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Introduction
Polyamines, including putrescine,
spermidine, and spermine, are required for cell proliferation and
homeostasis. The intracellular pool of polyamines is precisely
regulated through their biosynthesis, degradation, uptake, and
excretion[1]. The disorder of intracellular polyamines
plays an important role in carcinogenesis. Polyamines can promote
the neoplastic transformation of normal cells, stimulate the
proliferation of tumor cells, and facilitate angiogenesis in tumor
tissues. Therefore, their metabolism pathway is an interesting
anticancer drug target[2].
Agmatine, one of the analogs of
polyamines, is the product of L-arginine decarboxylation and
was initially believed to be present only in bacteria, plants, and
invertebrates. Now it has been shown to be present in mammals[3].
The accumulated results show that agmatine has some important
biological activities[4]. Among them, the inhibitory
effect of agmatine on cell proliferation is of great interest.
The current results show that
agmatine is able to modulate the cellular concentration of
polyamines[5]. Agmatine can be hydrolyzed to putrescine
and urea. Putrescine is then converted into spermidine and spermine
by spermidine/spermine synthases[6]. So agmatine might
have the capacity to increase the level of intracellular polyamines.
In addition, agmatine has been postulated to decrease the cellular
level of polyamines. There is much evidence to support this
hypothesis. First, because agmatine and polyamines are structurally
analogous and derived from same precursor, L-arginine[7],
administration of exogenous agmatine would be able to reduce the
synthesis of polyamines by a back-feed way. Second, as a competitor,
agmatine can retard putrescine intake by the same carrier[8].
Most importantly, besides polyamines, agmatine is the only known
molecule that has the capacity to induce antizyme[9].
Antizyme is the only known endogenous protein that binds to
ornithine decarboxylase, inhibiting its activity and accelerating
its degradation. Indeed, when tested in vitro, agmatine
inhibited DNA synthesis and proliferation in some cell lines[10].
Moreover, Regunathan et al[11] reported that
agmatine inhibited proliferation of human coronary artery vascular
smooth muscle cells by stimulation of imidazoline receptors.
Satriano et al[12] claimed that agmatine
dramatically decreased the ratio of DNA synthesis on mouse kidney
proximal tubule cells by attenuation of the cellular polyamine
level. In 2003, Gardini et al[13] found that
agmatine inhibited the proliferation of rat hepatoma cells. These
results indicate that agmatine might be an endogenous
anti-proliferation factor, and whether the pharmacological effect of
exogenous agmatine on cells in vivo is the same as in
vitro is an interesting question.
In the present study, we
investigated the inhibitory effects of agmatine on several classical
tumor cells in vivo and in vitro and explored its
possible mechanisms in vitro.
¡¡
Materials and methods
Reagents and drugs
Agmatine sulfate was obtained from the
Beijing Institute of Pharmacology and Toxicology; cyclophosphamide
was manufactured by Hengrui Pharmaceutical Co (Lianyungang, Jiangsu,
China); spermine, spermi-dine, putrescine,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT),
and sodium dodecylsulfonate (SDS) were obtained from Sigma Chemical
(St Louis, MO, USA); Roosevelt Park Memorial Institute medium
(RPMI-1640) was purchased from Gibco (Carlsbad, CA, USA); and [3H]thymidine
was obtained from DuPont/NEN Company (Boston, MA, USA).
Animals Male Kunming,
Balb/c, and C57 mice [20¡À2 g, Grade II, Certificate No
SCXK (Jun) 2002-001, Experimental Animal Center of Academy of
Military Medical Sciences] were used. After transplanted with tumor
cells, animals were randomly distributed into different groups. The
control group was administered with saline alone and the others were
treated with different drugs. All drugs were dissolved in normal
saline and freshly prepared on the experimental day. Both normal
saline and agmatine were administered subcutaneously (sc) and
cyclophosphamide was injected intraperitoneally (ip) in a volume of
10 mL/kg. All of the animals were housed and maintained in a
temperature-controlled room (22 ¡ãC-24 ¡ãC) with free access to
qualified food and water at all times.
Cell culture MCF-7
human breast cancer cells were maintained in RPMI-1640 medium with
10% fetal bovine serum (FBS), 100 kU/L penicillin and 100 kU/L
streptomycin. Cells were incubated at 37 ¡ãC in a humidified 5% CO2
atmosphere and subcultured every 3 d.
Evaluation of agmatine's
inhibitory effects on the growth of tumor cells in vivo S180
sarcoma and B16 melanoma cells were subcultured in the
abdominal cavity of mice for 8 d. The resulting ascites were diluted
with saline to form a suspension containing 2¡Á1010 cells/L.
