various peripheral tissues including tumors (34, 35). The
receptors for GHRH also were detected in various extrapitu-
itary organs (36). These results suggest that another mecha-
nism of the tumor growth-suppressing effect of the antago-
nistic analogs of GHRH could be based on blocking the
autocrine regulatory pathway of IGF-II directly in the tumor
cells or in their immediate environment. Thus, the goal of this
study was to clarify whether the antagonistic analogs for
GHRH can interfere with the autocrine stimulatory function
of IGF-II in tumor cells. To exclude the participation of the
GHRH–GH–IGF-I axis operating in vivo, we designed in vitro
experiments. Cancer cells of human origin were studied in
culture and the effects of antagonistic analogs of GHRH on
growth, IGF-II production, and expression of IGF-II mRNA
were evaluated.
MATERIALS AND METHODS
Peptides. GHRH antagonists [Ibu-Tyr
1
,D-Arg
2
,Phe(4-
Cl)
6
,Abu
15
,Nle
27
,Agm
29
]hGHRH(1–29) (MZ-4–71) and
[PhAc-Tyr
1
,D-Arg
2
,Phe(4-Cl)
6
,Abu
15
,Nle
27
,Agm
29
]hGHRH(1–
29) (MZ-5-156) and hGHRH(1–29), used as a standard for in
vitro experiments, were synthesized and characterized in our
laboratory as reported (15, 16). Other organic and inorganic
chemicals were purchased from Sigma.
Tissue Cultures. Tumor cell lines were obtained from the
American Type Culture Collection. The media for routine
culture (GIBCOyBRL) varied depending on the cell line. The
type of tissue culture medium varied according to the require-
ments of the cell lines: RPMI medium 1640 (RPMI) 110%
fetal bovine serum (FBS) were used for Capan-2, DU-145,
H-23, H-69, JAR, HEC-1A, and LNCaP cells; RPMI 15%
FBS for H-345 and PC-3 cells; RPMI 110% newborn calf
serum (NCS) for H-157 and H-510 cells; McCoy 5A Medium
110% FBS for HT-29 and SKOV-3 cells; F12 120% FBS for
LoVo cells; improved minimal essential medium (IMEM) 1
dextran-coated charcoal-treated FBS for MCF-7 cells; DMEM
110% NCS for MDA-MB-231 cells; IMEM 110% FBS for
MDA-MB-468 cells; DMEM 110% FBS for Panc-1 cells; L15
110% FBS for SW-1990 cells; RPMI 110% FBS supple-
mented with insulin for T47D cells; minimal essential medium
(MEM) 110% FBS supplemented with pyr uvate for U373MG
cells; RPMI 110% FBS supplemented with pyruvate and
glucose for ZR-75–1 cells; and RPMI 110% FBS 1pyruvate
and MEM vitamins for OV-1063 cells. The cultures were
maintained in a humidified atmosphere containing 5% CO
2
y
95% air at 37°C. The cells were passaged weekly and routinely
monitored for the presence of mycoplasma by using a test kit
from Boehringer Mannheim.
Colorimetric Tests. Crystal violet assay was performed as
described (37). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide] test is based on a method
described by Plumb (38) and was carried out as previously
reported (18, 20, 21).
[
3
H]Thymidine Incorporation Test. Cells were seeded into
96-well microplates in the appropriate medium. When cells
were 50% confluent (adherent cells) or after 24 hours (sus-
pension cells), the compounds to be tested and control media
were added, and the plates were incubated under standard
conditions for another 20 hours. All subsequent steps were
performed as described (17, 21).
Radioimmunoassay of IGF-II. Because only tissue culture
media of defined composition were used, the acid–ethanol
extraction method to eliminate IGF-binding proteins was
unnecessary. The radioimmunoassay for IGF-II was carried
out as reported (17–20).
RNA Extraction. Harvested tumor cells were washed once
with PBS, and total RNA was isolated by using the RNAzol B
reagent (Tel-Test, Friendswood, TX) following the manufac-
turer’s instructions.
Reverse Transcription (RT). One microgram of total RNA
was reverse-transcribed into cDNA by using Moloney murine
leukemia virus reverse transcriptase according to manufactur-
er’s instructions (Perkin–Elmer).
