N-Cadherin of SW-480 and HCT-116 Cells (Luminturahardjo W, et al.)
Indones Biomed J. 2021; 13(3): 289-94

DOI: 10.18585/inabj.v13i3.1562

RESEARCH ARTICLE

N-Cadherin as An Important Marker in Colorectal Cancer: An investigation of
b-Catenin and Cadherin Expressions of SW-480 and HCT-116 Cell Lines
Winarko Luminturahardjo1, Djoko Wahono Soeatmadji2, Karyono Mintaroem3,
Pudji Rahajoe4, Ferry Sandra5,
1
Doctoral Programme, Faculty of Medicine, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia
Department of Internal Medicine, Division of Endocrinology and Metabolic, Faculty of Medicine, Universitas Brawijaya, Jl. Veteran, Malang 65145,
Indonesia
3
Department of Pathology Anatomy, Faculty of Medicine, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia
2
Department of Otorhinolaryngology, Faculty of Medicine, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia
5
Department of Biochemistry and Molecular Biology, Section of Oral Biology, Faculty of Dentistry, Universitas Trisakti, Jl. Kyai Tapa No. 260, Jakarta,
Indonesia
2



Corresponding author. E-mail: ferry@trisakti.ac.id

Received date: Mar 22, 2021; Revised date: Jul 16, 2021; Accepted date: Jul 19, 2021

B

Abstract

ACKGROUND: The absence of potential
biomarkers to detect the metastatic process at an
early stage will consequently delay colorectal
cancer (CRC) treatment. Some biomarkers including
β-Catenin, E-Cadherin and N-Cadherin have been
suggested as potential markers. However, there were
opposite reports regarding expressions of these markers.
Therefore, current study was conducted using CRC cell
lines for early stage (SW-480 cells) and late stage (HCT116 cells) of CRC.
METHODS: SW-480 and HCT-116 cells were cultured
and seeded on coverslip glasses for immunofluorescence
staining to detect β-Catenin, E-cadherin, and N-cadherin.
Expressions of β-Catenin, E-cadherin, and N-cadherin were
observed and documented under a fluorescent microscope
and analyzed with Image J software. Measured results were
then statistically analyzed.

RESULTS: All β-catenin, E-Cadherin and N-Cadherin
expressions were observed in SW-480 and HCT-116 cells.
β-catenin MFI averages of SW-480 (47.157±3.479) and
HCT-116 (47.240±4.107) cells were similar. E-Cadherin
MFI average of SW-480 cells (45.104±4.107) was higher
than the one of HCT-116 cells (40.191±3.702). N-Cadherin
MFI average of HCT-116 cells (43.702±8.219) was
significantly higher (p=0.009) than the one of SW-480 cells
(72.506±5.297).
CONCLUSION: Taken together, N-Cadherin could be
suggested as an important metastasis marker in CRC since
the N-Cadherin expression was significantly higher in HCT116 cells as the late-stage CRC model than SW-480 as the
early-stage of CRC model. Further research is still needed
by comparing several biomarkers from various clinical
samples at all clinical stages of CRC.
KEYWORDS: CRC, β-Catenin, E-Cadherin, N-Cadherin,
Metastasis, Biomarker
Indones Biomed J. 2021; 13(3): 289-94

Introduction

Colorectal cancer (CRC), a malignant tumor arising from
the colon and rectum epithelium, is the 4th leading cause of

death from all types of cancers.(1,2) In 2013, the prevalence
of CRC worldwide was 9% of all types of cancer.(3,4) If
the tumor remains localized, the five-year survival rate
can reach 90%, but it decreases to 66% when transforming
into regional metastasis.(5,6) The metastasis of 10% of