Aliquots of cell suspensions (0.2 mL) were injected (sc) into the
right armpit of the mice. From the 1st day after implantation,
saline, agmatine (5-40 mg/kg, tid, sc), or cyclophosphamide
(20 mg/kg, qd, ip) were administered for 10 d. At d 10, the animals
were killed and the tumors were chipped from their armpits. The
tumor weights (g) were measured and the mean tumor weight of every
group was calculated. The anti-tumor activities of the drugs were
determined by a comparison between the inhibitory ratios obtained
from the treated groups and the control group. The inhibitory ratio
of drugs was expressed as [(average tumor weights in saline group-average
tumor weights in drug treated group)/(average tumor weights in
saline group)¡Á100].
Measurement of proliferation
in vitro in [3H]thymidine incorporation assay
Proliferation of the MCF cells was assessed by [3H]thymidine
incorporation assay. Briefly, cells suspended in RPMI-1640 medium
with 10% FBS were seeded into a 96-well cell culture plate
(80 µL/well) at a density of 6000 cells/well. Then they were treated
with saline (control), or different concentrations of agmatine (1,
10, 100, 200, 500, or 1000 µmol/L), respectively, at a volume of
20 µL. Drugs were added for a total period of 48 h and [3H]thymidine
(3.7¡Á104 Bq/well) was added at 36 h of incubation. The
medium was removed and the cells were washed three times
with phosphate-buffered saline and then twice with ice-cold 10%
trichloroacetic acid. Fixed cells were then solubilized
in 0.2 mol/L NaOH (100 µL/well) and sonicated for 15 min.
After mixing with scintillant liquid (1 mL) for 24 h, an aliquot
(90 µL/well) was used for scintillation counting. Then the
radioactivity was determined with a Multi-purpose Scintillation
Counter (Columbus Instruments, Columbus, OH, USA). The mean cpm
value of every group was calculated. The anti-proliferation potency
of the drugs was determined by a comparison between the inhibitory
ratios obtained from the treated groups and the control. The
inhibitory ratio of the drugs was expressed as [(average cpm value
in control group-average cpm value in drug treated group)/(average
cpm value in control group)¡Á100%].
Measurement of proliferation
in vitro Cell proliferation was also confirmed again
by measuring with MTT assay based on the colorimetric
measurement of formazan dye formed from MTT by
mitochondrial dehydrogenases. Exponentially growing cells were
plated at a seeding density of 7.5¡Á104 cells/mL in
96-well plates (80 µL/well). Then they were treated with saline
(control), or different concentrations of agmatine (100, 200, 500,
or 1000 µmol/L), respectively, at a volume of 20 µL. After they were
incubated with or without drugs for 44 h, 20 µL of MTT reagent
(0.5 g/L) was added to each well. The plates were incubated at 37 ¡ãC
for an another 4 h. At the end of the incubation, the formazan
crystals formed by MTT metabolism were solubilized by the addition
of 100 µL of 10% SDS to each well. After 16 h, the absorbance of the
solubilized product was measured at 570 nm in a Micro-plate Reader
(Molecular Devices Corporation, Sunnyvale, CA, USA). The
anti-proliferation potency of the drug was determined by a
comparison between the inhibitory ratios. The percentage of growth
inhibition was calculated by comparison of the absorbance of the
treated group versus the control [(average absorbance value in
control group-average absorbance value in drug treated
group)/(average absorbance value in control group)¡Á100].
Lactate dehydrogenase release
assay To assess whether the reduction of cell numbers was
attributable to the cellular toxicity of polyamines or agmatine, we
measured the release of lactate dehydrogenase (LDH) in
cell medium after drug treatment. Cells were cultured at a seeding
density of 7.5¡Á104 cells/mL in 24-well plates
(800 µL/well). They were treated with saline, a range of
concentrations of agmatine, spermidine, or spermine, respectively,
at a volume of 200 µL and incubated at 37 ¡ãC and 5% CO2
for 48 h. Then 0.8 mL of the supernatant of each well was used for
analysis by an Automatic Biochemical Analyzer (Hitachi7020, Tokyo,
Japan). The release of LDH from the treated cells was compared to
the control.
Statistical analysis Data
were expressed as mean¡ÀSD. SAS software (SAS Inc, Raleigh, NC) was
used to conduct a one-way ANOVA. P<0.05 was considered
statistically significant.
