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Research article
Effect of c-Met inhibitor SU11274 on human colon cancer cell growth
Shuohui GAO1, Chao LIU2, Jun WEI1,Ye FENG1
1. The department of general surgery, the third hospital of Jilin University, 130033
China
2. The department of neurosurgery, the second hospital of Jilin University, 130021
China
Correspondence to: XXXXX
Keywords:
Colon cancer, SU11274, cell growth, c-Met
Abstract
Background: Colon cancer is one of the major malignancies worldwide and still
remains resistant to much of the currently available chemotherapy. Downregulation of
HGF/c-Met signaling pathway is an emerging therapy for cancer treatment.
Methods: In this study, the inhibitory effects of c-Met phosphorylation were observed
with SU11274 on different colon cancer cell lines in vitro.
Results: The results revealed the significant inhibitory effects of SU11274 on cell
proliferation and cell survival, in a time and dose-dependent manner. Furthermore, the
inhibitory effects of SU11274 on different subgroups of colon cancer cells via the
signaling pathway were implicated in this study.
Conclusions: The results suggested the possible selective therapeutic effects of c-Met
inhibitor on colon cancer
Colon cancer represents one of the most common malignancies worldwide, and
despite advances in chemotherapy, this cancer still remains a major cause of death.
The risk of severe adverse effects are also associated with currently available
anti-cancer drugs [1]. Tumor invasion and lymph node metastasis are important
factors determining the prognosis of the disease. Therefore, novel individualized
treatment options are needed to improve patients’ survival, and the prevention of
cancer invasion and metastasis. The receptor tyrosine kinase c-Met, a receptor to
Hepatic Growth Factor (HGF) which activates the c-Met signaling pathway by
phosphorylation, was first identified by chemical rearrangement in vitro [6]. The
c-Met protein contains a tyrosine kinase domain that initiates a range of signals to
regulate various cellular functions, including cell proliferation, motility, adhesion and
tumor cell invasion [3]. In the past two decades, accumulating evidence suggests that
overexpression of c-Met protein is strongly associated with poor patient survival or
metastasis in numerous human malignancies [2,5]. Inappropriate HGF/c-Met
signaling in cancers can resemble developmental transitions between epithelial and
mesenchymal cell types. Thus, inactivation of c-Met phosphorylation has been
considered a potential therapeutic target for influencing the events of tumor
progression and some agents are currently in clinical trials [7]. Furthermore, c-Met
signaling is complex, and how the regulation of its receptor subunits via HGF/c-Met
signaling axis influences its oncogenic behavior is not fully understood. A small
molecule inhibitor SU11274 has been shown to inhibit c-Met phosphorylation, and
consequently decrease cell growth, motility, invasion and proliferation in lung cancer
and ovarian carcinoma [4, 9]. These results support the therapeutic potential of
targeting c-Met in cancers where c-Met plays a crucial role in tumor growth and
metastasis. The overexpression of c-Met in colon cancer is an important prognostic
marker for early stage invasion and regional metastasis, and the development of new
targeted therapy inhibiting the c-Met signal pathway promises a new modality for
treatment [8]. The aim of this present study was to study the inhibitory effect that
SU11274 had on colon cancer cell growth via HGF/c-Met signal pathway.
1. Methods
Cell lines and cell culture
Four well-differentiated colon cancer cell lines (K-ras wild type HT-29, Hct-8, and
K-ras mutant type Hct-116 and DLD-1) were purchased from the American Type
Culture Collection (ATCC, Rockville,MD). Cells were maintained in RPMI 1640
medium (Cellgro), supplemented with 10% FCS, and 1% penicillin-streptomycin.
After incubation at 37°C with 5% CO2, the cells were harvested during log phase
growth. For analysis all the cultured cells were flash frozen in liquid nitrogen and
stored at -80℃.
Reagents and antibodies
Rabbit anti-human Met, Akt, mTor, erk, ps6 and beta-actin monoclonal antibodies, as
well as rabbit anti-human phospho-Met, phospho-Akt, phospho-mTor, phospho-erk,
and phospho-ps6 monoclonal antibodies were all purchased from SIGMA, Inc.
(Beverly, MA, USA), and were used as primary antibodies. Peroxidase-conjugated
goat anti-rat monoclonal antibodies were used as secondary antibodies and were
purchased from Jackson Immunnoresearch Inc. Hepatocyte growth factor (HGF) was
purchased from Rocky Hill company. c-Met specific inhibitor SU11274 was
purchased from SUGEN, Inc (South San Francisco, CA, USA).
Cell viability assay
Cell viability was measured by the acid phosphatase assay. Continuously cultured
cells were harvested and seeded on 96-well plates at a density of 8x103 cells per 200
ul in medium containing 10% FBS and cultured for 24 h. Then, cells were treated
with 30ng/ml HGF and with 0.01uM, 0.1uM, 0.5uM and 2.5uM of SU117274
separately. After incubation at 37˚C for 1.5 h, the reaction was stopped by adding 10
μl of 1 M NaOH and cell viablilty was evaluated using a microplate reader (Bio-Rad
Laboratories, Hercules, CA) at 405 nm.