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Duke's A stage and 15-30% of Duke's B stage CRC cells
was reported as locoregional.(6) In fact, micrometastasis
occurs in tumor cells sizing 0.2-2 mm and is frequently
undetected in histopathological examinations.(7,8) Some
markers are important to detect micrometastasis, including
c-mesenchymal-epithelial transition factor (c-Met),
universal melanoma antigen family (uMAGE), and β-human
chorionic gonadotropin (β-HCG).(9)
There are proteins affect the adhesion between cells
and the translocation of protein into the nucleus, such as
Wingless-related integration site (Wnt)/β-Catenin.(8,9)
Wnt/β-Catenin signal is a classic pathway involved in
modulating the development of cancer cells, which include
proliferation, resistance, differentiation, motility, adhesion,
and apoptosis of cancer cells.(10) While β-catenin is a
multifunctional protein with a central role in the homeostasis
process in the human body. Wnt/β-Catenin signals and
adhesion process are mediated by Cadherins, which are
involved in both the embryonal development process
and cancer progression. Among the Cadherins, there are
E-Cadherin and N-Cadherin.
E-Cadherin is a complex existent in the cytoplasm
of cancer cells that functions to form cytoskeleton for
strengthening the adhesion between cells. E-Cadherin
is responsible for maintaining cell polarity; therefore, it
is considered as a marker of cancer cells that have
not undergone metastasis.(11) Meanwhile, N-Cadherin
is assumed as a marker for detecting micrometastasis
in cancer as well.(12,13) N-Cadherin will experience
escalating regulation in mesenchymal cells, in which these
cells are more motile and less polarized than epithelial
cells. N-Cadherin is expressed in several cell types, such as
neuron cells, endothelial cells, stromal cells, and osteoblasts.
(14,15)
In advanced stages of CRC, most will undergo a
process called Epithelial Mesenchymal Transition (EMT).
(11) In this process, Cadherin switching occurs, which
means a reduction in E-Cadherin regulation followed by
an escalation in N-Cadherin regulation.(14) However,
in some circumstances, E-Cadherin expression does not
change significantly, but the cells experience an increase
in expression of N-Cadherin.(15) Moreover, in some types
of cancer, E-Cadherin will switch to N-Cadherin, but for
other types, N-Cadherin will switch to E-Cadherin.(14,16)
Therefore, in this current study the micromestatic markers
(β-Catenin, E-Cadherin and N-Cadherin) were investigated
using stable cell model, SW480 and HCT-116 cell lines,
which are known as the early (Duke's B) and late (Duke's
D) stages of CRC, respectively.
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Methods

Cell Culture
SW-480 cell line (CCL-228, ATCC, Gaithersburg, MD,
USA) was cultured in L-15 medium (Sigma-Aldrich, St.
Louis, MO, USA) containing 2mM Glutamine (SigmaAldrich), 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich)
and Antibiotic Antimycotic Solution (Sigma-Aldrich).
HCT-116 cell line (CL-247, ATCC) was cultured in
McCoy′s 5a medium (Sigma-Aldrich), containing 2 mM
Glutamine, 10% FBS and Antibiotic Antimycotic Solution.
Both cell lines were cultured in humidified-37°C-5%
CO2 incubator. Upon reaching confluent, the cells were
subcultured using 0.25% Trypsin-EDTA (Sigma-Aldrich).
Immunofluorescence
SW-480 and HCT-116 cells were seeded on coverslip
glasses with their mediums in culture dishes overnight. Cells
were rinsed with phosphate buffered saline (PBS) and fixed
with fresly prepared 4% paraformaldehyde (Wako, Osaka,
Japan) at neutral pH. Fixed-cell were then rinsed in PBS and
permeabilized with 0.1% Triton X-100 (Merck Millipore,
Burlington, MA, USA) in PBS. After rinsed with PBS, then
the fixed-permeabilized cells were incubated in 1% Bovine
Serum Albumin (BSA) in PBS. Primary antibodies used in
this study were mouse monoclonal IgG β-Catenin (12F7)
(sc-53488, Santa Cruz, Dallas, TX, USA), E-Cadherin
(5F133) (sc-71007, Santa Cruz) and N-Cadherin (8C11)
(sc-53488, Santa Cruz) antibodies. The primary antibodies
were added on the cells overnight at 4oC. The cells were then
rinsed and labelled with FITC-conjugated goat anti-mouse
IgG (H&L) secondary antibody (610-1202-0500, Rockland,
Limerick, PA, USA). Then the cells were rinsed and stained
with 14.3 mM 4', 6-diamidino-2-phenylindole (DAPI)
solution (Sigma-Aldrich) and mounted with ProLong Gold
Antifade Mountant solution (Thermo Fisher Scientific,
Waltham, MA, USA) on slide glasses. Expressions of
β-Catenin, E-Cadherin and N-Cadherin were observed
and captured under an immunofluorescence microscope
(Olympus, Tokyo, Japan). Each group with 8 images were
analysed with ImageJ bundled with Java 1.8.0_172 software
(NIH, Bethesda, MD, USA) with 8-bit color. Intensities
of β-Catenin, E-Cadherin, N-Cadherin expressions were
shown as Mean Fluorescence Intensity (MFI) unit.
Statistical Analysis
All calculated datas of β-catenin, E-Cadherin, and
N-Cadherin were presented as mean±SD for three

