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 [UCI]I804:45008-200000601399

2021 學年度

탄닌산의 5-FU 내성 대장암 세포의 증식 및

전이능력 억제를 통한 5-FU 내성 극복 효과

Effects of tannic acid on overcoming 5-fluorouracil

(5-FU) resistance by inhibiting proliferation and
metastatic phenotypes of 5-FU-resistant colorectal
cancer cells

圓 光 大 學 校 大 學 院

韓 藥 學 科

文 正 健
탄닌산의 5-FU 내성 대장암 세포의 증식 및
전이능력 억제를 통한 5-FU 내성 극복 효과

Effects of tannic acid on overcoming 5-fluorouracil

(5-FU) resistance by inhibiting proliferation and
metastatic phenotypes of 5-FU-resistant colorectal
cancer cells

指 導 敎 授 洪 承 憲

이 論文을 韓藥學博士 學位 論文으로 提出함

2021年 10月

圓 光 大 學 校 大 學 院

韓 藥 學 科

文 正 健
 Contents

<국문초록>……………………………………………………………………………… 1

<ABSTRACT>………………………………………………………………………… 3

1. Introduction………………………………………………………………………… 5

2. Materials and Methods……………………………………………………… 10

2.1. Reagents and antibodies……………………………………………… 10

2.2. Cell culture……………………………………………………………… 10

2.3. Establishment of 5-FU-resistant CRC cells……………………… 11

2.4. Cell viability assay……………………………………………………… 11

2.5. Colony formation assay………………………………………………… 12

2.6. Annexin V assay………………………………………………………… 1 2

2.7. Mitochondrial membrane potential assay…………………………… 12

2.8. Western blot analysis…………………………………………………… 13

2.9. Cell cycle analysis……………………………………………………… 13

2.10. Real-time reverse transcription-polymerase chain reaction… 14

2.11. Wound healing assay………………………………………………… 17

2.12. Tumor spheroid migration assay …………………………………… 17

2.13. Invasion assay…………………………………………………………… 17

2.14. Gelatin zymography…………………………………………………… 18

2.15. Tumorigenicity assay…………………………………………………… 18

2.16. Statistical analysis……………………………………………………… 19

3. Results…………………………………………………………………………… 20

3.1. Establishment of 5-FU-resistant CRC cell lines………………… 20

3.2. Effect of TA on the proliferation of 5-FU-resistant CRC cells…………… 23

3.3. Effect of TA on colony-forming ability of 5-FU-resistant CRC cells․ 25

3.4. Effect of TA on the apoptosis in 5-FU-resistant CRC cells… 27

3.5. Effect of TA on the mitochondrial membrane potential in

5-FU-resistant CRC cells… … … … … … … … … … … … … … … … 30

3.6. Effect of TA on cell cycle arrest in 5-FU-resistant CRC cells…………… 33

3.7. Effect of TA on EMT of 5-FU-resistant cells…………………… 36

3 . 8 . E ff e ct of TA o n t h e m i g r a to r y a n d i n v a s i ve a bi l i ty o f
5-FU-resistant CRC cells……………………………………………… 38

3.9. Effect of TA on ABC transporters of 5-FU-resistant CRC cells……… 40

3.10. Effect of TA and 5-FU combination on the proliferation of

5-FU-resistant CRC cells……………………………………………… 42

3.11. Effect of TA on the growth of CT26/5-FU cells implanted in mice‥ 46

4. Discussion……………………………………………………………………… 49

5. References……………………………………………………………………… 54
<국문초록>

탄닌산의 5-FU 내성 대장암 세포의 증식 및

전이능력 억제를 통한 5-FU 내성 극복 효과

文正健

圓光大學校 大學院 韓藥學科

指導敎授 洪承憲

共同指導敎授 奇知藝

5-플루오로우라실은 여러 암종의 치료에 광범위하게 사용되고 있으며, 주로

대장암 치료에 사용되는 대표적인 항암제이다. 하지만, 5-플루오로우라실의 반응률은
약 10-15%에 불과하고, 5-플루오로우라실 투여 시 발생하는 내성은 여전히
대장암의 성공적인 치료를 위해 극복해야 할 점이다. 따라서, 5-플루오로우라실
내성을 극복하는 한편, 5-플루오로우라실의 치료 효과를 향상시킬 수 있는 치료제의
개발이 필요하다. 탄닌산은 한약재인 오배자, 작약, 그리고 커피, 레드 와인 등에
함유되어있는 폴리페놀 성분으로 항비만, 항당뇨, 항산화, 항균, 항염 등의 효과를
보이는 것으로 알려져 있다. 항종양 효과와 관련하여서는 폐암, 유방암, 간암, 난소암,

