Introduction
olorectal
cancer is one of the major causes of cancer-related deaths in the world. Over
the last decade, much progress has been made in the treatment of this
malignancy and new chemicals have been introduced. The survival of people with
metastatic colorectal cancer has improved with new therapeutics. Recently,
selective serotonin reuptake inhibitors (SSRIs) have
been suggested as preven-tive
agents for people at high risk of developing colorectal cancer,1 and
is based on reports indicating that SSRIs could decrease the risk of
colorectal cancer in humans.2 Laboratory studies have shown that Fluoxetine,
a novel SSRI drug, at doses of 10 – 100 µM may reduce the growth of cancer
cells in vitro. This effect may be mediated by increasing serotonin
levels in the peripheral nerve endings of the gastrointestinal tract.3
However; other reports suggest that SSRIs may induce mammary tumors in animals
and breast cancer in humans.4, 5
Serotonin (5-hydroxytryptamine; 5HT) is a
neurotransmitter that mediates a wide variety of physiological effects (both
peripherally and centrally) by binding to multiple receptor subtypes. The major
sites of 5HT synthesis and storage are located in the periphery, the gut enterochromaphin
cells and blood platelets, respectively. The great diversity of 5HT functions
is due to different 5HT receptors and the pharmacological complexity of these
receptors, reflecting the second messenger system to which the receptors are
coupled. The 5HT family, including 5HT1 and 5HT5 subtypes
of receptors negatively interact with adenylate cyclase and the 5HT2 subfamily
of receptors is coupled to the activation of phospholipase C-β. Adenylate
cyclase is activated by the 5HT4, 5HT6, and
5HT7 subtypes of receptors.6
5HT3 receptors are cation
channels that tend to depolarize the membrane when activated.7 5HT3
receptors contain four transmembrane domains and exist as homo- or
hetero-pentamers in the membrane. Two 5HT3 receptor subunits have
been identified: 5HT3A and 5HT3B.8,9 The 5HT3A
subunit can form a functional channel as a homo-pentamer, but the 5HT3B
subunit alone does not. In addition to two known 5HT3 subunits, two
splice variants of 5HT3A have been identified by Brüss et al.10
which further increases the functional diversity of these receptors. It is
possible that additional, undiscovered 5HT3 receptor subunits exist.
The 5HT4 receptor was first characterized
in mouse collicular neurons.11 It is also found in the hippocampus
and in peripheral tissues such as the guinea pig ileum, rat esophagus, and
human atrium.12 The 5HT4 receptor has been suggested to
contribute to tumor cell progression by secretion of hormones and other growth
factors.13
5HT3 receptor is expressed in the enteric
nervous system14,15 and plays an important role in enteric
physiology. Although there is some evidence regarding the mitogenic effects for
5HT in certain malignancies such as breast, prostate, and bladder cancer16–18;
to date, however, there have been no studies specifically assessing the role of
5HT and expression of 5HT3 and 5HT4 receptors in colon
cancer. There are some reports concerning the mitogenic role of serotonin in
colon cancer. For example, in one study, the influence of serotonin in dimethylhydrazine-induced
adenocarcinoma of the colon has been reported.19,20 However, few
studies have been conducted that examine the possible role of 5HT3 and
5HT4 receptors’ agonists and antagonists in tumor regulation. In the
present study we have determined the effect of 5HT, 5HT3, and 5HT4
agonists and antagonists on the proliferation of the HT29 human colon
adenocarcinoma cell line. We also studied the expression of 5HT receptor
subtypes 3 and 4 in this cell line and in colon cancer tissue.
Materials and Methods
Tissues and cell line
Human colon cancer tissue
samples were obtained during surgery from five patients (Imam Hospital, Tehran,
Iran). Tissue samples were used for western blot analysis.
