Dichloromethane fractions of Calystegia soldanella induce S‑phase arrest and apoptosis in HT‑29 human colorectal cancer cells

Abstract

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT‑29 was examined. First, five solvent fractions [n‑hexane, dichloromethane (DCM), ethyl acetate, n‑butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose‑ and time‑dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT‑29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose‑dependent manner, which was associated with the protein expression levels of apoptosis‑related proteins, including increased Fas, Bad and Bax, and decreased pro‑caspase‑8, Bcl‑2, Bcl‑xL, pro‑caspase‑9, pro‑caspase‑7 and pro‑caspase‑3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose‑dependent manner in HT‑29 cells treated with DCM. In addition, cell cycle analysis confirmed S‑phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle‑related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT‑29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S‑phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT‑29 colorectal cells. Further research on the composition of the DCM fraction is warranted.

Full text

MOLECULAR MEDICINE REPORTS 25: 60, 2022
Abstract. Calystegia soldanella is a halophyte and a peren-
nial herb that grows on coastal sand dunes worldwide.
Extracts from this plant have been previously revealed to
have a variety of bioactive properties in humans. However,
their effects on colorectal cancer cells remain poorly under-
stood. In the present study, the potential biological activity of
C. soldanella extracts in the colorectal cancer cell line HT-29
was examined. First, ve solvent fractions [n‑hexane, dichlo-
romethane (DCM), ethyl acetate, n-butanol and water] were
obtained from the crude extracts of C. soldanella through
an organic solvent extraction method. In particular, the
DCM fraction was demonstrated to exert marked dose- and
time-dependent inhibitory effects according to results from
the cell viability assay. Data obtained from the apoptosis assay
suggested that the inhibition of HT-29 cell viability induced
by DCM treatment was attributed to increased apoptosis. The
apoptotic rate was markedly increased in a dose-dependent
manner, which was associated with the protein expression
levels of apoptosis-related proteins, including increased Fas,
Bad and Bax, and decreased pro-caspase-8, Bcl-2, Bcl-xL,
pro-caspase-9, pro-caspase-7 and pro-caspase-3. A mitochon-
drial membrane potential assay demonstrated that more cells
became depolarized and the extent of cytochrome c release
was markedly increased in a dose-dependent manner in
HT-29 cells treated with DCM. In addition, cell cycle analysis
conrmed S‑phase arrest following DCM fraction treatment,
which was associated with decreased protein expression levels
of cell cycle-related proteins, such as cyclin A, CDK2, cell
division cycle 25 A and cyclin dependent kinase inhibitor 1.
Based on these results, the present study suggested that the
DCM fraction of the C. soldanella extract can inhibit HT-29
cell viability whilst inducing apoptosis through mitochondrial
membrane potential regulation and S-phase arrest. These
results also suggested that the DCM fraction has potential
anticancer activity in HT-29 colorectal cells. Further research
on the composition of the DCM fraction is warranted.
Introduction
Colorectal cancer is prevalent and a leading cause of death
worldwide (1). The mortality rate of colorectal cancer has
been declining over the past number of decades due to early
diagnosis using improved screening and treatment strategies.
However, the incidence remains high (2). Over the past several
decades, in the United States, the incidence and mortality rates
of colorectal cancer have been steadily decreasing among
those aged >50 years, but the number of those aged between
20 and 49 years is increasing. It is estimated that the incidence
and mortality rates of colorectal cancer according to these
age groups increase uniformly with economic development
due to environmental changes, such as lifestyle, increased
obesity and overall lifespan extension, and the consumption
of processed foods, alcohol and meat (1,2). To date, colorectal
cancer treatment involves radiotherapy and traditional thera-
pies, including surgery and chemotherapy (3). However, these
treatments are limited by toxicity, adverse events and drug
resistance (3). A number of studies have previously reported
that colorectal and colon cancer is negatively associated with
dietary factors, including plants, seaweeds, vegetables and
fruits, which contain a variety of phytochemicals (4-6). These
phytochemicals have been demonstrated to protect cells from
damage that leads to cancer (7-13).
The halophyte Calystegia soldanella (Linnaeus) Roem.
et Schult (Convolvulaceae) is a perennial herb that grows
on coastal sand dunes worldwide (14). This plant has been
extensively used in traditional medicine for general consump-
tion and as a type of herbal treatment, since it is considered
to confer bioactive effects against rheumatic arthritis, sore
throat, dropsy, scurvy, fever and diarrhea (15-17). In partic-
ular, fractions of C. soldanella have been reported to exhibit
anti‑inammatory (18‑20), antifungal (21), antiviral (22‑25),
anticancer (26,27) and analgesic effects (28). Although the
various bioactivities of C. soldanella have been assessed, its
effects on colon cancer have not been explored.