Western blotting
For stimulation studies with HGF, continuously cultured cells were deprived of
growth factors by incubating the cells without FBS for 24 h and cells were
homogenized in lysis buffer. Cells were treated with different doses of SU11274. The
cells were tested for protein tyrosine phosphorylation in response to 30 ng/ml HGF
for 15 minutes. Proteins were quantified by BioRad RC-DC assay. Proteins were
resolved using SDS-PAGE gel electrophoresis and blots were incubated overnight at
4°C with specific primary antibodies, followed by 1 h incubation with appropriate
peroxidase-coupled secondary antibodies and visualized. Western blotting band
intensities were expressed in arbitrary units and normalized to fold increases
compared with control. The same blot was also incubated with an anti--actin
antibody (Sigma, St. Louis, MO) for the normalization of protein loading. The
membranes were stripped between hybridizations.
Cell cycle analysis by flow cytometry
Cells were synchronized by serum deprivation, and the experimental cells were
treated 24 h with 0.5 M SU11274 and untreated cells were used as controls. Cells
were fixed with 75% ethanol in PBS, cells were treated with 30 ng/ml HGF in PBS at
37°C for 15 min and stained on ice with propidium iodide solution (20 mg/ml)
(Invitrogen) containing RNase A (100 g/ml) (Promega, Madison, WI, USA) at 4°C
for 30 min. The distribution of cells in the cell cycle was assessed by a FACSscan
flow cytometer
(Becton Dickinson, San Diego, CA, USA). The result is the mean of
three independent experiments and was processed using Excel (Microsoft, Inc.).
2. Results
Inhibition of cell proliferation
Each cell line was treated with different concentrations of SU11274 (0, 0.1, 0.5, and
2.5 uM) for 24 h, 48h and 72h separately, and cell growth was determined by the acid
phosphatase assay (Figure 1). Decreased proliferation was observed in all colon
cancer cells in response to SU11274 in a dose and time dependent manner. The dose
of 0.5 M SU11274 was sufficient to achieve the similar maxim inhibition after
treatment for 72 h as the dose of 2.5 M, but in cell line DLD-1, the inhibition was
similar after treatment with different doses of SU11274 for 48 h. These results suggest
that the inhibitory effects of SU11274 correlate with the dose and the time of
treatment.
HT-29
HCT-8
120
120
Cell prolifiration (%)
100
80
60
40
20
0
HT-29
0
24
48
SU11274-0.1M
SU11274-0.5M
SU11274-2.5M
100
Cell prolifiration (%)
SU11274-0.1M
SU11274-0.5M
SU11274-2.5M
80
60
40
20
0
HCT-8
72
0
Time effect (h)
HCT-116
48
SU11274-0.1M
SU11274-0.5M
SU11274-2.5M
100
120
SU11274-0.1M
SU11274-0.5M
SU11274-2.5M
Cell prolifiration (%)
100
80
60
40
20
0
HCT-116 0
72
DLD-1
120
Cell prolifiration (%)
24
Time effect (h)
24
48
72
80
60
40
20
Time effect (h)
0
DLD-1
0
24
48
72
Time effect (h)
Figure 1:
Graphs representing the effect of SU11274 on colon cancer cell viability.
Different colon cancer cells were treated with increasing concentrations of SU11274.
Cell viability was measured at different time points. The experiments were repeated 3
times and the results represent mean±SD.
Expression of c-Met signaling in cell lines with or without SU11274
A monoclonal antibody against c-Met was used to detect the c-Met expression.
Expression of Met proteins downstream c-Met signaling pathway in all the cells was
strongly detected. In response to HGF (30 ng/ml, 15min; Figure 2), there was
increased tyrosine phosphorylation of multiple proteins in the four cell lines used.
SU11274 effectively decreased the amount of activated c-Met proteins to a detectable
level within 30 min of treatment without significantly affecting the total c-Met protein
levels. With the concentration of 0.1 uM SU11274 in all the cell lines, the tyrosine
phosphorylation of Met was inhibited totally, and tyrosine phosphorylation in the rest
of the downstream proteins like Akt, mTor, rpS6 and Erk were weakened. With the
concentration of 0.5 uM SU11274, the tyrosine phosphorylation was totally inhibited
for Akt in cell lines HCT-8 and HCT-116 and for rpS6 in cell line DLD-1. With the
concentration of 2.5 uM SU11274, total inhibition of tyrosine phosphorylation was
seen for Akt, rpS6 and Erk in cell line HT-29, and in other cell lines the activated
c-Met signaling proteins were weakened significantly.
HT-29
HCT-8
HCT-116
DLD-1
Figure 2: Western blot analysis showing the expression levels of phosphorylated
proteins downstream of the c-Met signaling pathway. P42????
Modulation of cell cycle progression by SU11274
The effect of SU11274 on cell cycle progression in cells was tested by flow cytometry.