PCR Amplification. One microliter of the cDNA was am-
plified in a 50-
m
l solution containing 10 mM TriszHCl (pH 8.3),
50 mM KCl, 1.5 mM MgCl
2
, 200
m
M of each dNTP, 2.5 units
of Taq DNA polymerase (Perkin–Elmer) and 0.4
m
M each
primer. The primers used were 59-TCCTCTGACTTCAA-
CAGCGACACC-39(sense) and 59-TCTCTCTTCCTCTTGT-
GCTCTTGG-39(antisense) for hGAPDH and 59-AGTC-
GATGCTGGTGCTTCTCACCTTCTTGGC-39(sense) and
59-TGCGGCAGTTTTGCTCACTTCCGATTGCTGG-39
(antisense) for IGF-II (19). PCR consisted of 1 cycle at 95°C
for 3 min, 62°C for 1 min, and 72°C for 1 min and subsequently
26 cycles for GAPDH or 26 to 33 cycles for IGF-II, depending
on the endogenous levels of expression, at 95°C for 35 sec, 62°C
for 40 sec, and 72°C for 40 sec. The reactions were performed
by using a Stratagene Robocycler 40 system (Stratagene). The
number of cycles for each cell line was determined in prelim-
inary experiments to be within the exponential range of PCR
amplification (data not shown). Five microliters of each PCR
product was separated by electrophoresis in 2% agarose gel
and stained with ethidium bromide, or for quantitative anal-
ysis, in 8% polyacrylamide gel and stained with silver. The
intensity of the bands was analyzed by a scanning densitometer
(Model GS-700, Bio-Rad) coupled with the Bio-Rad PC
analysis software.
Statistical Analysis. For statistical analysis, two-tailed Stu-
dent’s ttest was used. The difference was considered signifi-
cant at P,0.05 tail probability.
RESULTS
IGF-II Production of Cancer Cell Lines. Twenty-six human
cancer cell lines were grown in vitro until they covered '70%
of the surface of the culture dish, the experiment lasting for 3
to 7 days. IGF-II concentration of samples from the culture
media taken before and after the culture period was deter-
mined by radioimmunoassay (Table 1). Because of wide
variations in experimental conditions, including the rate of cell
proliferation, type of medium, form of cell growth—
monolayer or floating cell-clusters—only semiquantitative
data are given (Table 1). Most of the media contained IGF-II
(5–15 ngyml) at the beginning of the experiments because of
their bovine serum content. IGF-II production of the cells was
considered negative, if IGF-II concentration of the sample
taken at the end of the experiment was similar to or lower than
that at the beginning.
Thirteen of the twenty-six cell lines showed an increase in
IGF-II concentration. In six cell lines, the increase was sub-
stantial (2–12 ngyml). In some cell lines, a considerable
decrease in IGF-II content of the medium was found (Table 1).
Subsequent experiments described below then were per-
formed with tumor cells found to secrete IGF-II.
Effect of Antagonistic Analogs of GHRH on IGF-II Release
from Human Cancer Cell Lines. Precultured human cancer
cells were incubated in seeding medium for 24–48 hours and
(at time T0) the medium was replaced by test medium. Each
of the cell lines was divided into five groups. One group of cells
served as a control and the other four groups were incubated
Medical Sciences: Csernus et al. Proc. Natl. Acad. Sci. USA 96 (1999) 3099
in medium containing MZ-4-71 or MZ-5-156 at 300 nM and 3
m
M concentrations for 42–97 hours (T1). The actual duration
of each experiment depended on the growth rate of the
respective cell-line. IGF-II content of media at T0 and T1 times
was determined. Changes in IGF-II concentrations as com-
pared with those of the respective control groups are presented
in Fig. 1. GHRH analogs induced a significant decrease in
IGF-II concentrations in most of the cell lines tested. DU-145
prostate cancer cells and MDA-MB-468 and ZR-75–1 breast
cancer cells responded to lower (300 nM) concentrations of
both analogs. Capan-2 pancreatic cancer was more sensitive to
MZ-5-156, whereas PC-3 prostate cancer responded better to
MZ-4–71. IGF-II secretion of SW-1990 pancreatic carcinoma
was inhibited significantly only by higher (3
m
M) concentration
of the analogs. H-69 small-cell lung carcinoma and OV-1063
ovarian cancer did not respond to the analogs under the
experimental conditions.