전립선암, 치주암, 담낭암, 대장암에 대한 억제 효과가 보고되어 있다. 본 연구에서는

5-플루오로우라실에 대한 내성을 갖는 대장암 세포를 확보하고, 5-플루오로우라실
내성 대장암에 대한 탄닌산의 억제 효과를 알아보고자 하였다. 탄닌산은 5-
플루오로우라실 내성 대장암 세포인 CT26/5-FU와 HCT116/5-FU 세포의 생존율을
감소시켰다. 이러한 세포 생존율 감소 효과는 내인적 및 외인적 경로를 통한
세포자멸사와 세포 주기 억제의 유도에 기인한 것임을 밝혀냈다. 탄닌산은 암세포의

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항암제 내성 획득과도 밀접한 관련이 있다고 알려진 상피-간엽 전환 관련 인자들의
발현을 조절하였을 뿐만 아니라, MMP-2와 MMP-9의 발현 및 활성을 억제하여
암세포의 이동성 및 침윤성을 약화시켰다. 또한 탄닌산은 세포 외 약물 유출과
관련된 ABC 수송체의 발현량을 조절하여, 5-플루오로우라실과 병용하였을 때
세포자멸사를 더 유도하는 것으로 확인되었다. 이러한 결과를 토대로, 탄닌산은 5-
플루오로우라실 내성 대장암의 치료 및 5-플루오로우라실에 대한 민감성 증가에
기여할 수 있을 것으로 사료된다.

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<ABSTRACT>

Effects of tannic acid on overcoming 5-fluorouracil

(5-FU) resistance by inhibiting proliferation and
metastatic phenotypes of 5-FU-resistant colorectal
cancer cells

Jeong-Geon Mun

Department of Oriental Pharmacy

Graduate School of Wonkwang University

Directed by Professor Seung-Heon Hong

Colorectal cancer (CRC) is the second most common cause of

cancer-associated mortality in the world. Drug resistance and

consequent ineffectiveness of cancer chemotherapy contribute to up to

90% of cancer related deaths. 5-fluoruracil (5-FU) is a conventional
treatment used for patients with CRC, but acquired resistance to 5-FU
is an obstacle to successful treatment and a major cause of
chemotherapy failure. Tannic acid (TA), polyphenolic compound, has
shown various biological effects including anti-obesity, anti-oxidative,

anti-viral, anti-microbial, anti-inflammatory, and anti-tumor effects.

However, the effects of TA on 5-FU-resistant CRC cells has not been
elucidated. This study aimed to investigate inhibitory and reversal

effect of TA on 5-FU-resistant CRC cells and the underlying

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mechanisms. TA decreased the viability of 5-FU-resistant CRC cell

lines by inducing apoptosis through the activation of extrinsic and

intrinsic pathways, cell cycle arrest through inactivation of CDK2/cyclin

A complex. Non-cytotoxic concentrations of TA suppress migration and

invasion of 5-FU-resistant CRC cells by inhibiting

epithelial-mesenchymal transition (EMT) which also contributes to drug

resistance by down-regulating the expression of mesenchymal markers

N-cadherin, vimentin, snail, and twist and activity of matrix
metalloproteinase (MMP)-2 and MMP-9. Additionally, non-cytotoxic

concentrations of TA can inhibit the expression of ABC transporters,

including ABCB1, ABCC1, and ABCG2. In conclusion, TA not only
inhibited proliferation of 5-FU-resistant CRC cells by inducing

apoptosis and cell cycle arrest, but also improved sensitivity by

suppressing apoptosis evasion, EMT, and drug efflux.

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 1. Introduction

Colorectal cancer (CRC), cancer of the colon or the rectum, is

the third most common cancer with 1,148,515 new cases and the
second most common cause of cancer-associated mortality, with an

estimated 576,858 deaths in 2020 worldwide [1]. In particular, over

90% of cancer related deaths are attributed to drug resistance and
consequent ineffectiveness of cancer chemotherapy [2]. Furthermore,
the 5-year survival rate for metastatic CRC is only 14% [3] and
almost 50% of metastatic CRC patients are resistant to 5-fluorouracil
(5-FU)-based chemotherapies [4].
5-fluoruracil (5-FU) is a chemotherapeutic drug that induces
cytotoxicity by inhibiting the nucleotide synthesis enzyme thymidylate
synthase and misincorporating fluoronucleotides into RNA and DNA,
resulting in interference with essential biosynthetic activity.
5-FU-based chemotherapy is a first-line and conventional treatment
used for CRC patients. However, the response rate of 5-FU for
advanced CRC is still only 10-15% [5,6]. One of the causes of
treatment failure is the innate or acquired resistance [4]. Therefore,
strategies for overcoming chemoresistance and improving the outcomes

of 5-FU treatment are needed.

