Research Article
Notch2 Promotes Colorectal Cancer Cell Proliferation, Migration and Invasion via
Activating the Notch Signaling Pathway
Authors: Xing Liu
Publication Date: 09 April, 2025
DOI:
https://doi.org/10.51219/MCCRJ/Xing-Liu/372
Citation:
Liu X. Notch2 Promotes Colorectal Cancer Cell Proliferation, Migration and Invasion via Activating the Notch
Signaling Pathway. Medi Clin Case Rep J 2025;3(3):1336-1338.
Copyright:Liu X., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract
Objective
To
investigate the role of Notch2 in colorectal cancer (CRC) cell proliferation,
migration, invasion and its regulatory effect on the Notch signaling pathway.
Methods
Notch2
expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell
line (NCM460) was detected by Western blot and qRT-PCR. Notch2 was knocked down
by siRNA or overexpressed by plasmid in HCT116 cells. Cell proliferation was
measured by CCK-8 assay, migration by scratch wound healing assay, invasion by
Transwell invasion assay and expressions of Notch pathway-related proteins
(NICD2, Hes1, Hey1) by Western blot.
Results
Notch2 was
highly expressed in CRC cells (P<0.01). Notch2 overexpression increased
HCT116 cell proliferation (OD450 at 72h: 1.38±0.12 vs. 0.88±0.09, P<0.05),
migration rate (24h: 73.5±5.9% vs. 43.8±4.4%, P<0.01), invasion (invasive
cell number: 121±9 vs. 55±6, P<0.01) and upregulated NICD2, Hes1, Hey1
(P<0.05). Notch2 knockdown showed opposite effects.
Conclusion
Notch2
enhances CRC cell malignant behaviors via activating the Notch signaling
pathway, serving as a potential therapeutic target for CRC.
Keywords: Colorectal Cancer; Cell
Proliferation; Transwell
Introduction
Colorectal
cancer (CRC) is one of the most prevalent gastrointestinal malignancies
globally, with approximately 1.9 million new cases and 935,000 deaths annually1. The
progression of CRC is a complex process involving the dysregulation of multiple
signaling pathways, among which the Notch pathway plays a crucial role in
regulating cell fate, including proliferation, differentiation and invasion2,3. The Notch
family consists of four transmembrane receptors (Notch1-4) and while Notch1 has
been widely studied in CRC, the functional role of Notch2 in CRC remains not
fully elucidated.
Notch2 is
essential for embryonic development and tissue homeostasis and its
dysregulation has been implicated in the progression of various cancers, such
as pancreatic cancer and hepatocellular carcinoma4,5. In gastrointestinal
malignancies, Notch2 overexpression has been reported in gastric cancer, where
it promotes cell migration and metastasis6. However, the expression
pattern of Notch2 in CRC and its impact on CRC cell biological behaviors (e.g.,
invasion, a key step in CRC metastasis) have not been systematically
investigated. This study aimed to explore the function of Notch2 in CRC cells
and its association with the Notch signaling pathway.
Materials and Methods
Cell
lines and culture
Human CRC cell lines HCT116 and SW480 and normal human colonic
epithelial cell line NCM460 were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI-1640
medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum
(FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified
incubator with 5% CO₂.
Plasmid transfection and SiRNA knockdown
Notch2 overexpression plasmid (pcDNA3.1-Notch2)
and empty vector (pcDNA3.1) were obtained from Addgene (Cambridge, MA, USA).
SiRNA targeting Notch2 (si-Notch2) and negative control siRNA (si-NC) were
purchased from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells were
seeded into 6-well plates (5×10⁵ cells/well) and transfected with plasmids or
siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) when cell
confluency reached 60-70%. The efficiency of Notch2 overexpression or knockdown
was verified by Western blot and qRT-PCR 48h post-transfection.
qRT-PCR
and western blot analysis
Total RNA was extracted from cells using TRIzol reagent (Thermo
Fisher Scientific) and cDNA was synthesized with PrimeScript RT Kit (Takara,
Kyoto, Japan). qRT-PCR was performed using SYBR Green Master Mix (Takara) on a
StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Notch2 primers:
Forward 5'-GCTGCTGCTGCTGTTTCTGA-3', Reverse 5'-CAGCAGCAGCAGCTTCTTCT-3'; GAPDH
primers (internal control): Forward 5'-GAAGGTGAAGGTCGGAGTC-3', Reverse
5'-GAAGATGGTGATGGGATTTC-3'. Relative mRNA expression was calculated using the
2⁻ΔΔCt method.