Effect of GHRH Antagonists on the Rate of Proliferation of
Cancer Cell Lines. In experiments similar to those described
above, we tested the effect of the antagonistic analogs of
GHRH on the proliferation of the cancer cells. At the begin-
ning (T0) and end (T1) of the incubation periods, colorimetric
tests were performed. In the case of H-69 cells that mostly f loat
in culture, the thiazolyl-blue (MTT) test was carried out, but
for the majority of the cells that attach well to the surface of
the wells, crystal violet tests were done. The differences in
optical densities measured at T1 and T0 times for cell groups
treated with the analog compared with those of respective
controls are shown in Fig. 2. As can be seen, the analogs
decreased the growth of most of the cancer cell lines tested.
The reduction in growth was significant at both doses of
analogs in the case of DU-145 and PC-3 prostate cancers and
ZR-75-1 and MDA-MB-468 breast cancers (except for the
lower concentration of MZ-5-156). In these experiments, the
growth of H-69 small-cell lung carcinoma was inhibited only at
high concentration of MZ-5-156, and the growth reduction of
Capan-2 pancreatic cancer was not significant.
The effect of the analogs of GHRH antagonists on the
proliferation of various human cancer cells also was investi-
gated by using [
3
H]thymidine incorporation assay. The exper-
imental design was similar to those described above. Groups of
cancer cells were exposed to MZ-4-71 or MZ-5-156 at 300 nM
and 3
m
M concentrations for 24 hours and 0.2
m
Ci [
3
H]thy-
midine (1 Ci 537 GBq) was then added to each well. After 4
hours exposure time, the cells were washed and their radio-
activities were counted. The incorporation of radioactiv ity into
the cells treated with analog was compared with that of the
control group. The results are shown in Fig. 3. Antagonistic
analogs of GHRH significantly reduced the incorporation of
[
3
H]thymidine into DNA in MDA-MB-468 breast cancers,
OV-1063 ovarian cancers (except for 3
m
M MZ-4–71), and
FIG. 1. The effect of antagonistic analogs of GHRH (MZ-4-71 and
MZ-5-156) on IGF-II production of human cancer cell lines in culture.
Each group consisted of 4 –8 wells with 3,000–5,000 cells per well. The
cells were exposed to the analogs at 300 nM or 3
m
M concentration for
42–97 hours depending on the rate of proliferation of the cell line.
Control cultures received medium alone. Changes of IGF-II concen-
tration in the tissue culture media as compared with control groups are
plotted as mean 6SEM of 4– 6 experiments. Significant differences
from the control groups are indicated: p, 0.05 .P.0.01; pp,P,0.01,
two-tailed Student’s ttest.
FIG. 2. The effects of antagonistic analogs of GHRH on growth of
human cancer cells as determined by colorimetric tests (MTT for H-69
and crystal violet for other cells). The experimental conditions were
similar to those in Fig. 1. p, 0.05 .P.0.01; pp,P,0.01, two-tailed
Student’s ttest.
Table 1. Semiquantitative evaluation of the IGF-II production
(increase in the concentration of IGF-II in the medium) of some
human cancer cell lines in tissue culture
Cancer
cell line
Increase by
2–12 ng/ml
Increase by
,2 ng/ml No increase
Breast
MDA-MB-468 ZR-75-1
MCF-7-M-III†
MDA-MB-231
MCF-7-LCC1†
T47D
Prostatic PC-3 DU-145 LNCaP
Lung
SCLC H-69 H-510 H-345†
non-SCLC H-23 H-157†
Pancreatic MiaPaCa-2
SW-1990 Capan-2 Panc-1†
Ovarian OV-1063
SKOV-3
Colorectal LoVo HT-29
Choriocarcinoma JAR†
Osteosarcoma MG-63
Glioma U-373-MG
Endometrial HEC-1A
Total 6 7 13
The cell lines were grouped according to the increase in the amount
of IGF-II in the medium. The incubation time varied from 2 to 5 days
depending on the rate of proliferation of the cells.