The concentration of 0.5 M SU11274 was used to evaluate the effect of the
compound on cell cycle progression. The data demonstrated that all the cells exhibited
a dramatic reduction in DNA synthesis (Figure 3). The percentage of cells in S phase
was reduced significantly (from 24% to 8% in HT-29, 25.3% to 6.3% in HCT-8,
25.4% to 13.9% in HCT-116, and from 27.9% to 16.7% in DLD-1 cells, respectively).
This decrease in S-phase population was accompanied by a concomitant increase of
the G0/G1 fraction (increasing from 58.2% to 80.5% in HT-29, 59.6% to 81.3% in
HCT-8, 59.4% to 71.5% in HCT-116 and from 55.6% to 70.3% in DLD-1 cells,
respectively) ,suggesting a strong G0-G1 cell cycle arrest. Furthermore, SU11274
treatment also led to a reduction in the G2-M phase (from 17.8% to 11.5% in HT-29,
15.1% to 12.4% in HCT-8, 15.2% to 14.6% in HCT-116 and from 16.5% to 13% in
DLD-1 cells, respectively).
Figure 3: Cell cycle analysis of different colon cancer cells treated with 0.5 M
SU11274.
3. Discussion
Normal epithelial cells express c-Met widely, including normal colorectal epithelium,
and overexpression of c-Met has been shown to correlate with metastasis and poor
patient prognosis in various human malignancies [7]. So it is suggested that c-Met
expression plays an important role in the progression of the cancer. Targeting
HGF/c-Met signaling pathway provides a potential therapy for cancer treatment, and
the small molecule inhibitor SU11274 specific for c-Met has been used to test this
hypothesis in vitro [9]. However, the effect of c-Met inhibition on colon cancer has
not investigated widely. We endeavored to study the effects of SU11274 on the
oncogenic behavior of c-Met in colon cancer.
Four cell lines of colon cancers were
used in this study, all of which were treated with HGF and displayed proliferation in
the presence of HGF, indicating the initiation of HGF/c-Met signaling pathway, which
is dependent on HGF. To our knowledge, HGF is the only ligand for c-Met and is used
widely in targeting c-Met signaling pathway [10]. In the four cell lines used, the cell
proliferation showed inhibition with increasing concentrations of SU11274 and with
prolonged time periods as shown in Figure 1, indicating the blockage of HGF/c-Met
signal pathway by SU11274 is both time-dependent and dose-dependent.
The
inhibition rate of cancer cells by chemotherapy differs significantly with different
dosages and the administration conditions. A recent study on HCT-116 cell line
reflected this efficacy of chemotherapy [19]. Similar results were also seen in our
study.
In this study, we did not test the inhibition of SU11274 in response to
different doses of HGF, which might reflect the autocrine loops of HGF/c-Met which
is responsible for increase in cell proliferation [11, 12]. Our study also demonstrates
the differential effect of SU11274 on different colon cancer cell lines, so future
studies are required to analyze the response of different colon cncer cell types toward
chemotherapies [13].
HGF/c-MET signaling pathway regulates multiple cellular process, including cell
proliferation, apoptosis and angiogenesis, and in recent years efforts have been made
to investigate the functional effects of this pathway on the progression of human
cancer [20]. Blockage of the downstream c-Met signaling pathways plays a potent
role in inhibiting the c-Met oncogenic behavior [16,18]. Proteins downstream of this
pathway include Erk, c-Met, Akt and mTor, which induce proto-oncogenic expression
of c-Met. Studies targeting c-Met in vivo or in vitro hae demonstrated attenuated
expression of these proteins leading to the inhibition of cellular proliferation and
differentiation [21-22]. Western blotting in our study revealed the significant
inhibition of phosphorylation of these proteins following treatment with SU11274, as
shown in Figure 2.
The tyrosine phosphorylation of c-Met was inhibited totally by
0.1 uM SU11274 in all the cell lines, while the phosphorylation of the remaining
proteins were weakened with this dose and displayed decreasing expression levels in
response to increasing amount of SU11274 with time. The differential effects of
SU11274 on the inhibition of these proteins did not show any obvious significance in
the different cell lines. Total inhibition of phosphorylation of mTor was not detected
in this study, implicating that there might be a possible mechanism for regulation of
c-Met signal in tumor microenvironments [17].
It is known that c-Met is expressed more strongly in primary colorectal cancers than
in normal mucosa, and there is lack of evidence to show the correlation between the
c-Met overexpression and cancer stages [14]. In our study, the percentage of cells in S
phase arrested to G0/G1 phase was seen with SU11274 treatment,
suggesting the
inhibitory effect of this agent on the initiating events in the progression of cancer [15].
The significant reduction of DNA synthesis of cancer cells promises a therapeutic role
of c-Met inhibitor in cancer treatment. However, modulation of cell cycle was not
tested with different doses of SU11274 in our study.
We analyzed the effects of c-Met inhibitor SU11274 on different colon cancer cell
lines with proliferation, signal expression and motility in vitro. Our study strongly
suggests that SU11274 suppresses the oncogenic behavior of c-Met via its signaling
pathway, but different cancer subtypes have different responses to this inhibitor, and
the possible mechanisms underlying the resistance of cancer to chemotherapies are
yet to be elucidated.
Disclosure
The authors declare no conflicts of interest in this work.
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