Apoptosis, a form of programmed cell death, is important in
controlling cell proliferation and eliminating cells with DNA damage
[7]. Apoptotic cells are characterized by morphological and biochemical
changes, including cell shrinkage, membrane blebbing, chromatin

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condensation, and nuclear fragmentation without inducing inflammation

[8]. Reduction of apoptosis or resistance to apoptosis is necessary for

carcinogenesis and malignant transformation [9]. In addition, resistance

to apoptosis is one of the factors contributing to the development of

5-FU resistance, and 5-FU-resistant cancer cell lines showed reduced

apoptosis. Induction of apoptosis is not only one of the efficient

anti-tumor mechanisms in current cancer treatment strategies, but also

plays a vital role in the sensitivity of cancer cells to chemotherapy
[10].

Mitochondrial membrane potential, a key indicator of

mitochondrial activity, is made up of a proton gradient generated by
the mitochondrial respiratory chain. The mitochondrial membrane

potential is the main force that drives protons into the mitochondrial
matrix and generates ATP. The mitochondria of many cancer cells
exhibit significantly increased potential compared to healthy normal
cells, suggesting therapeutic strategies that target mitochondrial
function. During apoptosis, mitochondria undergo changes, such as
mitochondrial membrane depolarization and the permeabilization of the
outer mitochondrial membrane that is regulated by Bcl-2 family
[11,12].
The arrest of cell cycle progression is another mechanism for

the treatment of cancer. When DNA damage occurs in cells, they

arrest the cell cycle progression to initiate recovery or promote cell
death, preventing transmission of damaged genome to daughter cells.

Defects in cell cycle checkpoint mechanisms are often found in cancer

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cells [13]. Cyclin-dependent kinases (CDKs) are serine/threonine

kinases and their catalytic activities are controlled by binding to

regulatory subunits, known as cyclins to form complexes. The

progression through each phase of the cell cycle is mediated by each

CDK/cyclin complexes [14]. Drugs targeting cell cycle checkpoints

arrest the cell cycle progression by modulating the CDK/cyclin

complexes, resulting in the suppression of cell proliferation.

Epithelial-mesenchymal transition (EMT) is a complex
morphogenetic process that allows cells with epithelial properties to

undergo functional and biochemical changes that enable them to acquire

a mesenchymal cell phenotype. The acquisition of mesenchymal
phenotypes leads to the loss of cell polarity and adherent tight

junction, resulting in the increase of motility and invasiveness [15].

EMT is necessary for the malignant transformation and metastasis of
cancer cells. In addition, accumulating evidence shows that EMT is not
only associated with cancer metastasis, but also acquired resistance to
cancer drugs [16].
ATP-binding cassette (ABC) transporters utilize the energy of
ATP hydrolysis to translocate substrates across cellular membranes,
which is known to play a central role in multidrug resistance in cancer.
The ABC transporters consist of transmembrane domains, the site at

which the substrate binds to the transporter, and nucleotide-binding

domains that exerts ATPase activity to provide the energy required for
the efflux of substrates. ABCB1, ABCC1, and ABCG2 are prominent

efflux transporters known to mediate chemoresistance in cancer cells

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[17,18].

Tannic acid (TA) is polyphenolic compound composed of a

central core of glucose esterified to digallic acid residues linked by

depside bonds between gallic acids [19]. TA is found in gallnuts of

rhus chinensis, Caesalpinia spinose, Paeonia officinalis, rhus coriaria,

Quercus infectoria, coffee, and red wine [20,21]. TA has
multi-pharmacological activities including anti-obesity [22,23],
anti-diabetic [24], anti-oxidative [25], anti-viral, anti-microbial [26],
anti-inflammatory [27,28], and anti-tumor effects. Previous studies
have been observed that TA showed anti-tumor actions in lung
[29,30], breast [31,32], liver [33,34], ovarian [35], prostate
[20,36,37], gingival cancer [38], and CRC [19,21,39,40] cells. In
particular, TA inhibits proliferation of HCT116 human CRC cells by
inducing cell cycle arrest and apoptosis dependently of p53 [39], and
inducing senescence and DNA damage that are capable of suppressing
tumor development independent of p21 and p53 [40]. In addition, TA
inhibits proliferation of CRC cells by targeting PKM2 activity [21] and
growth of HT29 and HCT116 human CRC cells via suppression of the
NF-κB pathway [19]. A recent study also demonstrated that TA

inhibits the lung metastasis of CRC cells by inducing apoptosis, cell

cycle arrest and autophagy, and suppressing EMT, migration and
invasion [41]. However, the anti-tumor effect of TA on 5-FU-resistant

CRC cells has not been elucidated. In the present study, the
anti-proliferative and anti-metastatic properties of TA against
5-FU-resistant CRC cell lines as well as the effect of enhancing 5-FU

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sensitivity and underlying mechanisms were investigated.