†Cell lines showing a decrease in IGF-II content in the medium.
SCLC, small-cell lung carcinoma; non-SCLC, non-small-cell lung
carcinoma.
3100 Medical Sciences: Csernus et al. Proc. Natl. Acad. Sci. USA 96 (1999)
PC-3 prostate cancers (except for 300 nM MZ-5-156). In
MiaPaCa-2 pancreatic cancer, the inhibition occurred only at
higher doses of both analogs. In the case of lung carcinoma
cells (H-69 and H-23), only MZ-5-156 was active.
Expression of IGF-II mRNA in Cancer Cell Lines. mRNA
extracted from various cultured cancer cells, including MDA-
MB-468 breast cancer, PC-3 prostate cancer, MiaPaCa-2 pan-
creatic cancer, OV-1063 ovarian cancer, and H-23 and H-69
lung cancer cells was reverse-transcribed, and IGF-II and
hGAPDH cDNA was amplified by using RT-PCR (31 and 26
cycles, respectively). The product was separated by electro-
phoresis in 2% agarose and stained with ethidium bromide
(Fig. 4) or in 8% polyacrylamide and stained with silver. All of
the cells tested expressed IGF-II mRNA. The intensities of the
silver-stained bands were analyzed with a computer-controlled
densitometer. From the relative intensities (mRNA of IGF-II
normalized for mRNA of GAPDH), semiquantitative results
on the levels of IGF-II mRNA were obtained (data not shown).
The highest expression of IGF-II mRNA was shown by lung
carcinoma cells (H-69 and H-23), followed by MDA-MB-468
breast cancer and PC-3 prostate cancer (Fig. 4).
Effect of GHRH Antagonists MZ-4-71 and MZ-5-156 on
Expression of IGF-II mRNA in Tumor Cells. Tumor cells of
human origin were cultured for 4 hours in the presence of 3
m
M
antagonists MZ-4-71 or MZ-5-156. Control cultures received
medium alone. The cells were then harvested, and their RNA
was extracted. IGF-II mRNA was amplified by using RT-PCR.
After electrophoresis on polyacrylamide gel, optical densities
of the silver-stained bands were measured and the levels of
IGF-II mRNA were assessed by semiquantitative RT-PCR.
Optical densities of IGF-II cDNA bands (corrected by the
density of the hGAPDH band) relative to those of the un-
treated controls, which were arbitrarily considered as 100%,
are shown in Table 2. As can be seen, antagonistic analogs of
GHRH substantially reduced the levels of IGF-II mRNA in
MDA-MB-468 breast cancers, PC-3 prostate cancers, Mi-
aPaCa-2 pancreatic cancers, and OV-1063 ovarian cancers
(MZ-5-156 only). Neither analog showed a significant effect on
IGF-II mRNA levels in H-23 and H-69 lung cancer cells.
In a similar experiment, electrophoresis of RT-PCR product
was performed on 2% agarose gel, and the bands were stained
with ethidium bromide (Fig. 5). The results were similar to
those obtained in the previous experiment. In this case, a
visible reduction in the density of IGF-II cDNA bands of the
Capan-2 and SW-1990 pancreatic cancer cells treated with
GHRH antagonists could also be observed.
DISCUSSION
Malignant tumors are among the leading causes of death in
western countries. Because of the heterologous nature of many
malignancies, the treatment of cancer requires a variety of
approaches aimed at different elements of the intracellular
machinery responsible for cell growth. Some strategies can be
directed at the signaling pathways controlled by growth factors
that are necessary for the proliferation of most cancers. IGF-I
and IGF-II are among the key growth factors involved in tumor
growth (1–7). A reduction in the concentration of IGF-I or
IGF-II in the microenvironment of the tumor cells could
decrease the growth of various tumors. Because IGF-I is
mostly produced by the liver and is under the control of the
GHRH–pituitary GH axis, serum concentration of IGF-I can
be substantially reduced by inhibition of GH release (2, 5, 19).