Dichloromethane fractions of Calystegia soldanella induce S‑phase
arrest and apoptosis in HT‑29 human colorectal cancer cells
IN-HYE KIM1*, TAEKIL EOM1*, JOON-YOUNG PARK2, HYUNG-JOO KIM1 and TAEK-JEONG NAM1
1Future Fisheries Food Research Center, Institute of Fisheries Sciences, Pukyong National University, Busan 46041;
2Department of Food Science and Nutrition, Pukyong National University, Busan 48513, Republic of Korea
Received September 8, 2021; Accepted November 11, 2021
DOI: 10.3892/mmr.2021.12576
Correspondence to: Professor Taek-Jeong Nam, Future Fisheries
Food Research Center, Institute of Fisheries Sciences, Pukyong
National University, 474 Ilgwang-ro, Busan 46041, Republic of Korea
E-mail: namtj@pknu.ac.kr
*Contributed equally
Key word s: apoptosis, Calystegia soldanella, dichloromethane
fraction, apoptosis, HT-29 cells

KIM et al: DICHLOROMETHA NE FRACTION INDUCES S-PHASE ARREST AND APOPTOSIS
2
In a previous study, the viability of numerous cancer
cell lines was assessed, including the hepatocarcinoma
HepG2, gastric cancer AGS, colorectal cancer HT-29 and the
breast cancer cell line MCF-7, following treatment with the
C. soldanella crude extract (27). Similar effects, including a
decrease in cell viability, were observed in HT-29 and HepG2
cells (27). Therefore, the aim of the present study was to
evaluate the mechanism underlying any changes in HT-29 cell
physiology after treatment with C. soldanella extract fractions.
Materials and methods
Sample collection and preparation. Whole-plant C. soldanella
samples were collected from Gijang, Busan, Korea. A voucher
specimen was deposited at the Herbarium of the Division of
Marine Environment and Bioscience, Korea Maritime and
Ocean University (Busan, Korea). The entire plant samples
were briey air‑dried at room temperature for 1 month, ground
into a ne powder using a blender and stored at ‑20˚C.
Extraction and fractionation. The crude extract of the plant
samples (500 g) was eluted in 99% ethanol for 3 h at room
temperature before being filtered and concentrated three
times. The concentrated crude extracts were evaporated under
reduced pressure at 40˚C using rotary vacuum evaporator
and partitioned between H2O-methanol (9:1) and n-hexane
(4.3 g). The organic layer was further partitioned into dichloro-
methane (DCM; 15.2 g) and ethyl acetate (2.3 g). The aqueous
layer was also fractionated into n-butanol (14.6 g) and water
(16.4 g). Each fraction used was completely removed using a
reux condenser and was subsequently freeze‑dried for use
in experiments. All solvent reagents used for extraction were
of analytical grade. The DCM fraction was diluted to a nal
concentration of 0.2% DMSO so as not to induce toxicity. For
the control, an equivalent volume of 0.2% DMSO was added
to the culture medium.
Ultra‑performance liquid chromatography coupled with
electrospray ionization quadrupole time‑of‑flight mass
spectrometry (UPLC‑ESI‑Q‑TOF‑MS) analysis. The DCM
fractions were analyzed using UPLC-ESI-Q-TOF-MS.
The UPLC system (Agilent innity 1260 series; Agilent
Technologies Deutschland GmbH), with an incorporated
photodiode array detector (DAD) and Impact II Q-TOF
mass spectrometer (Bruker Corporation), was equipped
with an ESI source that operated on the negative ion
mode. A reverse phase Kintex core-shell C-18 column
(100x2.1 mm, 1.7 µm, Phenomenex) was used at a flow
rate of 0.5 ml/min. The mobile phase consisted of water
containing 0.1% TFA (A) and 0.1% TFA containing aceto-
nitrile (B) using the following gradient conditions: 0-1 min,
10% B; 1-4 min, 10-20% B; 4-6 min, 20-25% B; 6-8 min,
25% B; 8-9 min, 25-30% B; 9-11 min, 30% B; 11-12 min,
30-50% B; 12-14 min, 50-60% B; 14-15 min, 60-80% B;
and 15-17 min, 80% B. The injection volume was 2 µl. Mass
spectra in positive-ion or negative-ion mode were recorded
within 20 min. The UPLC profiles of the extracts were
measured using a DAD. The analyses were conducted in the
negative ion mode in a mass range from m/z 50 to 1,000.
The ESI source parameters were: Capillary voltage, 4.5 KV;
nebulizing gas pressure, 1.5 bar; drying gas temperature,
200˚C, drying gas ow, 9.0 l/min; Funnel 1RF 250.0 Vpp;
transfer time, 50.0 µs; and prepulse storage, 2.0 µs. The
MS data were analyzed using Data Analysis 4.2 software
(Bruker Corporation).