The HT29 cell line was kindly
provided by the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran. It
must be noted that HT29 is a current human cell line with potent metastatic potential
which has been widely used in colorectal carcinoma studies. With respect to
this and according to other studies,21 we selected this cell line
for our study. The cells were maintained
in exponential growth phase in 25-cm2 flasks in RPMI 1640 (Gibco, Germany)
medium supplemented with 10% heat-inactivated fetal calf serum (Gibco,
Germany), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin
(all from Sigma, Germany) in a humidified atmosphere of 5% CO2 at a
constant temperature of 37°C. Cells were split when they reached a confluency
of approximately 90 – 95%.
MTT proliferation assay
HT29 cells were washed with Phosphate
buffer saline (PBS) and harvested with a 0.5% trypsin (Sigma, Germany) solution
at 50 – 60% confluency. Cells were then added to wells at a density of 104
cells/well in a 96-well plate to a final volume of 100 μL/well.
After 24 hours of incubation at 37°C in a 5% CO2 atmosphere, the culture
medium was replaced with 200 μL fresh culture medium containing 5HT
hydrochloride (Sigma,Germany),1-phenylbiguanide hydrochloride (5HT3 agonist), cisapride (5HT4 agonist), Y-25130 hydrochloride (5HT3
antagonist), or RS
23597-190 (5HT4 antagonist;
all from Tocris Laboratories, UK) at concentrations of: 3.125, 6.25, 12.5, 25,
50, and 100 µM. Cells cultured solely in media served as negative controls.
After 48 hours of incubation at 37°C in a 5% CO2 atmosphere,
the culture medium was removed and 8 µL MTT reagent (diluted in PBS at a
concentration of 4 mg/mL; Sigma, Germany) was added to 50 μL of fresh
culture medium at a final concentration of 0.55 mg/mL. The optimum incubation
period time was determined in a pilot study.
After incubating the plates again for a 4
hour at 37°C in 5% CO2 atmosphere, the resulting formazan product
was resolved in 50 μL dimethyl sulfoxide (Merck Germany) and measured
spectrophotometrically at 570 nm (with a reference wavelength of 690 nm). The mitogenic
effect was determined using the following formula: (mean absorbance (570 – 690 nm)
of treated wells/mean absorbance (570 – 690 nm) of control wells) ×100.
Results were expressed as the
mean of five assays (three replicates for each concentration of each assay).
Protein extraction
HT29 cells were harvested with
a 0.5% trypsin solution at 90 – 95% confluency and resuspended in lysis buffer
containing a complete protease inhibitor (Roche, Germany), 1% SDS, tris 50 mM,
pH 7.4; 0.5% sodium deoxycholate (Sigma, Germany). The lysate was incubated for
30 minutes at 4○C. Subsequently, the suspension was
centrifuged at 12000 g for 20 minutes at 4○C and supernatant
was kept at -70°C.
Frozen samples of colon cancer
tissue and rat brain as positive controls were homogenized in lysis buffer and
the proteins were extracted as described above. Protein concentrations of
supernatants were determined by the Bradford assay, with BSA as a standard.
Western blot analysis
Equal amounts of
protein samples were heated at 100°C for 5 minutes in 6× SDS
gel-loading buffer consisting of 375 mM Tris-HCl (pH 6.8), 12% SDS, 60%
glycerol, 30% 2-mercaptoethanol, and 0.6% bromophenol blue. Proteins were separated
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 5% stacking and 10%
separating gels. Proteins were then transferred to an Immobilon-P PVDF membrane
(BioRad, Germany). Nonspecific binding
sites were blocked by 1% bovine serum albumin in PBS (BSA-PBS). The
membrane was subsequently incubated for 1 hour with goat anti-5HT3
or goat anti-5HT4 antibody (Santa Cruz, USA). After
incubation with antibody, the membrane was washed three times, for 15 minutes
each, with 50 mL of PBS containing 0.05% tween 20 (PBS-T). The membrane was
then incubated for 1 hour with peroxidase conjugated donkey anti-goat IgG
(Santa Cruz, USA) and was washed as mentioned above. The membrane was developed with diaminobenzidine (DAB;
Sigma, Germany). Normal marginal tissues from the same patients were used as
negative controls.