DOI: 10.18585/inabj.v13i3.1562

independent experiments. Statistical analyses were
perfomed using SPSS for Windows software version 20
(IBM, Armonk, NY, USA). The significance level was set at
confidence level of 95%, p<0.05 was significant.

Results

β-catenin Expressions of SW-480 and HCT-116 Cells
β-catenin expressions were observed in SW-480 and HCT116 cells in cytoplasm and nucleus (Figure 1A). β-catenin
MFI averages of SW-480 (47.157±3.479) and HCT-116
(47.240±4.107) cells were similar (Figure 1B) and not
statistically different (p=0.595) with independent T-test.
E-Cadherin Expressions of SW-480 and HCT-116 Cells
E-Cadherin expressions were also observed in SW-480
and HCT-116 cells in cytoplasm (Figure 2A). E-Cadherin
MFI average of SW-480 cells (45.104±4.107) was higher
than the one of HCT-116 cells (40.191±3.702) (Figure 2B),

N-Cadherin of SW-480 and HCT-116 Cells (Luminturahardjo W, et al.)
Indones Biomed J. 2021; 13(3): 289-94

although it was not statistically different (p=0.843) with
independent T-test.
N-Cadherin Expressions of SW480 and HCT-116 Cells
N-Cadherin expressions were clearly observed in SW-480
and HCT-116 cells in cytoplasm and nucleus. Especially
for HCT-116, high N-Cadherin expression was observed
as purplish white colour in the merged figure (Figure 3A).
In accordance, N-Cadherin MFI average of HCT-116 cells
(43.702±8.219) was significantly higher (p=0.009) than
the one of SW-480 cells (72.506±5.297) (Figure 3B) with
Mann-Whitney test.

Discussion

Translocation of cytoplasmic β-Catenin into nucleus in CRC
was correlated with the change of benign to malignant.(17)
Moreover, the loss of cytoplasmic β-Catenin expression
was suggested to indicate a poor prognosis of CRC.(18)

A

B

Figure 1. β-catenin expression of SW-480 and HCT-116 cells. A:
SW-480 and HCT-116 cells were cultured and immunofluorescence
stained with b-Catenin and DAPI. B: β-catenin MFI averages of
SW-480 and HCT-116 cells were measured and statistical analysis
was performed using independent T-test. White bar = 50 mm. Each
experiment was performed in triplicate and conducted twice.

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Print ISSN: 2085-3297, Online ISSN: 2355-9179

A

B

Figure 2. E-Cadherin expression of SW-480 and HCT116 cells. A: SW-480 and HCT-116 cells were cultured and
immunofluorescence stained with E-Cadherin and DAPI. B:
E-Cadherin MFI averages of SW-480 and HCT-116 cells were
measured and statistical analysis was performed using independent
T-test. White bar = 50 mm. Each experiment was performed in
triplicate and conducted twice.