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 2. Materials and methods

2.1. Reagents and antibodies

TA (1401-55-4; purity ≥95%) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). 5-FU, tween-20, and crystal

violet solution were purchased from Sigma-Aldrich (St Louis, MO,

USA). Bovine serum albumin (BSA) was purchased from Millipore
(Billerica, MA, USA). Anti-caspase-3, cleaved caspase-3, caspase-8,
caspase-9, poly ADP-ribose polymerase (PARP), cleaved PARP, Bcl-2,
Bcl-xL, Bax, N-cadherin, matrix metalloproteinase (MMP)-2, and
ABCG2 were purchased from Cell Signaling Technology, Inc. (Danvers,
MA, USA). Anti-cyclin A, cyclin E, CDK2, vimentin, snail, twist,
ABCB1, ABCC1, and GAPDH were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA). MMP-9 antibody was
purchased from Invitrogen (Carlsbad, CA, USA).

2.2. Cell culture

The murine CRC cell line colon 26 (CT26) and human CRC cell
line HCT116 were purchased from Korean Cell Line Bank (Seoul,
Korea). CT26 cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM) and HCT116 cells were grown in Roswell Park

Memorial Institute (RPMI) 1640. The mediums were supplemented with
10% heat-inactivated fetal bovine serum (FBS) and 1%

penicillin-streptomycin. Both cell lines were incubated at 37 ℃ in a

humidified atmosphere containing 5% CO2.

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2.3. Establishment of 5-FU-resistant CRC cells
5-FU was purchased from Sigma-Aldrich (St Louis, MO, USA)
and dissolved in dimethyl sulfoxide (DMSO). To established acquired

resistant cell lines, CT26 and HCT116 cells were cultured with
gradually elevating concentrations of 5-FU for more than 6 months.
Briefly, CT26 and HCT116 cells were seeded into 10 cm dish at a
density of 1 × 105 cells and exposed to 5-FU for 72 h. The cells
were then grown in fresh medium without 5-FU until 70% confluence

to allow recovery. The treatment process was repeated with increasing

concentrations of 5-FU from 0.1 μM to 30 μM in CT26 and from 0.1 μ
M to 50 μM in HCT116 cells. Thereafter, the 5-FU-resistant CRC cells
were maintained in medium supplemented with 30 μM and 50 μM of
5-FU once every 2 weeks to maintain resistance.

2.4. Cell viability assay

CT26, HCT116, 5-FU-resistant CT26 (CT26/5-FU) and
5-FU-resistant HCT116 (HCT116/5-FU) cells were seeded into
96-well plates at a density of 2 × 103 cells/well and cultured
overnight. The cells were treated with various concentrations of 5-FU
(2.5, 5, and 10 μM) and TA (5, 10, 15, and 20 μM). After incubation

for 24, 48, and 72 h, water-soluble tetrazolium salt-8 reagent (WST-8;

Enzo Life Sciences, Farmingdale, NY, USA) was mixed with a fresh
medium and added to each well. The absorbance was measured at 450

nm using a microplate reader.

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2.5. Colony formation assay
Cells were seeded into a 12-well plate at a density of 5 × 102
cells/well for CT26/5-FU and 1 × 103 cells/well for HCT116/5-FU.

After stabilization, colony expansion of single cells progressed with TA

(5-20 μM) for 7 days. The colonies were fixed with 3.7%

formaldehyde for 15 min and washed with phosphate-buffered saline

(PBS). Colonies were stained with 0.1% crystal violet solution for 20
min and then rinsed with PBS. The stained colonies were photographed
after air-drying.

2.6. Annexin V assay

Apoptotic cells were measured using Muse Annexin V & Dead
Cell Assay Kit (Luminex Corporate, Austin, TX, USA) according to the
manufacturer’s protocols. Briefly, the cells treated with TA for 48 h

were collected and suspended in a fresh serum-containing medium to

achieve a final concentration of 5 × 104 cells/100 μL. Harvested cells
were mixed with the same volume of Muse Annexin V & Dead cells

Reagent for 20 min at room temperature in the dark. The apoptotic

cells were measured using MuseTM Cell Analyzer.