Cell culture. The human colorectal HT-29 cell line
(cat. no. 30038) was purchased from the Korean Cell Line
Bank, Korean Cell Line Research Foundation. The STR
prole of the HT‑29 cell line was as follows: D3S1358, 15/17;
von Willebrand factor type A, 17/19; fibrinogen α chain,
20/22; amelogenin, X; tyrosine hydroxylase 1, 6/9; thyroid
peroxidase, 8/9; CSF1P0, 11/12; D5S818, 11/12; D13S317,
11/12; and D7S820, 10. The cells were cultured at 37˚C with
5% CO2 in RPMI-1640 medium (Welgene, Inc.) supplemented
with 10% FBS (Welgene, Inc.) containing 100 U/ml penicillin
and 100 µg/ml streptomycin (cat no. CA005-10; GenDEPOT,
LLC). The culture medium was refreshed every 2 days and the
cells were subcultured for use in subsequent experiments.
Cell viability assays. Cell viability was analyzed using
a EZ-Cytox Kit (cat. no. EZ-1000; DoGenBio Co., Ltd.)
according to the manufacturer's protocol. Cells were seeded
into 96-well plates at 4x104 cells/well and allowed to attach
for 24 h. First, cell viability was examined for each fraction
(hexane, DCM, ethyl acetate, butanol, water) at concentra-
tions of 0, 25, 50 or 100 µg/ml for 24 h. Next, attached
cells were treated with 0, 10, 20, 40, 60, 80 or 100 µg/ml
of the DCM fraction in serum-free medium for 24 or 48 h.
Subsequently, cells were incubated with the EZ-Cytox
solution (100 µl/well) for 30 min at 37˚C before absor-
bance at 450 nm was quantied using the FilterMAX F5
microplate reader (Molecular Devices LLC.). In addition,
morphological cell changes were subsequently observed
using a light microscope (magnification, x200; Eclipse
TS100-F; Nikon Corporation).
Apoptosis assay. Apoptosis was assessed using the Muse®
Annexin V and Dead Cell Kit (cat. no. MCH100105; Luminex
Corporation) according to the manufacturer's protocol. Cells
were seeded into six-well plates at 1x105 cells/well and
treated with 20, 40 or 80 µg/ml concentrations of the DCM
fraction for 20 h. The cells were then harvested at a density
of 5x104 cells/well and washed twice with PBS and stained
with FITC-Annexin V and dead cell reagent for 20 min at
room temperature in the dark. The percentage of apoptotic
cells was determined using the Guava® Muse® Cell Analyzer
(2012model; Luminex Corporation).
Assessment of mitochondrial membrane potential (MMP).
The MMP was assessed using the Muse® MitoPotential Kit
(cat. no. MCH100110; Luminex Corporation.) according to
the manufacturer's protocol. Cells were seeded into six-well
plates at 1x105 cells/well and treated with 0, 20, 40 or 80 µg/ml
concentrations of the DCM fraction for 20 h. The cells were
harvested at a density of 5x104 cells/well, washed twice with
PBS, stained with MitoPotential working solution containing
MitoPotential dye and incubated for 20 min in a 37˚C CO2
incubator. The MMP was determined using the Guava Muse
Cell Analyzer (2012 model).

MOLECULAR MEDICINE REPORTS 25: 60, 2022 3
Cell cycle assay. The cell cycle was assessed using the
Muse® Cell Cycle Assay Kit (cat. no. MCH100106; Luminex
Corporation) according to the manufacturer's protocol. Cells
were seeded into six-well plates at 1x105 cells/well and treated
with 0, 20, 40 or 80 µg/ml concentrations of the DCM fraction
for 20 h. The cells were harvested, washed twice with PBS,
xed in ice cold 70% ethanol and frozen at ‑20˚C for 3 h. The
xed cells were stained at a density of 5x104 cells/ml with
200 µl Muse cell cycle reagent for 30 min at room temperature.
The cell cycle phase was determined using the Guava Muse
Cell Analyzer (2012 model).
Preparation of total cell lysate. HT-29 cells were treated
with 0, 20, 40 or 80 µg/ml of the DCM fraction in serum-free
medium for 24 h at 37˚C. The cells were washed with PBS
and lysed in M-PER Mammalian Protein Extraction Reagent
(cat. no. 78501; Thermo Fisher Scientific, Inc.) containing
phosphate inhibitor cocktail (cat. no. 1862495; Thermo Fisher
Scientic, Inc.) and ProteaseArrest™ protease inhibitor cock-
tail (cat. no. 786-108; G-Biosciences; Geno Technology, Inc.)
on ice for 30 min. The extracts were centrifuged at 12,000 x g
for 10 min at 4˚C and the supernatants were subsequently
used for western blotting. The mitochondrial and cytosolic
fractions were extracted using a Mitochondria Isolation Kit
(cat. no. 89874; Thermo Fisher Scientic, Inc.), according to
the manufacturer's protocols. Protein concentrations were
measured using a BCA Protein Assay Kit (cat. no. 23225;
Thermo Fisher Scientic, Inc.).