However, in another study, β-catenin was reported to not
correlated with CRC progressivity.(19) In our current results,
β-Catenin expressions of both SW-480 as the early-stage of
CRC model and HCT-116 cells as late-stage CRC model,
were accordingly observed in the cytoplasm and nucleus.
The expressions of β-catenin in SW-480 and HCT-116 cells
were similar. Accordingly, both SW-480 and HCT-116 cells
were CRC models with clear expressions of b-Catenin.
E-Cadherin is the main component to strengthening
the bonds between epithelial cells and is vital for the
development, differentiation, and defense of cancer tissue.
(20) In CRC, E-Cadherin was reported as a tumor suppressor
with opposite function to Wnt signals.(21) Decreased
regulation of E-cadherin expression was associated with
poor prognosis of CRC, and the loss of E-cadherin expression
was associated with CRC cell metastasis.(22,23) However
there are two forms of E-cadherin (membrane-tethered and
soluble); as a result, E-cadherin has two properties, as an
inhibitor of cancer progression and a trigger for metastasis.
(16) E-cadherin can be expressed on several CRC cell lines,
292

including HCT-116 and signet ring cell carcinoma (SRCC).
(20) In accordance, in the current study, both SW-480 and
HCT-116 cells expressed E-cadherin. E-cadherin expression
of SW-480 cells was just slightly higher than E-cadherin
expression of HCT-116 cells. Therefore, SW-480 as the
early-stage CRC and HCT-116 cells as the late-stage of
CRC, could not be determined clearly based on single
marker of E-Cadherin.
N-cadherin expression was associated with the
development of carcinoma.(15,16) In the opposite of
E-cadherin, expression of N-Cadherin in CRC was higher at
Duke's C/D stage than that of Duke's A/B stage.(24) Based
on current results, HCT-116 cells, as the late-stage CRC
model, expressed significantly higher N-Cadherin than SW480 cells. However, since the SW-480 cells also expressed
N-Cadherin, this suggested that the SW-480 might have low
metastatic property as well. N-cadherin could be considered
as a candidate biomarker along with E-cadherin as an
independent promoter for EMT processes, such as Snail and
Twist.(20,25)

DOI: 10.18585/inabj.v13i3.1562

N-Cadherin of SW-480 and HCT-116 Cells (Luminturahardjo W, et al.)
Indones Biomed J. 2021; 13(3): 289-94

A

B

Figure 3. N-Cadherin expression of SW-480 and HCT116 cells. A: SW-480 and HCT-116 cells were cultured and
immunofluorescence stained with N-Cadherin and DAPI. B:
N-Cadherin MFI averages of SW-480 and HCT-116 cells were
measured and statistical analysis was performed using MannWhitney test. White bar = 50 mm. Each experiment was performed
in triplicate and conducted twice.

Conclusion

Since the N-Cadherin expression was significantly higher
in HCT-116 cells as the late-stage CRC model than SW480 as the early-stage of CRC model, it could be confirmed
that N-Cadherin as an important metastasis marker in CRC.
Meanwhile, b-catenin and E-Cadherin could be suggested
as confirmative markers. Further research is still needed by
comparing several biomarkers from various clinical samples
at all clinical stages of CRC.

Acknowledgements

The authors thank to the Postgraduate Program of
Universitas Brawijaya for their support especially to the
Parasitology Laboratory and the Biomedical Laboratory
of the Faculty of Medicine, Universitas Brawijaya for
laboratory facilities.

Authors Contribution

WL constructed research problem, baseline theory,
hypothesis, methodology, doing research, discussion and
conclusion. DWS contributed in the colorectal oncogenesis
theory, methodological and statistical analysis and submission
of this research. KM contributed in immunofluorescence
and histopathologial theory and method and interpretation
in this research. PR contributed in the transduction signaling
(include Wnt/β-catenin, E-cadherin and N-cadherin) and
construct the theory/research pathways. FS contributed in
result and statistical analyses, interpretation and manuscript
revision.

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