2.7. Mitochondrial membrane potential assay

Mitochondria potential was measured using Muse MitoPotential
Kit (Luminex Corporate, Austin, TX, USA) according to the
manufacturer’s protocols. After TA treatment for 48 h, the cells were

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harvested and suspended at 5 × 104 cells/100 μL using 1 X Assay

Buffer. Each samples were incubated with MitoPotential working

solution for 20 min at 37 ℃ in a CO2 incubator. Thereafter,

MitoPotential 7-AAD reagent was added and incubated for 5 min at

room temperature. Mitochondrial membrane potential changes were
analyzed using MuseTM Cell Analyzer.

2.8. Western blot analysis

After treatment, the cells were lysed with PRO-PREPTM Protein
Extraction Solution (iNtRon Biotech, Seoul, Korea). Lysates were mixed

with 2X buffer (ElpisBiotech, Inc., Daejeon, Korea) after determination

of total protein concentration using the Lowry method and boiled at 95
℃ for 5 min. The proteins were separated using sodium dodecyl

sulfate polyacrylamide gel electrophoresis using acrylamide gels and

transferred onto polyvinylidene fluoride (PVDF) membranes. After
saturation with 5% BSA, the membranes were incubated with primary
antibodies, followed by their treatment with horseradish peroxidase
(HRP)-conjugated secondary antibodies (Jackson ImmunoResearch

Laboratories, Inc., Pennsylvania, PA, USA). The protein blots were

detected using an enchanced chemiluminescence (ECL) solution

(ElpisBiotech, Inc., Daejeon, Korea) and visualized using the FluorChem
M System (ProteinSimple, Sna Jose, CA, USA).

2.9. Cell cycle analysis

Cell cycle analysis was conducted using Muse Cell Cycle Kit

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(Luminex Corporate, Austin, TX, USA) according to the manufacturer’s

protocols. The cell samples were collected and fixed with 70% ethanol
for at least 3 h at –20 ℃. Ethanol-fixed cells were centrifuged at 300

× g for 5 min at room temperature and suspended in PBS. After

centrifugation, the supernatant was removed and the cell pellet was re
suspended in Muse Cell Cycle Reagent for 30 min at room temperature

in the dark. The cell cycle arrest was analysed using the MuseTM cell

analyzer.

2.10. Real-time reverse transcription-polymerase chain reaction

(RT-PCR)
RNA extraction was performed using easy-spinTM Total RNA
Extraction Kit (iNtRon Biotech, Seoul, Korea) in accordance with the

manufacturer’s protocol. High Capacity RNA-to-cDNA kit (Applied

Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was
used for the synthesize first-strand cDNA. After quantification of the

total RNA, reverse transcription was performed at 37 ℃ for 60 min

and at 95 ℃ for 5 min. Real-time RT-PCR with SYBR green
(Real-Time qPCR 2X Master Mix; ElpisBiotech, Inc., Daejeon, Korea)

was carried out using a StepOnePlusTM Real-Time PCR System

(Applied Biosystems by thermo Fisher Scientific, Inc., Waltham, MA,

USA). The thermal cycling conditions were as follows: one cycle at 95

℃ for 10 min, then 40 cycles of 95 ℃ for 15 sec, 60 ℃ for 1 min, 95
℃ for 15 sec and melt curve at 60 ℃ for 1 min, 95 ℃ for 15 sec.
The primer sequences were described in Table 1 and Table 2.

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 Table 1. Sequences for real-time RT-PCR human primers.

Gene Forward (5’-3’) Reverse (5’-3’)

TCAGTACCTTAGGGAAGC CCAGTCCACCAGAATCGT
Cyclin A
TGAAA G
GGGTCTGCACAGACTGCA
Cyclin E GGCCAAAATCGACAGGAC
T
AAAGCCAGAAACAAGTTG GTACTGGGCACACCCTCA
CDK2
ACG GT
TTGCTGCTTACATTCAGG AGCCTATCTCCTGTCGCA
ABCB1
TTTCA TTA
CTCTATCTCTCCCGACAT AGCAGACGATCCACAGCA
ABCC1
GACC AAA
CAGGTGGAGGCAAATCTT ACCCTGTTAATCCGTTCG
ABCG2
CGT TTTT
GGAGCGAGATCCCTCCAA GGCTGTTGTCATACTTCT
GAPDH
AAT CATGG

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 Table. 2. Sequences for real-time RT-PCR mouse primers.