Results
Inhibitory effects of agmatine on
the growth of S180 sarcoma tumor cell lines in Kunming
and Balb/c mice In Kunming
mice transplanted with S180 sarcoma tumor cell lines, the
S180 cells grew well and the average tumor weight reached
1.6 g in the normal, saline-treated group. Meanwhile,
cyclo-phosphamide, a clinically approved anticancer agent, exhibited
significant anti-tumor activity. In the cyclophosphamide-treated
group (20 mg/kg, qd, ip), the average tumor weight was only 0.7 g
and the inhibitory ratio was 56.3%. Agmatine also exerted a
remarkable inhibitory effect on tumor growth. In the agmatine-treated
groups (5-40 mg/kg, tid, sc), the tumor weights were significantly
reduced (n=27, P<0.05) in a dose-dependent manner. The
inhibitory ratio of tumor growth reached 31.3% at a dose of 40 mg/kg
(Table 1).
In Balb/c mice transplanted with S180
sarcoma tumor cell lines, we obtained similar results. The average
tumor weight was 1.0 g in the normal, saline-treated group.
Cyclophosphamide (20 mg/kg, qd, ip) inhibited tumor growth
significantly, and the inhibitory ratio reached 50.0%. Agmatine also
inhibited the growth of tumors in a dose-dependent manner (n=10,
P<0.05). The inhibitory ratio of agmatine on tumor growth was
50.0% at a dose of 40 mg/kg (Table 2).
Inhibitory effects of agmatine on
the growth of B16 melanoma tumor cell lines in C57
mice In C57 mice transplanted with B16
melanoma tumor cells, the tumor weight was 1.8 g in the normal,
saline-treated group. In the cyclophosphamide-treated group
(20 mg/kg, qd, ip), the tumor weight decreased to 1.0 g and the
inhibitory ratio reached 44.4%. Agmatine
(2.5-20.0 mg/kg, tid, sc) significantly suppressed the growth of the
tumor (n=10, P<0.05), but the effect did not exhibit
an obvious dose-dependent relationship (Table 3).
Inhibitory effect of agmatine on
the proliferation of MCF cells in vitro In the [3H]thymidine
incorporation assay, the MCF cells grew well and the average cpm
value was 3143.8 in the normal, saline-treated group after a 48-h
incubation. Agmatine showed anti-proliferation activity compared
with the saline-treated group in a concentration-dependent manner.
The cpm value was significantly reduced (n=8, P<0.05)
after pretreatment with agmatine (1-1000 µmol/L). The inhibitory
ratio of cell proliferation was 50.3% at a concentration of
1000 µmol/L (Table 4).
This effect of agmatine on cellular
proliferation was further proved with the MTT assay. The MCF cells
grew well and the absorbance value was 0.99 in the normal,
saline-treated group after a 48-h incubation. Agmatine showed
anti-proliferation activity compared with the saline-treated group
in a concentration-dependent manner. The absorbance value was
significantly reduced (n=8, P<0.05) after the cells
were administered with agmatine (100-1000 µmol/L). The inhibitory
ratio of cell growth was 23.8% at a concentration of 1000 µmol/L
(Table 5).
We then investigated the
time-dependent effect of agmatine on MCF cell proliferation. MCF
cells were cultured for different lengths of time in the presence of
1 mmol/L agmatine, and cell viability was evaluated by a [3H]thymidine
incorporation assay. Over 48 h, the inhibitory potency of agmatine
strengthened gradually with prolonged time (n=8, P<0.01).
The inhibitory ratio was 10%, 17%, 38%, and 62% at 12 h, 24 h, 36 h
and 48 h, respectively (Figure 1).
Effect of agmatine on LDH release
in the medium To assess whether the reduction of cell numbers
was attributable to toxicity of polyamines or agmatine, we measured
the release of LDH in cell medium after drug treatment.
Administration on MCF cells for 48 h, spermine (20 µmol/L) or
spermidine (20 µmol/L) significantly increased the activity
of LDH in the medium, but agmatine (1-1000 µmol/L) did not. At
a concentration of 1000 µmol/L, agmatine decreased the LDH activity
significantly (n=8, P<0.05; Table 6).
Putrescine reverses agmatine's
inhibitory effect on MCF cell proliferation As polyamines are
essential growth factors, their dramatic intracellular decrease may
be the main mechanism of the anti-proliferation action of agmatine.
To check whether this is the mechanism involved, MCF cells were
treated simultaneously with agmatine (1 mmol/L) and putrescine
(12.5-100.0 µmol/L). Putrescine counteracted the inhibitory effect
of agmatine on MCF cell proliferation in a concentration-dependent
manner (n=8, P<0.05; Table 7).