Western blotting. Total protein (20-40 µg protein/la n e) wa s
electrophoresed via SDS-PAGE on a 8-15% acrylamide gel
and transferred onto polyvinylidene fluoride immobilon-P
membranes (cat. no. MLP.IPVH00010; MilliporeSigma). The
membranes were blocked with 1% bovine serum albumin
(BSA; cat. no. A0100; GenDEPOT, LLC) in TBS with 0.1%
Tween-20 (TBST; 5 mM Tris, 20 mM sodium chloride,
pH 7.4) and incubated with primary antibodies (1:1,000) in
1% BSA /TBST with gentle agitation at 4˚C overnight. The
membranes were then washed twice for 15 min in TBST
each and incubated with the corresponding HRP-conjugated
secondary antibodies (1:10,000) for 2 h at room temperature,
before being washed again using TBST. Immunoreactive
bands were detected using the WesternBright® ECL HRP
Substrate (cat. no. K12045; Advansta, Inc.) and visualized
using the GeneGnome 5 (model 75000; Syngene). Differences
in protein levels were determined by semi-quantifying the
western blotting band densities using ImageJ software version
IJ.146r (National Institutes of Health).
The antibodies used were as follows: Anti-Fas
(cat. no. sc-7886; rabbit; Santa Cruz Biotechnology,
Inc.), anti-caspase-8 (cat. no. sc-7890; rabbit; Santa Cruz
Biotechnology, Inc.), anti-Bcl-2 (cat. no. sc-7382; mouse;
Santa Cruz Biotechnology, Inc.), anti-Bcl-extra-large (xL;
cat. no. sc-7195; rabbit; Santa Cruz Biotechnology, Inc.),
anti-Bad (cat. no. sc-8044; mouse; Santa Cruz Biotechnology,
Inc.), anti-Bax (cat. no. sc-7480; mouse; Santa Cruz
Biotechnology, Inc.), anti-caspase-9 (cat. no. sc-7885;
rabbit; Santa Cruz Biotechnology, Inc.), anti-caspase-7
(cat. no. sc-6138; rabbit; Santa Cruz Biotechnology,
Inc.), anti-caspase-3 (cat. no. sc-7148; rabbit; Santa Cruz
Biotechnology, Inc.), anti-X-linked inhibitor of apop-
tosis protein (XIAP; cat. no. 2045; rabbit; Cell Signaling
Technology, Inc.), anti-cellular inhibitor of apoptosis protein
(cIAP)-1 (cat. no. 7065; rabbit; Cell Signaling Technology, Inc.),
anti-cIAP-2 (cat. no. 3130; rabbit; Cell Signaling Technology,
Inc.), anti-cytochrome c (cat. no. 4272; rabbit; Cell Signaling
Technology, Inc.), anti-cytochrome c oxidase subunit IV (COX
IV; cat. no. 4844; rabbit; Cell Signaling Technology, Inc.),
anti-cyclin A (cat. no. BS-0571R; rabbit; BIOSS), anti-CDK2
(cat. no. sc-163; rabbit; Santa Cruz Biotechnology, Inc.),
anti-cell division cycle 25 A (Cdc25A; cat. no. sc-7389; mouse;
Santa Cruz Biotechnology, Inc.) and anti-cyclin dependent
kinase inhibitor 1 (p21; cat. no. sc-271532; rabbit; Santa
Cruz Biotechnology, Inc.). Anti-β-actin (cat. no. sc-47778;
mouse; Santa Cruz Biotechnology, Inc.) antibody was used
as the loading control. The secondary antibodies used were
HRP-conjugated anti-mouse IgG (cat. no. 7076; Cell Signaling
Technology, Inc.) and anti-rabbit IgG (cat. no. 7074; Cell
Signaling Technology, Inc.).
Statistical analysis. All data are presented as the mean ± SD
of three independent experiments. Means between >2 groups
were compared using one-way or two-way ANOVA followed
by Bonferroni's multiple comparison test using GraphPad
Prism version 7 software (GraphPad Software, Inc). P<0.05
was considered to indicate a statistically signicant difference.
Results
Solvent fractions of C. soldanella reduce HT‑29 cell viability.
To determine the effects of the C. soldanella fractions on cell
viability, HT‑29 cells were treated with each of the ve solvent
fractions (n-hexane, DCM, ethyl acetate, n-butanol and water)
at different concentrations (0, 25, 50 and 100 µg/ml) for 24 h,
after which cell viability was examined. Cell viability was
signicantly decreased following treatment with the DCM
fraction compared with that in the 0 µg/ml group (Fig. 1A).
Therefore, the DCM fraction with the highest dose-dependent
effect was selected for further study.
Reductions in the viability of HT‑29 cells was conrmed
following treatment with different concentrations (0-100 µg/ml)
of DCM fraction for 24 and 48 h (Fig. 1B). HT-29 cell viability
appeared to be decreased following DCM fraction treat-
ment in a time- and dose-dependent manner compared with
those in the 0 µg/ml group. After treatment with 0, 10, 20,
40, 60, 80 and 100 µg/ml DCM, cell viability was 100±4.7,
89.0±3.5, 74.6±6.7, 73.2±2.0, 53.5±7.1, 26.7±1.6 and 25.4±1.7%
at 24 h, respectively, whereas it was 100±3.4, 92.6±8.3, 57.9± 6.6,
35.4±3.6, 20.2±1.6, 9.3±0.5 and 8.7±0.2% at 48 h, respectively
(Fig. 1B). In addition, it was observed that the morphological
changes conrmed via microscope were reduced in the same
way as the results of the cell viability assay (Fig. 1C).