Gene Forward (5’-3’) Reverse (5’-3’)

CTTGGCTGCACCAACAGT CAAACTCAGTTCTCCCAA
Cyclin A
AA AAACA
TTTCTGCAGCGTCATCCT TGGAGCTTATAGACTTCG
Cyclin E
C CACA
CTGCATCTTTGCTGAAAT GATCCGGAAGAGTTGGTC
CDK2
GG AAT
TGGAGAACCCCATTGACA TGATCCCTCAGGAACTGT
N-cadherin
TT CC
CGGAAAGTGGAATCCTTG CACATCGATCTGGACATG
Vimentin
CA CTG
TCCAAACCCACTCGGATG TTGGTGCTTGTGGAGCAA
Snail
TGAAGA GGACAT
AGCTACGCCTTCTCCGTC TCCTTCTCTGGAAACAAT
Twist
T GACA
CCCCATGAAGCCTTGTTT TTGTAGGAGGTGCCCTGG
MMP-2
ACC AA
AGACCAAGGGTACAGCCT GGCACGCTGGAATGATCT
MMP-9
GTTC AAG
CCATCAGCCCTGTTCTTG TCCCCAGCCTTTTAGCTT
ABCB1
GAC CTT
GACCGGGGCTACATCCAG TGTTGGGCTGACCAGTAA
ABCC1
AT CAC
AAATGCTGTTCAGGTTAT TCCGACCTTAGAATCTGC
ABCG2
GTGGT TACTT
GACATGCCGCCTGGAGAA AGCCCAGGATGCCCTTTA
GAPDH
AC GT

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2.11. Wound healing assay

CT26/5-FU cells were seeded in a SPL ScarTM Block (SPL Life

Sciences, Pocheon, Korea). When the cells formed a confluent
monolayer, the block was removed and serum-free medium containing

TA (1.25, 2.5, and 5 μM) was added. After 24 h, images were captured
using EVOSⓇ XL Core Imaging System (Thermo Fisher Scientific, Inc.,
Waltham, MA, USA).

2.12. Tumor spheroid migration assay

CT26/5-FU cells (1×106 cells/ml) were seeded by 10 μL onto
the inverted lid of 10 cm dish and the bottom of the dish was filled
with PBS to serve as a hydration chamber. The cells were incubated
until they formed spheroids [42]. The 96-well plate is coated with
0.1% gelatin solution (50 μL/well) for 2 h, washed twice with PBS, and
then blocked with 1% BSA (100 μL/well) for 1 h [43]. The spheroids
were transferred to the pre-coated migration plate filled with 200 μL
of medium supplemented with 2% FBS, and treated with TA. Images
were captured using EVOSⓇ XL Core Imaging System (Thermo Fisher
Scientific, Inc., Waltham, MA, USA).

2.13. Invasion assay

Matrigel-coated SPL InsertTM Hanging cell culture chamber was

used to estimate the invasion of cancer cells. CT26/5-FU cells (5 ×

104 cells) in 200 μL serum-free medium were seeded into the upper

chamber, and the chambers were placed in a 24-well plate that filled

- 17 -
with 750 μL of medium containing 10% FBS. After treatment with TA

(1.25, 2.5, and 5 μM) for 24 h, the chambers were fixed with 3.7%
formaldehyde and permeabilized with methanol. The cells on the

surface of the chamber membrane were stained with giemsa solution.

Non-invading cells were removed by gently rubbing the inner sides fo

the chamber with a swab. Images were captured using EVOSⓇ XL Core

Imaging System (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

2.14. Gelatin zymography

CT26/5-FU cells (1 × 105 cells/well) were cultured in a 6-well
plate and treated with TA for 24 h. The supernatant from TA-treated
cells was mixed with 2× zymogram sample buffer (KOMA Biotech,
Seoul, Korea). The samples were electrophoresed on an 8% SDS
polyacrylamide gel containing 0.1% gelatin in the zymogram running
buffer (KOMA Biotech). The gel was washed with zymogram renaturing
buffer (KOMA Biotech) for 30 min, then incubated with zymogram
developing buffer (KOMA Biotech) at 37 ℃ for 24 h. The gel was
stained with Coomassie Blue R-250 solution (Enzynomics, Daejeon,
Korea) to reveal the gelatinolytic activitiy of MMP-2 and MMP-9.

2.15. Tumorigenicity assay

BALB/c mice (female, 4 weeks old, 17-18 g) were obtained

from Samtako (Osan, Korea). CT26/5-FU cells were subcutaneously
(s.c.) injected into the right flank of each mouse (3 × 106 cells/100 μ

L/mouse). When tumors were palpable, TA (25 and 50 mg/kg) was

- 18 -
orally administered and repeated 5 days a week for 4 weeks. Tumor

growth was measured twice a week with a caliper and the volume was
calculated according to the following formula: V=

1/2(diameterlong×diametershort2). The mice were euthanized by cervical

dislocation, and tumors were resected. Aspartate aminotransferase

(AST), alanine aminotransferase (ALT), creatinine, and blood urea

nitrogen (BUN) levels were analyzed by the Seoul Medical Science

Institute (Seoul Clinical Laboratories, Seoul, Korea).