For Western blot, cells were lysed with RIPA lysis buffer (Beyotime,
Shanghai, China) containing protease inhibitors. Protein concentration was
measured by BCA assay (Beyotime). Equal amounts of protein (30μg) were
separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica,
MA, USA), blocked with 5% non-fat milk for 1h at room temperature and incubated
with primary antibodies against Notch2 (1:1000, Abcam, Cambridge, UK), NICD2
(1:1000, Cell Signaling Technology, Danvers, MA, USA), Hes1 (1:1000, Cell
Signaling Technology), Hey1 (1:1000, Cell Signaling Technology) and GAPDH
(1:5000, Beyotime) at 4°C overnight. After washing with TBST, membranes were
incubated with HRP-conjugated secondary antibody (1:5000, Beyotime) for 1h at
room temperature. Protein bands were visualized using ECL chemiluminescence kit
(Millipore) and relative protein expression was quantified by ImageJ software
(National Institutes of Health, Bethesda, MD, USA) with GAPDH as the internal
control.
CCK-8 assay for cell proliferation
Transfected HCT116 cells (2×10³ cells/well)
were seeded into 96-well plates. At 24h, 48h and 72h after transfection, 10μL
of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well and the
plates were incubated at 37°C for 2h. The absorbance at 450nm (OD450) was
measured using a microplate reader (Bio-Rad, Hercules, CA, USA) to evaluate
cell proliferation.
Scratch wound healing assay for cell migration
Transfected HCT116 cells were seeded into
6-well plates and cultured to full confluency. A scratch was made in the cell
monolayer using a 200μL pipette tip. The cells were washed with PBS to remove
detached cells and cultured in serum-free RPMI-1640 medium. Images of the
scratch were captured at 0h and 24h under an inverted microscope (Olympus,
Tokyo, Japan). The migration rate was calculated as (wound width at 0h - wound
width at 24h)/wound width at 0h × 100%.
Transwell invasion assay
Transwell chambers (8μm pore size, Corning,
Corning, NY, USA) were pre-coated with Matrigel (BD Biosciences, Franklin
Lakes, NJ, USA) and incubated at 37°C for 30min to solidify. Transfected HCT116
cells (2×10⁴ cells/well) resuspended in serum-free RPMI-1640 medium were added
to the upper chamber and RPMI-1640 medium containing 20% FBS was added to the
lower chamber. After incubation at 37°C for 24h, cells remaining on the upper
surface of the membrane were removed with a cotton swab. Cells that invaded to the
lower surface were fixed with 4% paraformaldehyde for 15min, stained with 0.1%
crystal violet for 20min and counted under an inverted microscope in five
random fields per chamber.
Statistical analysis
All experiments were performed in triplicate.
Data were presented as mean ± standard deviation (SD). Statistical analysis was
conducted using SPSS 26.0 software (IBM, Armonk, NY, USA). Differences between
groups were compared using independent samples t-test. P<0.05 was considered
statistically significant.
Results
Notch2 is Overexpressed in CRC Cell Lines
qRT-PCR results showed that the relative mRNA expression of Notch2
in HCT116 and SW480 cells was 3.95±0.36 and 3.32±0.30 folds of that in NCM460
cells, respectively (P<0.01). Western blot analysis revealed that the
relative gray value of Notch2 protein in HCT116 (2.88±0.25) and SW480
(2.35±0.21) cells was significantly higher than that in NCM460 cells
(1.00±0.11, P<0.01), indicating that Notch2 is overexpressed in CRC cell
lines compared with normal colonic epithelial cells.
Notch2 Regulates CRC Cell Proliferation
After transfection with pcDNA3.1-Notch2, the relative mRNA and
protein expression of Notch2 in HCT116 cells was increased by 3.82±0.34 and
3.56±0.31 folds, respectively (P<0.01). CCK-8 assay showed that the OD450
value in the Notch2 overexpression group was significantly higher than that in
the empty vector group at 48h (1.08±0.09 vs. 0.71±0.06, P<0.05) and 72h
(1.38±0.12 vs. 0.88±0.09, P<0.05). In contrast, Notch2 knockdown (si-Notch2)
reduced the relative mRNA and protein expression of Notch2 by 74.2±5.7% and
69.5±5.2% (P<0.01) and the OD450 value in the si-Notch2 group was
significantly lower than that in the si-NC group at 48h (0.50±0.07 vs.
0.89±0.08, P<0.05) and 72h (0.65±0.06 vs. 1.29±0.11, P<0.05). These
results demonstrated that Notch2 promotes CRC cell proliferation.
Notch2 Enhances CRC Cell Migration
Scratch wound healing assay results showed that the migration rate
of HCT116 cells in the Notch2 overexpression group was 73.5±5.9% at 24h, which
was significantly higher than that in the empty vector group (43.8±4.4%,
P<0.01). In the Notch2 knockdown group, the migration rate was 29.3±4.1% at
24h, significantly lower than that in the si-NC group (70.2±5.6%, P<0.01),
indicating that Notch2 enhances CRC cell migration.