Statistical analysis
The results were expressed as the mean±SD.
Statistical differences were evaluated by one-way ANOVA. P≤0.05 was considered significant.
Results
MTT assay
The MTT
assay was used to determine the effects of 5HT, 5HT3, and 5HT4
receptor agonists and antagonists on human colon cancer cell line
proliferation. Cells were treated with increasing concentrations of drugs and
the numbers of cells after incubation were determined spectrophotometrically
using the MTT reagent.
As shown
in Figure 1, 5HT, 5HT3 agonist (1-phenylbiguanide hydrochloride), and the 5HT4 agonist (cisapride)
caused a dose dependent proliferation of HT29 cells after 48 hours incubation.
The maximum proliferation was at a 5HT concentration of 12.5 μM (P≤0.01). 5HT3 agonists significantly stimulated the
growth of cells at concentrations of 3.125 μM (P≤0.05) and 6.25 μM (P≤0.01).
Stimulation of cell growth with 5HT4 receptor agonist (cisapride)
needed a higher concentration of related agonist. A significant growth
stimulatory effect was observed at 100 μM (P≤0.05).
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Figure
1. Effects of 5HT, 5HT3, and 5HT4
agonists on human colon cell proliferation. Cells were treated with
increasing concentrations of drugs and cell proliferation was determined by
MTT proliferation assay. Results represent the mean±SD values. (*P
≤0.05, **P≤0.01)
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5HT3
receptor antagonist (Y-25130 hydrochloride) and 5HT4 antagonist
(RS 23597-190) had dose-dependent inhibitory effects on cell
growth (Figure 2). The 5HT3 antagonist significantly inhibited
proliferation of HT29 cells at concentrations of 50 and 100 μM (P≤0.01). The 5HT4 receptor antagonist inhibited
proliferation of the colon cell line at only the 100 μM concentration (P≤0.01).
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Figure 2. Antagonist to receptor 5HT3 (Y-25130
hydrochloride) significantly inhibited proliferation of HT29 cells at
concentrations of 50 and 100 µM (P≤0.01).
5HT4 antagonist (RS 23597-190) inhibited proliferation
of colon cell line at 100 µM (P≤0.01)
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Western blot analysis
Western
blot analysis of HT29 cells and human colorectal tumor tissues has confirmed
expression of 5HT3 and 5HT4 receptor proteins. At least
three different western blots were performed with total proteins extracted from
each sample. Polyclonal anti-5HT3 IgG recognized a single band of
approximately 48 kDa, and anti-5HT4 IgG showed a band of
approximately 44 kDa in the HT29 cell line and cancer tissues. Proteins
isolated from the rat brain (positive control) revealed similar bands to those
of proteins isolated from the HT29 cell line and human colorectal tumor tissues
(Figure 3).
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Figure
3. Western blot analysis showing expression of 5HT3 and
5HT4 receptors in HT29 cell line and human colon cancer tissue.
Polyclonal anti-5HT3 IgG recognized a single band of approximately
48 kDa (A), and anti-5HT4 IgG showed a band of approximately 44
kDa in HT29 cell line and cancer tissues.
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Discussion
In
the present study, for the first time, we have demonstrated that 5HT3
and 5HT4 serotoninergic receptors are expressed in a human colon
cancer cell line (HT29) and in the tumor tissues from patients with colon
carcinoma.