2.16. Statistical analysis

SPSS Satistics v18 (IBM Corporation, Armonk, NY, USA) was
used as the statistical analysis software. The student’s t-test was used

to determine the statistical significance. Data were presented as the

mean ± standard deviation (SD), and p < 0.05 was considered to

indicate a statistically significant difference.

- 19 -
 3. Results

3.1 Establishment of 5-FU-resistant CRC cell lines

To compare the difference in the cytotoxic effects of 5-FU on

parental and 5-FU-resistant CRC cells, cell viability was examined

using WST-8 assay. Following 5-FU treatment, CT26 and HCT116
cells showed reduced cell viability, whereas CT26/5-FU and
HCT116/5-FU exhibited greater viability (Fig. 1A). As shown in Fig.
1B, treatment with 5-FU for 48 h decreased the number of cells and
showed morphological changes in CT26 and HCT116 cells and had no
effect on CT26/5-FU and HCT116/5-FU cells. Unlike in CT26 and
HCT116 cells, 5-FU-induced apoptosis was also inhibited in

CT26/5-FU and HCT116/5-FU cells (Fig. 1C). The accumulation of

sub-G1 represents DNA fragmentation, which occurs during apoptosis
[44]. 5-FU treatment induced the accumulation of sub-G1 phase in

CT26 cells and arrest of G2/M phase in HCT116 cells, whereas there
was no change in cell cycle distribution of CT26/5-FU and
HCT116/5-FU cells (Fig. 1D).

- 20 -
Fig. 1. 5-FU reduces cell viability through induction of apoptosis and
cell cycle arrest in parental CRC cells, but not 5-FU-resistant CRC
cells. (A) Cell viability of 5-FU-treated CT26, CT26/5-FU, HCT116,
and HCT116/5-FU cells. Cells were treated with 5-FU at final
concentration of 2.5-10 μM, and cell viability was determined after 24
and 48 h of treatment. (B) Morphology of CT26, CT26/5-FU, HCT116,
and HCT116/5-FU cells with 5-FU treatment for 48 h.

- 21 -
(C) CT26, CT26/5-FU, HCT116, and HCT116/5-FU cells were
incubated with 5-FU (5 μM) for 48 h and stained with Annexin
V/7-AAD. (D) Cell cycle phase distribution of 5-FU-treated CT26,
CT26/5-FU, HCT116 and HCT116/5-FU cells. The images are
representative of three independent experiments. Results are expressed
as the mean ± SD of three independent experiments. *p < 0.05, **p <
0.01, and ***p < 0.001.

- 22 -
3.2 Effect of TA on the proliferation of 5-FU-resistant CRC cells

To evaluate whether TA can exert cytotoxicity on

5-FU-resistant CRC cells, various concentrations of TA (0-20 μM)

were treated to CT26/5-FU and HCT116/5-FU cells at different
intervals (24-72 h). The viability of TA-treated cells was reduced in a
dose- and time-dependent manner (Fig. 2A). Reduced cell number and
morphological changes of CT26/5-FU and HCT116/5-FU cells were
induced by treatment with TA for 48 h (Fig. 2B).

- 23 -
Fig. 2. TA decreases cell viability in 5-FU-resistant CRC cells. (A)
Cell viability of TA-treated CT26/5-FU and HCT116/5-FU cells. The
cells were treated with TA (5-20 μM). The cell viability was measured
after 24, 48, and 72 h of treatment. (B) Morphology of TA-treated
CT26/5-FU and HCT116/5-FU cells. Images were captured by
microscope after 48 h incubation with TA. Results are expressed as
the mean ± SD of three independent experiments. *p < 0.05, **p <
0.01, and ***p < 0.001.

- 24 -
3.3 Effect of TA on colony-forming ability of 5-FU-resistant CRC cells

Infinite proliferation is the typical characteristic of cancer cells,

and a way to measure the proliferative ability of cells is to evaluate

colony formation by single cells [45]. To evaluate whether TA inhibits
the ability of a single 5-FU-resistant cell to grow into a colony, TA
(0-20 μM) was treated to CT26/5-FU and HCT116/5-FU cells for 7
days. Treatment with TA reduced the colony-forming ability of
CT26/5-FU and HCT116/5-FU cells (Figs. 3A and 3B).