Discussion
The present study demonstrated the
inhibitory properties of agmatine toward S180 sarcoma and
B16 melanoma cells in vivo. We found that, at doses of
5-40 mg/kg, agmatine suppressed S180 and B16
cell growth in three kinds of mice in vivo. The highest
inhibitory ratio was more than 50.0%. We applied for a Chinese
patent with these results in 2002 (02125495.8). Although Gardini
et al reported that agmatine inhibited the proliferation of rat
hepatoma cells in vitro by modulation of polyamine metabolism
in 2003[13], they did not report anything related to the
inhibitory effects of agmatine on the growth of transplanted tumors
in vivo; in addition, the paper by Gardini et al[13]
was published much later than when we applied for the Chinese
patent. It is reasonable to state, therefore, that our current
results demonstrate for the first time that agmatine has an
inhibitory effect on S180 and B16 cell lines
in vivo.
Polyamines play an essential role in
proliferation, differentiation, and neoplastic transformation in
mammalian cells[1]. Indeed, cellular polyamine levels are
higher in tumor cell lines. Conversely, the depletion of polyamines
results in growth arrest of neoplastic cells in vitro. The
polyamine-biosynthetic pathway is an inviting target for the
development of agents inhibiting carcinogenesis and tumor growth.
The present therapeutic agents acting on this pathway are
¦Á-difluoromethylornithine (DFMO) and polyamine analogs[2].
They influence both polyamine synthesis and degradation and are now
being used in clinical trials.
Agmatine is an analog of polyamines
and can modulate the cellular concentration of polyamines[14].
As an intermediate of putrescine, agmatine may be a factor for
increasing the cellular concentration of polyamines. Although tumor
cells and tissues have been reported to have increased polyamines
levels compared with normal cells, this increase is often in the
range of 2- to 3-fold. When putrescine levels are approximately
10-fold higher than those present in cancer cells, the cells undergo
apoptosis[15]. It has also been reported that overloaded
polyamines have toxic effects on some normal cells. The toxicity of
polyamines was studied in a well-characterized neuronal system of
cerebellar granule cells in vitro. Twenty-four-hour exposure
to spermine (1-500 µmol/L) resulted in a concentration-dependent
death of granule cells, with the half of lethal dose (LD50)
being reached at a concentration below 50 µmol/L. Putrescine was
moderately toxic, with the LD50 at a concentration of
only 500 µmol/L. The LD50 of spermidine was tested
between concentrations of 50 and 100 µmol/L and its toxicity has
been evaluated to be approximately 50% of that of spermine[16].
This was consistent with our results (data not shown). In contrast,
agmatine has been postulated to decrease the cellular level of
polyamines by inducing antizyme, competing with putrescine on
transporter and other mechanisms.
So, in theory, agmatine might have
double-edged effects on cell growth. But, to date, there has been no
report that agmatine can enhance the proliferation of cells.
Conversely, there is much evidence that agmatine can suppress cell
proliferation, including different cells and malarial para-
sites[17,18]. Consistent with these studies, the present
study proves agmatine has a significant inhibitory effect on cell
proliferation in several classical solid tumors in a transplanted
model in vivo and in an MCF model in vitro. Putrescine
prevented the effect of agmatine on [3H]thymidine
incorporation in MCF cells. This effect of agmatine is similar to
that of DFMO, which can block the synthesis of polyamines
significantly. These results suggest that the effect of agmatine
might be related to its influence on the synthesis of polyamine.
In addition, polyamines can interact
with DNA directly[19], so, in our experiments, the
decreased [3H]thymidine incorporation may be caused by
the inhibitory synthesis of DNA. As putrescine plays a partial role
in energy supply[20], the results of the MTT assay
indicate that agmatine might inhibit MCF cell proliferation by
influence on energy metabolism. In addition, the effect on LDH
activity in the medium showed that agmatine (1-1000 µmol/L)
exhibited no cellular toxicity, whereas spermidine (20 µmol/L) and
spermine (20 µmol/L) did. To sum up, these results partly
demonstrate that agmatine does not increase the level of cellular
polyamines and supports the conclusion that the anti-proliferation
effect of agmatine is a result of polyamine limitation.
Currently, we know that agmatine's
toxicity is low and its effects on tumor cells are not similar to
those of classical chemotherapeutical drugs. Regarding its low
toxicity, its enhancement of opioid analgesia, and its
antidepressant effect[21,22], we hope that agmatine could
efficiently improve life quality of cancer patients. There are still
many issues to be explored, and further experiments should be
carried out to confirm our results.
In conclusion, agmatine has
significant inhibitory effects on transplanted tumor growth in
vivo and proliferation of tumor cells in vitro. The
possible mechanisms might be related to inducing decrease of
intracellular polyamine contents.
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