Moreover, MTT
proliferative assays which measure cell viability through the reductive
activity of mitochondria revealed that 5HT, 5HT3, and 5HT4
agonists stimulate HT29 cell proliferation. It has been shown that 5HT acts as
a growth factor for several types of non-tumor and tumor cells including the choriocarcinoma,
breast, prostate, and bladder cancer cell lines.16–18,22 Given the wide distribution of 5HT3 and 5HT4 receptors
in the alimentary tract, we were interested to determine if the mitogenic effect of 5HT was mediated by these
receptors. The proliferation of the HT29 cell line in response to 5HT3
and 5HT4 agonists suggested that the mitogenic effect of 5HT on this
cell line was mediated in part by 5HT3 and 5HT4
receptors. Siddiqui et al. have shown that 5HT1 but not 5HT2
and 5HT3 exert cell growth in bladder cancer cells.16
This effect is mediated by 5HT2 in a breast cancer cell line.17
Although 5HT1 and 5HT2 receptors are known as
mitogenic receptors, the mitogenic activity of 5HT3 receptors is a
novel finding. The mitogenic effect of 5-HT via a 5HT2A receptor has
been described as being mediated by different signal transduction pathways,
including the Jak/STAT23 and Erk/MAPK activation.24,25
The signal transduction pathway of the mitogenic effect of 5HT3
receptor remains to be revealed.
Likewise, the 5HT4
receptor agonist results in HT29 cell line proliferation. Receptor subtype 4
has been shown to stimulate hormone secretion. The agonist to 5HT4
stimulates cortisol secretion in patients with adrenal tumors and IGF-1
secretion in mice fibroblasts.26 It has been suggested that 5HT4
may contribute to tumor cell progression by stimulating hormonal secretion
and other growth factors, probably via activation of adenylate cyclase.27
Moreover, 5HT3 and 5HT4
antagonists inhibited HT29 cell growth. In line with our findings, the
anti-proliferative effect of RS 23597-190 (5HT4 antagonist) has also been reported
in the DU145 prostate cancer cell line.28 However, the 5HT3
and 5HT4 antagonists had no significant inhibitory effects on cell
growth of the bladder cancer cell line 16 and PC3 prostate cancer cells.16,29
To further confirm
the role of 5HT3 and 5HT4 receptors in regulation of the colon
cell line growth, western blot analysis was carried out. In the present study
western blot analysis identified 5HT3 and 5HT4 receptor
proteins in the HT29 colon cell line and colon cancer tissues. A similar
profile with HT29 cells, colon tumor tissues, and rat brain demonstrated major
protein bands at 48 and 44 kDa for 5HT3 and 5HT4
receptors, respectively. Also this immunoblot expression for the 5HT3 receptor
was completely significant when compared with the negative control (normal
human marginal tissue) and as strong as the positive control (brain tissue). However,
for the 5HT4 receptor, this expression was mild. According to other
protein expression studies, it has been previously demonstrated that the 5HT2
receptor expressed profoundly in breast cancer.17 Other western blot
and immunohistochemistry studies have shown that 5HT1A and 5HT1B
receptors expressed in bladder and prostate cancer.16,18 In those
studies, immunohistochemistry assays showed expression of 5HT receptors in the cell
membrane and cytoplasm of colon epithelial cells which are the site of
localization of the G-protein coupled receptors (GPCR) as 5HT receptors. Therefore,
our study as long as previous researches, describes the expression of 5HT
receptors in cancer. But further studies are needed to clarify the pattern of
these receptor expressions.
In summary, our
results demonstrate for the first time that the 5HT3 and 5HT4
receptors are expressed in both human colon cancer tissue and cell line HT29. This
expression for the 5HT3 receptor is much stronger and more important
than 5HT4. Our data also show that 5HT, in part via the 5HT3
and 5HT4 receptors, has a mitogenic effect on HT29 cell
proliferation.
Acknowledgments
This study was supported by a research
grant from Pasteur Institute of Iran. We would like to acknowledge Dr. Fazeli
from Surgery department of Imam Hospital for providing tumor biopsies and Mr.
Davood Iravani for his technical assistance.
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Keywords: Colon adenocarcinoma · HT29 cell · serotonin · 5HT3 and
5HT4 receptors