In contrast, IGF-II is produced by a wide variety of cells
controlled by unknown factors and is considered to exert
mostly paracrine or autocrine effect (1, 2, 4, 24, 32).
Antagonistic analogs of GHRH developed in our laboratory
appear to be excellent candidates for inhibiting tumor growth
by suppressing the GHRH–GH–IGF-I axis (2, 15, 16). We
have already shown that analogs such as MZ-4-71 and MZ-5-
156 can inhibit growth of various tumors both in vivo and in
vitro (17–21). These GHRH antagonists reduced IGF-I con-
centration in serum, but their effects and in vitro activity could
FIG. 3. The effects of antagonistic analogs on [
3
H]thymidine
incorporation into DNA of cultured human cancer cell lines. The cells
were exposed to MZ-4-71 or MZ-5-156 for 24 hours, [
3
H]thymidine
was added, and the incubation was continued for 4 hours. Relative [
3
H]
activities of the washed cells (mean 6SEM) are plotted relative to
those of control groups. Significant differences from the control
groups are indicated. p, 0.05 .P.0.01; pp,P,0.01, two-tailed
Student’s ttest.
FIG. 4. IGF-II mRNA expression in cultured human cancer cell
lines. mRNA from each cell line was amplified by using RT-PCR. After
electrophoresis in 2% agarose gel, the product was stained with
ethidium bromide. The PCR product was of the expected size (538 bp)
for IGF-II. M, pUC18/MspI-digested cDNA marker.
Table 2. The effect of antagonistic analogs of GHRH on IGF-II
mRNA expression of human cancer cell lines
Cell lines Control, % MZ-4-71, % MZ-5-156, %
Capan-2 100 83 65
H-23 100 93 91
H-69 100 95 96
MiaPaCa-2 100 80 70
MDA-MB-468 100 59 58
OV-1063 100 98 73
PC-3 100 70 77
SW-1990 100 92 86
The cells were exposed to 3
m
M concentration of analogs MZ-4-71
or MZ-5-156 for 4 hours followed by semiquantitative multiplex
RT-PCR and electrophoresis on polyacrylamide gel. Optical densities
of silver-stained IGF-II and hGAPDH cDNA bands were measured.
Data for IGF-II bands were corrected by their hGAPDH values.
Relative corrected IGF-II band densities compared to those of the
control group are given in %.
Medical Sciences: Csernus et al. Proc. Natl. Acad. Sci. USA 96 (1999) 3101
not be explained merely by the suppression of hepatic IGF-I
production (17–21). This conclusion led to the demonstration
that in vivo these analogs also influence the local IGF-II
production in tumors (17–20). To assess the involvement of the
antagonists in blocking the IGF-II autocrine regulatory path-
way in tumor cells, in vitro experiments were performed.
Previous studies reported IGF-II production in several tumors
including sarcomas, adrenal, kidney, or brain tumors, as well
as pancreatic and prostatic cancers (1, 4, 23, 24, 27, 28). To
extend this list, we tested 26 human cancer cell lines for IGF-II
production in vitro. Six of these cell lines, including MDA-
MB-468, PC-3, H-69, H-23, MiaPaCa-2 and SW-1990, showed
a major and 7 showed a moderate increase in IGF-II levels in
the culture medium, indicating IGF-II production by these
cells (Table 1). Most of the cells tested could be cultured only
in the presence of bovine serum containing significant quan-
tities (5–15 ngyml) of IGF-II. In some cases, a considerable
decrease in IGF-II concentration was found during the culture
period. This may indicate enzymatic inactivation or cellular
uptake of IGF-II originally present in the medium. The
difference between the IGF-II contents present in the medium
before and after the culture period is a function of two factors,
which by themselves were not measurable in our experimental
conditions—the clearance of IGF-II originally present in the
medium and production of IGF-II by the cells. An increase in
IGF-II concentration definitely indicates IGF-II production by
the cells, whereas no increase or even a slight decrease may still
occur in the case of a modest IGF-II release accompanied by
an extensive reduction in IGF-II because of inactivation or
cellular uptake. This is why no numerical data were given in
Table 1.