DCM fraction from C. soldanella induces apoptosis in
HT‑29 cells. The Annexin V and Dead Cell Kit was used to
determine whether this decrease in cell viability induced by
DCM fraction treatment resulted from apoptosis. The rates
of early and late apoptosis were signicantly increased in a
dose-dependent manner following treatment with the DCM
fraction compared with those in the 0 µg/ml group (Fig. 2).

KIM et al: DICHLOROMETHA NE FRACTION INDUCES S-PHASE ARREST AND APOPTOSIS
4
The proportions of early apoptotic cells were 0.7±0.67,
38.2±1.81, 23.0±3.07 and 1.0±0.38%, whilst those of late apop-
totic cells were 0±0.05, 2.1±0.40, 12.9±7.12 and 35.1±0.58%,
following DCM fraction treatment at concentrations of 0,
20, 40 and 80 µg/ml, respectively.
Regarding the protein expression levels of apoptosis-related
proteins, DCM fraction treatment at 40 and 80 µg/ml signi-
cantly increased the protein expression levels of Fas protein
whilst significantly decreasing those of pro-caspase-8,
an extrinsic signaling pathway-related protein (Fig. 3),
compared with those in the 0 µg/ml group. Other intrinsic
apoptosis signaling pathway-related proteins that also
showed signicantly decreased protein expression levels after
40 and 80 µg/ml DMC treatment were Bcl-2 and Bcl-xL, whilst
those that showed signicantly increased protein expression
levels following 40 and 80 µg/ml DCM fraction treatment
were Bad and Bax (Fig. 3). Consequently, the Bax/Bcl-2 ratio
was significantly increased in the 40 and 80 µg/ml DCM
fraction treatment groups compared with that in the 0 µg/ml
group. In addition, 40 and 80 µg/ml DCM fraction treatment
also signicantly decreased the expression of pro‑caspase‑9,
pro-caspase-7 and pro-caspase-3 levels compared with those
in the 0 µg/ml group. The protein expression levels of XIAP,
cIAP-1 and cIAP-2, caspase inhibitors involved in apoptosis
inhibition, were signicantly decreased by 40 and 80 µg/ml
DCM treatment compared with those in the 0 µg/ml group.
These results suggest that the treatment of HT-29 cells with
DCM from C. soldanella may induce apoptosis by regulating
the expression of pro-apoptotic, pre-apoptotic and caspase
inhibitor proteins.
DCM fraction from C. soldanella induces MMP changes in
HT‑29 cells. Since MMP changes are also associated with
apoptosis (29-31), the effects of DCM fract ion treatment on the
MMP in HT-29 cells were investigated. The proportions of live
and dead cells with depolarized mitochondria were markedly
increased following DCM fraction treatment compared with
those in the 0 µg/ml group. The proportions of depolarized
Figure 1. DCM from Calystegia soldanella reduces the viability of HT‑29 colorectal cancer cells. (A) HT‑29 cells were incubated with the ve different solvent
fractions at 0‑100 µg/ml for 24 h. The letters displayed above the concentrations represent signicant differences (P<0.05) as determined by Bonferroni's
multiple comparisons test; groups with different letters are signicantly different to one another, whereas those with the same letter are not. (B) HT‑29 cells were
incubated with t he DCM fraction at 0-100 µg/ml for 24 and 48 h. Cell viability was examined using the EZ-Cytox proliferation assay. These data represent the
percentage of surviving cells compared with the control (0 µg/ml). (C) Cell mor phology changes were observed using light m icroscopy. Scale bar, 100 µm. Data
are presented as the mea n ± SD of three i ndependent exper iments. *P<0.05 vs. 0 µg/ml DCM; #P<0.05. DCM, dichloromethane; NS, not signicant.

MOLECULAR MEDICINE REPORTS 25: 60, 2022 5
live cells were 5.2±0.75, 43.6±1.49, 2.9±0.21 and 1.2±0.05%,
whereas the proportions of depolarized dead cells were
0.8±0.24, 10.5±1.48, 89.7±0.33 and 95.8±0.35%, at concen-
trations of 0, 20, 40 and 80 µg/ml, respectively (Fig. 4A).
The protein expression levels of MMP-related proteins were
examined using western blotting. DCM fraction treatment
at 40 and 80 µg/ml signicant ly incr eased the rele ase of cy to-
chrome c into the cytosol from the mitochondria compared
with that in the 0 µg/ml group (Fig. 4B). In addition, DCM
fraction treatment also resulted in the signicantly increased
translocation of Bax into the mitochondria from the cytosol
in a dose-dependent manner compared with the 0 µg/ml
group.
DCM from C. solda nella induces S‑phas e arrest in HT‑29 cells.
