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DEC1 and DEC2 are basic helix-loop-helix (bHLH)
transcription factors and are involved in the regulation of apoptosis, cell
proliferation, circadian rhythms and the response to hypoxia. Type I collagen
is the most abundant collagen of the human extracellular matrix (ECM) and is
known as an epithelial-mesenchymal transition (EMT) inducing factor. Here, we
demonstrated that DEC1, DEC2 and mesenchymal markers (snail and a-smooth muscle actin) were up-regulated in MCF-7 human breast cancer
cells cultured on the type I collagen-coated plates, while epithelial markers
(E-cadherin and claudin-1) were down-regulated in mRNA levels. Furthermore,
type I collagen increased cell proliferation and invasive potential and
affected cell morphology. These results indicated that type I collagen
up-regulated DEC1 and DEC2 concomitant with EMT processes in MCF-7 cells and
proposed a possibility that DEC1 and DEC2 participated in type I collagen
stimulated EMT.
Keywords: Type I collagen, Helix-loop-helix, MCF-7, Extracellular matrix
INTRODUCTION
Circadian rhythms are 24 h cycles of
biological processes including sleeping, moving and eating. The rhythms are
tightly regulated by molecular clock mechanisms based oscillations of clock
genes including period (Per 1, Per 2, Per 3), cryptochromes (Cry 1, Cry 2, Cry
3), clock, aryl hydrocarbon receptor nuclear translocator-like (Arntl/Bmal1),
casein kinase I (CKI/Tau) and differentiated embryo-chondrocyte I
(Dec1/Bhlhb2/Sharp2/Stra13) and Dec2 (Bhlhb3/Sharp1) in the normal and cancer
cells [1]. Disruption of circadian rhythms leads to various pathological
conditions such as insomnia, cardiovascular disorders and cancer progression
[2-4]. Recently, several reports have stated that the relationship between
cancer metastasis including epithelial-mesenchymal transition (EMT) and
circadian rhythms [5-7].
DEC1 and DEC2 are basic helix-loop-helix
(bHLH) transcription factors that are involved in the regulation of circadian
rhythms, apoptosis, cell proliferation and the response to hypoxia [8-17].
Previously, we reported that DEC1 mediated EMT which is the primary step
leading to invasion and migration of various tumor cells [18]. DEC1 expression
was increased during progression from normal to carcinoma in situ and invasive
carcinoma [19] and mutant DEC1 prevented metastasis of cancer in vivo [14]. Based on the findings,
DEC1 was thought to be a key factor of cancer invasion and metastasis. On the
other hand, DEC2 was associated with the regulation of apoptosis and cell
proliferation [20,21] and the expression of vascular endothelial growth factor
(VEGF) gene [10]. However, roles of DEC1 and DEC2 in cancer progression are not
well studied.
Extracellular matrix (ECM) is the
extracellular part of animal tissue that usually contributes to dynamic cell behavior,
pooling of growth factors, wound healing and tumor invasion. ECM is composed of
an interlocking mesh of glycosaminoglycans and
fibrous proteins such as collagens, elastins, fibronectins and laminins [22]. Type
I collagen is one of the major proteins in ECM and promotes EMT in both
physiological and pathological processes such
In the present study,
we investigated the effects of type I collagen on the expression of DEC1 and
DEC2 in human breast cancer MCF-7 cells. Our results indicated that type I
collagen up-regulated the expression of DEC1 and DEC2 and regulated
EMT-associated genes expressions. These findings suggested that EMT induced by
type I collagen might occur through DECs. An understanding of the interaction
between type I collagen and clock gene DECs will help in comprehending the
complex dynamics of tumor invasion and metastasis in cancer biology.
METHODS
Cell culture and treatment
MCF-7 human breast
cancer cells purchased from ATCC (American Type Culture Collection) were
maintained in Dulbecco’s Modified Eagle’s Medium (Sigma Chemical Co., St.
Louis, MO, USA) supplemented with 10% fetal bovine serum and 1% penicillin and
1% streptomycin in 5% CO2 at 37°C. Cells were cultured on Biocoat Cell
Environment Collagen I Cellware 6-well or 96-well plate (BD BioCoatTM, Belgium,
UK) for type I collagen treatment.
Reverse transcription-polymerase chain reaction (RT-PCR)
MCF-7 cells were
seeded on type I collagen-coated or non-coated 6-well plates for 24 h. Then
total RNA was isolated from the cells using RNeasy RNA isolation kit (QIAGEN,
Hilden, Germany). First-strand cDNA was synthesized from 1 μg of total RNA
using ReverTra Ace (TOYOBO, Tokyo, Japan). Then, RT-PCR was performed using the
aliquot of first-strand cDNA as a template under standard conditions using Taq
DNA polymerase (QIAGEN). The PCR products were separated on 1.5% (w/v) agarose
gels. The signal intensities were compensated by glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as internal controls. The sequences and product sizes of
the primer sets are shown in Table 1.
Cell morphology characterization
To observe the
morphology changes of MCF-7 cells by type I collagen treatment, cell staining
was performed using Cnt-ST-100 stain kit (CELLnTEC Advanced Cell Systems AG,
Bern, Switzerland). MCF-7 cells were seeded on type I collagen-coated or
non-coated 6-well plate and cultured for 24 h. Then, the medium of each well
was aspirated and fixed with 4% paraformaldehyde for 20 min before being
stained by Cnt-ST-II solution for 10 min. The cells were washed in PBS for
twice. Then the cells were covered with cover glasses and photographed.