Some of the cell lines, including MDA-MB-468, PC-3,
MiaPaCa-2, H-69, H-23, OV-1063, Capan-2, and SW-1990,
that release IGF-II into the tissue culture medium, also were
tested for the expression of IGF-II mRNA. All of these cells
showed different degrees of IGF-II mRNA expression. The
results are in good agreement with the measurement of IGF-II
release into the medium. The detection of an increase in
IGF-II in the media showed that IGF-II mRNA not only was
expressed in certain tumor cells, but also was translated and
secreted from the cells.
Antagonistic analogs of GHRH, MZ-4-71, and MZ-5-156,
which proved to be potent inhibitors of tumor growth in vivo,
also were active in inhibiting growth, IGF-II release, and
expression of IGF-II mRNA of MDA-MB-468 and ZR-75-1
breast cancers, PC-3 and DU-145 prostatic cancers, and Mi-
aPaCa-2, Capan-2, and SW-1990 pancreatic cancers in vitro.
Although H-69 and H-23 lung and OV-1063 ovarian cancer
cells synthesize and secrete high quantities of IGF-II, their
growth and IGF-II production were only marginally affected
by these analogs.
Our results indicate that antagonistic analogs of GHRH
reduce the concentrations of IGF-II in the microenvironment
of the tumor cells, at least in the case of some breast, prostate,
and pancreatic cancers. As has been reported previously, these
analogs reduce the blood level of IGF-I by inhibiting GH
release from the anterior pituitary (17–21). The present study
provides evidence that in the case of several cancers, these
analogs also interrupt the autocrine regulatory pathway of
IGF-II produced by the tumor cells. Because IGFs are neces-
sary for proliferation of most tumor cells, the antagonistic
analogs of GHRH appear to be potential candidates for
therapy of various tumors.
Much evidence exists that octapeptide analogs of soma-
tostatin such as Sandostatin (Octreotide) and RC-160 (Vap-
reotide) suppress GH release, lower the levels of hepatic
IGF-I, and, thus, reduce the proliferation of some IGF-I-
responsive neoplasms (reviewed in ref. 5). The inhibition of
hepatic IGF-I gene expression by Octreotide has also been
reported (5). However, analogs of somatostatin show selectiv-
ity for subtypes 2 and 5 of somatostatin receptors (SSTR-2 and
-5), which are not expressed by a significant subset of tumors
(5). In the absence of these receptors, somatostatin octapep-
tide analogs cannot exert major direct effects. Moreover, there
is so far no evidence that somatostatin analogs can suppress
tumor levels of IGF-I and IGF-II and expression of mRNAs of
IGF-I and IGF-II in tumors.
The results presented in this paper indicate that, in some
human cancer cell lines, the antagonistic analogs of GHRH
inhibit proliferation of tumor cells by interfering with the
autocrine regulatory effect of IGF-II. However, the mecha-
nism of action of GHRH antagonists remains to be clarified.
It was shown that several human cancers and cancer cell lines
produce GHRH (34, 35, 39). GHRH-sensitive receptors on
various tumor cells have been also described (19). Thus, the
GHRH antagonists may block these receptors that could be
responsible for controlling the IGF-II production of the cells.
Although the decrease in IGF-II expression and production
was relatively small under our experimental conditions, the
biological importance of these findings is supported by the
observation that the levels of IGF-II mRNA decreased after a
short exposure of only 4 hr to the GHRH antagonists. In
addition, the fact that MZ-4-71 and MZ-5-156 showed slightly
different potencies in inhibiting growth of various tumor cells
in vitro may indicate that different receptors or receptor
subtypes could be present in various tumors, providing further
support for the specificity of these findings.
The authors thank Dr. Kate Groot for performing the radioummu-
noassay for IGF-II. This work was supported by the Medical Research
Service of the Veterans Affairs Department and CaP CURE (Asso-
ciation for the Cure of Prostate Cancer) and by a grant from ASTA
Medica (Frankfurt am Main, Germany) to Tulane University School
of Medicine (all to A.V.S.).
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FIG. 5. The effect of antagonistic analogs of GHRH on IGF-II mRNA expression in cultured human cancer cell lines. The cells were exposed
to the analogs (MZ4 5MZ-4-71, MZ5 5MZ-5-156) at 3
m
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