To determine whether decreased cell viability was associated
with the cell cycle, HT-29 cell cycle progression was analyzed
using a cell cycle kit following DCM fraction treatment. DCM
significantly induced S-phase arrest in a dose-dependent
manner compared with the 0 µg/ml group, with S-phase cell
proportions of 20.7±1.8, 33.8±0.2, 39.9±5.6 and 45.3±0.2% at
concentrations of 0, 20, 40 and 80 µg/ml of the DCM fraction,
respectively (Fig. 5A). Subsequently, the relative protein expres-
sion levels of S-phase-related proteins were analyzed using
western blotting. DCM fraction treatment at 40 and 80 µg/ml
led to the signicant downregulation of cyclin A, CDK2 and
Cdc25A protein expression and the signicant upregulation of
p21 protein expression compared with those in the 0 µg/ml
group (Fig. 5B).
ESI‑Q‑TOF‑MS analysis of DCM f raction from C. soldanella.
UPLC-ESI-Q-TOF-MS analysis was applied to analyze the
polyphenolic compounds in the DCM fraction of C. soldanella
to screen for any anticancer substances. The HPLC-UV chro-
matogram (350 nm) and total ion current chromatogram in the
DCM fraction are presented in the Fig. 6A and B.
Molecular ion mass, MS/MS fragment ion mass and
MS-based compound analysis data are all provided in Table I.
UPLC-ESI-Q-TOF MS analysis demonstrated that the major
compounds in the DCM fraction were hydroxybenzoic acid,
hydrosinapinic acid and coumaric acid, which are phenolic
acid derivatives, and quercetrin, which is a avonoid quercetin
derivative.
Discussion
The ultimate aim of discovering novel chemotherapeutic
strategies is to overcome drug resistance (32). A number of
studies have previously investigated the potential anticancer
activity of natural compounds/products and suggested them
to be promising sources of new anticancer drugs (4-13). For
example, the natural compound S-adenosylmethionine has
Figure 2. DCM f raction treatment induces apoptosis in HT-29 cells. HT-29 cells were incubated with various concent rations of the DCM fraction for 20 h.
FITC‑Annexin V staining followed by ow cytometry was used to determine the percentage of apoptotic cells. Data are presented as the mean ± SD of three
independent experiments. The letters displayed above the concentrations represent signicant differences (P<0.05) as determined by Bonferroni's multiple
comparisons test; groups with different letters are signicantly different to one another, whereas those with the same letter a re not. DCM, dichloromethane;
7-AAD, 7-Aminoactinomycin D; apop, apoptotic.

KIM et al: DICHLOROMETHA NE FRACTION INDUCES S-PHASE ARREST AND APOPTOSIS
6
been found to exert anti-tumor properties, including reduction
of cell proliferation, induction of apoptosis, autophagy and
inhibition of invasion and metastasis, in various cancer cell
types, such as human hepatocellular carcinoma, human breast
cancer and head and neck squamous cancer cells, in previous
in vitro and in vivo stud ies (33 -36).
To date, anticancer-associated studies related to
C. soldanella have revealed cytotoxic effects of methanol
and chloroform extracts on the lung cancer A545 and colon
cancer Col2 cell lines (26) and the anticancer effects of an
85% aqueous methanol fraction on the liver cancer cell line
HepG2 (27). However, only a few studies have investigated the
anticancer effects of C. soldanella.
In the present study, solvent fractions were obtained from
C. soldanella, which have been previously reported to exert
anticancer activity in human liver cancer cell line HepG2 (27),
before an effective fraction that can exhibit anticancer effects
on the colorectal cancer cell li ne HT-29 was selected. Following
the crude extraction of C. soldanella with ethanol, the crude
extracts were fractionated into n-hexane, DCM, ethyl acetate,
n‑butanol and water. The effects of these ve fractions on
HT-29 cell viability were examined before the DCM fraction
was selected due to its signicant time‑ and dose‑dependent
effects.
Apoptosis serves a critical role in the regulation of cell
development and proliferation (37,38). This process has
become a target of cancer treatments due to its association
with a number of different types of cancer (39,40). Two major
pathways of apoptosis have been identied: i) The extrinsic
death receptor pathway; and ii) the endoplasmic reticulum
stress pathway and intrinsic mitochondrial apoptosis (41).
To determine the effect of DCM fraction treatment on cell
Figure 3. Effects of DCM fraction t reatment on the expression of apoptosis-related proteins in HT-29 cells. HT-29 cells were incubated with various concentra-
tions of the DCM fraction for 20 h. The expression levels apoptosis-related proteins Fas, pro-caspase-8, Bcl-2, Bcl-xL, Bad, Bax, pro-caspase-9, pro-caspase-7,
pro-ca spase-3, XIA P, cIA P-1 and cIAP-2 were then det ermined by western blott ing. β-acti n was used as the loading contr ol. The band s were semi- quantitatively
analyzed by ImageJ software and the relative protein expression levels are presented. All results were norma lized to the untreated control (0 µg/ml). Data are
presented as t he mean ± SD of three independent experiments. *P<0.05 vs. 0 µg/ml DCM. DCM, dichloromethane; xL, extra-la rge; XIAP, X-linked inhibitor
of apoptosis protein; cIAP-1, cellular inhibitor of apoptosis protein-1; cIAP-2, cellular inhibitor of apoptosis protein-2.