Cell invasion assay
Cell invasion assay
was performed using BD BioCoat Matrigel invasion Chamber kit (Becton Dickinson,
New Jersey, USA). MCF-7 cells were seeded on type I collagen-coated or non-coated
6-well plate. 24 h later, 1 × 105 cells/600 ml were
added to the top chamber of a cell culture insert in a 24-well companion plate.
After 48 h incubation, the chambers were collected and stained by Cnt-ST-II
solution and invaded cells numbers on the membrane were counted. The number of
cells that had migrated was quantified by counting them in ten random distinct
fields using a light microscope.
Cell proliferation assay
The cell
proliferation assay was performed using the MTS[3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay.
MCF-7 cells were seeded on type I collagen-coated or non-coated 96-well plate
(Becton Dickinson). These cells were cultured in 5% CO2 at 37°C for
indicated time (24, 48, 72 and 96 h). Then, the cells were added along with the
Cell Titer 96 AQueous One Solution Reagent (Promega Corporation, Medison, WI,
USA) to each well and were incubated at 37°C for additional 1 h. Absorbance at
490 nm was measured using a microplate reader.
RESULTS
Type I collagen significantly up-regulated the expression of both DEC1
and DEC2
Type I collagen
affected the mRNA levels of DEC1, DEC2 as well as EMT-associated markers, while
the basal expression of both DEC1 and DEC2 were low in mRNA levels (Figure 1). Type I collagen also
up-regulated the expression of mesenchymal markers such as snail and a-SMA, while type I collagen did not affect the expression of slug. The
mRNA levels of E-cadherin and claudin-1 were decreased by type I collagen
treatment. These results indicated that signaling pathways induced by type I
collagen regulated the expression of DEC1 and DEC2, as well as that of
EMT-associated genes in human breast cancer MCF-7 cells.
Type I collagen altered the morphology of MCF-7 cells and promoted
invasiveness of MCF-7 cells
Cancer cells with EMT are characterized by acquiring a fibroblast-like motile and invasive phenotype. We examined whether type I collagen induced EMT-like morphological changes in MCF-7 cells. MCF-7 cells cultured on the type I collagen-coated dish for 24 h showed a spindle-shaped morphology (Figure 2A). Next we examined whether type I collagen affected invasive potentials of MCF-7 cells using cell invasion assay. Cell invasion of MCF-7 cells was significantly increased by type I collagen treatment compared to non-treated condition (Figure 2B). These results indicated that type I collagen induced a mesenchymal phenotype in MCF-7 cells.
Type I collagen promoted cell proliferation of MCF-7 cells
To investigate whether type I collagen affects cell proliferation of MCF-7 cells, we performed the MTS assay after culture for 24, 48, 72 and 96 h. The MTS assay showed that the proliferation of MCF-7 cells was induced by type I collagen treatment in any culture periods. Especially, cultured for 24, 48, 96 h showed significantly increased in the proliferation of MCF-7 cells by type I collagen treatment (Figure 3). These results indicated that type I collagen promoted MCF-7 cells proliferation
DISCUSSION AND CONCLUSION
In the present study,
we demonstrated type I collagen induced EMT in human breast cancer MCF-7 cells
as follows:
i.
Decreased epithelial markers and increased mesenchymal
markers of MCF-7 cells on the type I collagen-coated plates,
ii.
MCF-7 cells morphology were changed to fibroblast-like
shapes on the type I collagen-coated plates, and
iii.
Invasive potential of MCF-7 cells were increased by
type I collagen treatment. This is the first report to describe the up-regulated
expression of DEC1 and DEC2, concomitant with EMT-associate factors.
EMT is a process by
which epithelial cells lose their cell polarity and cell-to-cell adhesion, and
gain invasive and migratory properties to become mesenchymal cells. EMT is
essential for numerous developmental processes including mesoderm formation and
neural tube formation. EMT has also been shown to occur in wound healing in
organ fibrosis and in the initiation of metastasis for cancer progression [31].
In the molecular levels, EMT is associated with up-regulated transcription
factors such as snail and slug, decreased adhesion molecules (e.g. E-cadherin, b-catenin, claudin) and increased mesenchymal markers (e.g. N-cadherin,
vimentin, a-smooth muscle actin). In our present study,
type I collagen induced EMT and the mRNA levels of slug showed no significant
changes, although slug has been known as one of the EMT-associated genes [32].
We have not found any reports that type I collagen up-regulates slug gene expression
and we speculated that the mRNA level of slug is not directly affected by type
I collagen.
We demonstrated that
type I collagen also up-regulated DEC1 and DEC2 expressions, concomitant with
inducing EMT-associated factors. The up-regulated DEC1 and DEC2 are thought to
participate in EMT processes in MCF-7 cells treated with type I collagen.
Previously, we reported that DEC1 knockdown inhibited EMT processes [18] and
DEC1 was the downstream factor of PI3K-Akt signaling pathway [33]. Type I
collagen has shown to promote EMT through ILK-Akt signaling pathway [29].
Therefore, DEC1 is thought to be up-regulated by crosstalk between PI3K-Akt and
ILK-Akt signaling pathways, and to promote EMT by type I collagen treatment. In
the near future, we will try to clarify the molecular mechanisms of DEC1, as
well as the significance of DEC2 in EMT process induced by type I collagen.
Elucidation of relationship between DECs and EMT may provide new insights into
breast cancer therapeutic strategies.
ACKNOWLEDGEMENT
This study was
supported by Grants-in-Aid for science from them ministry of Education,
Culture, Sports, Science and Technology of Japan and a grant for Hirosaki
University Institutional Research.
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