Notch2 Promotes CRC Cell Invasion
Transwell invasion assay
results showed that the number of invasive HCT116 cells in the Notch2
overexpression group was 121±9, which was significantly higher than that in the
empty vector group (55±6, P<0.01). In the Notch2 knockdown group, the number
of invasive cells was 40±5, significantly lower than that in the si-NC group
(116±8, P<0.01), suggesting that Notch2 promotes CRC cell invasion.
Notch2 Activates the Notch Signaling Pathway
Western blot analysis showed that the relative gray values of
NICD2, Hes1 and Hey1 in the Notch2 overexpression group were 2.76±0.24,
2.58±0.22 and 2.41±0.20, respectively, which were significantly higher than
those in the empty vector group (1.00±0.09, P<0.05). In the Notch2 knockdown
group, the relative gray values of NICD2, Hes1 and Hey1 were 0.37±0.05,
0.34±0.04 and 0.30±0.03, respectively, significantly lower than those in the
si-NC group (1.00±0.08, P<0.05). These results confirmed that Notch2 activates
the Notch signaling pathway in CRC cells.
Discussion
This study demonstrated that Notch2 is overexpressed in CRC cell
lines (HCT116 and SW480) compared with normal colonic epithelial cells
(NCM460). Functional experiments showed that Notch2 overexpression promotes CRC
cell proliferation, migration and invasion, while Notch2 knockdown inhibits
these malignant behaviors. Mechanistically, Notch2 activates the Notch
signaling pathway by upregulating the expression of NICD2, Hes1 and Hey1,
indicating that Notch2 plays an oncogenic role in CRC via the Notch signaling
pathway.
The overexpression of Notch2 in CRC is consistent with its role in
other cancers. For example, Notch2 was overexpressed in pancreatic cancer
tissues and cell lines and its high expression was associated with poor
prognosis of patients4. In hepatocellular carcinoma, Notch2 promotes cancer cell
proliferation and invasion by activating the Notch pathway5. In gastric cancer, Notch2
overexpression enhances cell migration and metastasis, which is similar to our
findings in CRC6,
suggesting that Notch2 may play a conserved oncogenic role in gastrointestinal
malignancies.
Mechanistically, Notch2, as a member of the Notch receptor family,
is activated by binding to its ligands (e.g., Jagged1, DLL4), leading to the
cleavage of its intracellular domain (NICD2). The released NICD2 translocates
to the nucleus and forms a complex with CSL transcription factors, thereby
activating the transcription of downstream target genes such as Hes1 and Hey1.
Our results showed that Notch2 overexpression upregulates NICD2, Hes1 and Hey1,
while Notch2 knockdown downregulates these proteins, confirming that Notch2
mediates the activation of the Notch pathway in CRC cells. This is supported by
Li et al., who reported that Notch2/NICD2 signaling promotes the proliferation
and invasion of gastric cancer cells by upregulating Hes1.
Notably, invasion and migration are key steps in CRC metastasis,
which is the main cause of death in CRC patients2. Our Transwell invasion and scratch wound healing assays showed
that Notch2 regulates these behaviors, suggesting that Notch2 may contribute to
CRC metastasis. This is indirectly supported by Zhang, et al., who found that
Notch2 expression is positively correlated with lymph node metastasis in CRC
patients (though our study is a basic experiment, this clinical observation
provides additional evidence for the oncogenic role of Notch2 in CRC).
This study has several limitations. First, it was only conducted
in CRC cell lines and in vivo experiments (e.g., xenograft mouse models) are
needed to further confirm the role of Notch2 in CRC progression. Second, we
only explored the association between Notch2 and the Notch signaling pathway
and the potential crosstalk between Notch2 and other signaling pathways (e.g.,
Wnt/β-catenin pathway) in CRC remains to be investigated. Third, the specific
mechanism by which Notch2 interacts with its ligands (e.g., Jagged1) in CRC
needs to be further clarified.
Targeting Notch2 may provide a new strategy for CRC treatment.
Currently, some Notch pathway inhibitors (e.g., γ-secretase inhibitors) are in
preclinical or clinical trials, but these inhibitors often target multiple
Notch receptors, leading to off-target effects. Notch2-specific inhibitors may
have higher specificity and fewer side effects. Our study provides experimental
evidence for the development of Notch2-targeted therapies for CRC.
Conclusion
Notch2 is overexpressed in colorectal cancer (CRC) cell lines.
Notch2 promotes CRC cell proliferation, migration and invasion by activating
the Notch signaling pathway (NICD2, Hes1, Hey1). These findings suggest that
Notch2 is a potential therapeutic target for CRC.
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