MOLECULAR MEDICINE REPORTS 25: 60, 2022 7
viability and apoptosis, apoptosis and apoptosis-related
protein expression levels in HT-29 cells that were treated with
the DCM fraction were investigated. The results demonstrated
signicantly increased apoptotic rates and marked changes
in the expression levels of apoptosis-related proteins in both
the extrinsic and intrinsic signaling pathways. Treatment with
DCM fraction increased Fas and decreased pro-caspase-8,
which corresponds to the extrinsic signaling pathway. In addi-
tion, treatment with DCM fraction increased Bad and Bax, and
decreased Bcl-2, Bcl-xL, pro-caspase-9, pro-caspase-7 and
pro-caspase-3, which corresponds to the intrinsic signaling
pathway.
The mitochondrial apoptosis pathway involves a number
of components, including pre-apoptotic proteins Bcl-2 and
Bcl-xL and pro-apoptotic proteins Bax and Bak (42-44).
The ratio of Bax/Bcl-2 determines the direction of apoptosis
regulation. An increased Bax/Bcl-2 ratio can lead to the loss
of mitochondrial membrane potentials, which is an important
process in the initiation of apoptosis (45). Furthermore, it has
been reported that an increased Bax/Bcl-2 ratio can activate
caspase-3 to in turn activate apoptosis (46,47). In the present
study, caspase-3 was also markedly activated following
DCM fraction treatment. Since the Bax/Bcl-2 ratio was also
markedly increased with DCM fraction treatment, it was
hypothesized that DCM may activate apoptosis by altering
the mitochondrial membrane potential.
Apoptosis involves the regulation of a series of proteins
mainly in the mitochondrial signaling pathway (48). The
Figure 4. DCM fraction treatment induces depolarization of mitochondria l membrane potential in HT-29 cells. (A) HT-29 cells were incubated with various
concentrations of the DCM fraction for 20 h. MitoPotential kit staining followed by ow cytometry was used to determine the percentages of depola rized cells.
The letters displayed above the concentrations represent signicant differences (P<0.05) as determined by Bonferroni's multiple comparisons test; groups with
different letters are signicantly different to one another, whereas those with the same letter are not. (B) Protein expression levels of MMP‑related proteins
cytochrome c and Bax were determ ined using western blotting analysis. β-actin and COX IV were used as loading controls for the cytosol and mitochon-
dria, respectively. All results were normalized to the untreated control (0 µg/ml). Data are presented as the mean ± SD of three independent experiments.
*P<0.05 vs. 0 µg/ml DCM. DCM, dichloromethane; 7-AAD, 7-Aminoactinomycin D; COX IV, cytochrome c oxidase subunit IV; mi, mitochondr ial; cy, cytosol.

KIM et al: DICHLOROMETHA NE FRACTION INDUCES S-PHASE ARREST AND APOPTOSIS
8
mitochondria maintain the cellular energy balance and regu-
late cell death processes (49). Cellular energy generated during
mitochondrial respiration is stored as an electrochemical
gradient across the mitochondrial membrane, which allows
the mitochondria to induce ATP synthesis (49). Changes
in the MMP are associated with apoptosis, necrosis and
caspase-independent cell death processes in addition to the
opening of mitochondrial transition pores, to release cyto-
chrome c into the cytosol and initiate apoptotic and caspase
cascades (31,49). In the present study, MMP changes following
DCM fraction treatment led to matrix condensation and expo-
sure of cytochrome c to the intermembrane space, which may
have activated apoptosis (Fig. 4).
Numerous proteins associated with cell cycle regulation
are known to be involved in apoptosis (50). In the present study,
cell cycle assay was performed and the protein expression
levels of several cell cycle-related proteins were investigated
to determine whether apoptosis induced by DCM fraction
treatment was due to cell cycle regulation in HT-29 cells. The
results demonstrated that DCM fraction treatment signicantly
increased the proportion of cells in the S-phase. Cyclins and
cyclin-dependent kinases serve important roles in cell cycle
regulation, where changes in the composition of cyclin/CDK
complexes can either increase or decrease cell proliferation
and/or differentiation through apoptosis (51,52). Since cyclin
A, CDK2 and Cdc25A serve critical roles in S-phase regula-
tion (53,54), their protein expression levels following DCM
fraction treatment were examined. The results showed marked
downregulation of cyclin A, CDK2 and Cdc25A expression
following DCM treatment. CDK1, p21 (Waf1/Cip1) have been
previously shown to induce cell cycle arrest by inhibiting CDK
activity (55,56). The results of the present study demonstrated
a dose-dependent increase in p21 protein expression levels in
response to DCM, which may have inhibited Cyclin A-CDK2
complex formation and contributed to cell cycle arrest between
the S and G2/M phases.
Figure 5. DCM fraction treatment induces S-phase arrest in HT-29 cells. (A) HT-29 cells were incubated with various concentrations of the DCM fraction
for 20 h. Flow cytometry was used to deter mine the percentages of cells in each phase of the cell cycle. The letters displayed above the concentrations
represent signicant differences (P<0.05) as determined by Bonferroni's multiple comparisons test; groups with different letters are signicantly different
to one another, whereas those with the sa me letter a re not. (B) Protein expression levels of S-phase-related proteins cyclin A, CDK2, Cdc25A and p21 were
measured using western blotti ng. All results were nor malized to the untreated control (0 µg/ml). Data are presented as the mean ± SD of three independent
experiments. *P<0.05 vs. 0 µg/ml DCM. DCM, dichloromethane; CDK2, cyclin- dependent kinase 2; Cdc25A, cell division cycle 25 homolog A; p21, cyclin
dependent kinase inhibitor 1.

MOLECULAR MEDICINE REPORTS 25: 60, 2022 9
It has been frequently reported that numerous compounds
can exert anticancer activity independent of p53 (57-60). The
tumor suppressor p53 has been found to be dysfunctional in
≥50% colorectal cancer cases (58,59). Colorectal cancer cells
and tumors with p53 mutation are reported to be more aggres-
sive and resistant to chemotherapy (60). In the present study,
the HT-29 cell line is a p53 mutant cell line (genotype R273H),
to which DCM exerted anticancer effects in the absence of p53.
Although not conducted in the present study, anticancer effects
following DCM fraction treatment are also hypothesized to be
present in p53 wild-type cell lines and warrants further study.
Previous studies have reported that C. soldanella contains
a variety of polyphenolic compounds, including avonoids,
avonoid glycosides and phenolic acid derivatives (56,61).
Molecular ion mass, MS/MS fragment ion mass and
MS-based compound analysis data are all provided in Table I.
UPLC-ESI-Q-TOF MS analysis demonstrated that the major
compounds in the DCM fraction were hydroxybenzoic acid,
hydrosinapinic acid and coumaric acid, which are phenolic
acid derivatives, and quercetrin, which is a avonoid quer-
cetin derivative. Various phenolic acids, such as coumaric
acid, caffeic acid and ferulic acid, function as secondary
plant metabolites (62). In particular, cinnamic acid-based
phenoic acid compounds, such as coumaric acid, caffeic acid,
ferulic acid and sinapinic acid, have been reported to exert
anticancer activity in various colon cancer cell lines (63).
Coumaric acid is reported to induce apoptosis in the colon
cancer HCT-115 cell line and induce G1/S arrest in the colon
cancer cell line Caco-2 (64-66). These aforementioned
phenolic acids also have inhibitory activities on the prolifera-
tion of various colorectal/colon cancer cell lines (67-69).
In conclusion, to the best of our knowledge, the present
study was the rst to report the anticancer effects of DCM
from C. soldanella on HT-29 colorectal cancer cells, which
possibly occurred by MMP alteration, S-phase arrest and
induction of apoptosis through the intrinsic/extrinsic signaling
pathways. The present study also demonstrated that phenolic
acid derivatives are the main components of the DCM frac-
tion, which exerted inhibitory activities on HT-29 colorectal
cancer cell by apoptosis and cell cycle arrest.
Acknowledgements
Not applicable.
Funding
The present study was part of the Future Fisheries Food
Research Center Project, funded by the Ministry of Oceans
and Fisheries (grant no. 201803932).
Figure 6. UPLC-ESI-Q-TOF-MS analysis of DCM fraction. (A) UV chromatogram at 350 nm a nd (B) UPLC-ESI-Q-TOF MS total ion chromatogram of the
dichloromethane fraction. intens, intensity; UPLC-ESI-Q-TOF-MS, ultra-performance liquid chromatography coupled with electrospray ionization quadru-
pole time‑of‑ight mass spectrometry.
Table I. Identied compounds in the DCM fraction.
Peak no. Compound Retention time, min Mass m/z MS fragment
1 Hydroxybenzoic acid 1.6 138.12 137.02 112.98
2 Hydrosinapinic acid 2.0 226.08 225.11 181.12, 121.02
3 Coumaric acid 2.2 164.04 163.03 119.05
4 Quercetrin 3.0 448.38 447.09 301.03, 146.93
Peaks correspond to peaks observed in Fig. 6B. DCM, dichloromethane; MS, mass spectrometry.

KIM et al: DICHLOROMETHA NE FRACTION INDUCES S-PHASE ARREST AND APOPTOSIS
10
Availability of data and materials
The datasets used and/or analyzed in the current study are
available from the corresponding author on reasonable request.
Authors' contribution
IHK, TE and TJN conceived and designed the experiments. IHK,
JYP and HJK performed the experiments. IHK, TE and HJK
analyzed and interpreted the results and wrote and revised the
manuscript. IHK, TE and TJN conrm the authenticity of all the
raw data. All authors have read and approved the na l manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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