- 25 -
Fig. 3. TA inhibits colony formation of 5-FU-resistant CRC cells. (A)
Colony formation of CT26/5-FU and HCT116/5-FU cells with TA
treatment for 7 days. (B) The stained cells dissolved in 1% SDS and
absorbance was measured at 570 nm. *p < 0.05, **p < 0.01, and ***p
< 0.001.

- 26 -
3.4 Effect of TA on the apoptosis in 5-FU-resistant CRC cells

To investigate whether TA-induced cell death was related to

apoptosis, Annexin V/7-AAD staining was performed. As shown in Fig.

3A, the percentages of apoptotic cells in CT26/5-FU and
HCT116/5-FU cells were significantly increased following treatment
with TA for 48 h in a dose-dependent manner (Figs. 4A and 4B). To
further explore the mechanisms underlying TA-induced apoptosis, the
expression of apoptosis-related proteins was detected by western blot
analysis following exposure of CT26/5-FU and HCT116/5-FU cells to
TA for 48 h. TA induced cleavage of caspase-3, caspase-8,
caspase-9, and PARP (Fig. 4C).

- 27 -
Fig. 4. TA induces apoptosis in 5-FU-resistant CRC cells thorugh
extrinsic and intrinsic pathway. (A) CT26/5-FU and HCT116/5-FU
cells were incubated with TA (10, 15, and 20 μM) for 48 h and stained
with Annexin V/7-AAD. The image is a representative of three
independent experiments. (B) The statistical graph of apoptotic cells.

- 28 -
(C) CT26/5-FU and HCT116/5-FU cells were treated with the
indicated concentrations of TA for 48 h. The expressions of
caspase-3, cleaved caspase-3, caspase-8, caspase-9, PARP and
cleaved PARP were detected by western blotting using corresponding
antibodies. Results are expressed as the mean ± SD of three
independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

- 29 -
3.5 Effect of TA on the mitochondrial membrane potential in

5-FU-resistant CRC cells

To elucidate whether TA-induced apoptosis was mediated via

mitochondrial dysfunction, the mitochondrial membrane potential was

evaluated using MuseTM MitoPotential Kit in TA-treated CT26/5-FU

and HCT116/5-FU cells. TA significantly increased the percentages of

total depolarization in CT26/5-FU and HCT116/5-FU cells, from 6.22
± 2.76% and 6.04 ± 1.76% in the untreated control to 16.38 ± 4.01%
and 14.71 ± 2.85% (10 μM), 22.52 ± 4.74% and 31.42 ± 10.27% (15
μM), and 25.55 ± 2.77% and 39.93 ± 8.91 (20 μM), respectively (Figs.
5A and 5B). These results suggest that TA-induced apoptosis may be
attributed to mitochondrial dysfunction.
To further confirm the role of TA in mitochondira-mediated
apoptosis, the expression of mitochondria-related apoptotic proteins
was detected using western blot analysis. The expression of
anti-apoptotic proteins such as Bcl-2 and Bcl-xL was decreased in
TA-treated CT26/5-FU and HCT116/5-FU cells, whereas expression
of pro-apoptotic protein such as Bax was increased in CT26/5-FU

cells treated with TA (Figs. 5C and 5D).

- 30 -
Fig. 5. TA reduces the mitochondrial membrane potential of
5-FU-resistant CRC cells. (A) CT26/5-FU and HCT116/5-FU cells
were incubated with TA (10, 15, and 20 μM) for 48 h and the
mitochondrial polarization was evaluated. The images are representative
of three independent experiments. (B) The percentage of cells with
depolarized mitochondrial membrane potential.

- 31 -
(C) CT26/5-FU and HCT116/5-FU cells were treated with the
indicated concentrations of TA for 48 h. The expressions of Bcl-2,
Bcl-xL, and Bax were detected by western blotting using
corresponding antibodies. (D) The relative density of western blot
bands was calculated by densitometric analysis using ImageJ software.
Results are expressed as the mean ± SD of three independent
experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

- 32 -
3.6 Effect of TA on cell cycle arrest in 5-FU-resistant CRC cells

To investigate whether TA reduced the proliferation of

5-FU-resistant CRC cells by inducing cell cycle arrest, the cell cycle
analysis was conducted using propidium iodide (PI) staining. Exposure
to TA for 48 h led to S phase arrest in CT26/5-FU and HCT116/5-FU
cells, increasing from 23.47 ± 0.60 % and 17.1 ± 1.11 to 36.53 ±
1.70% and 31.97 ± 3.02%, respectively (Figs. 6A and 6B). In the S
phase, cyclin E is degraded, inactivating the CDK2/Cyclin E complex,
and the free CDK2 binds to cyclin A, activating of CDK2/Cyclin A
complex, which leads further S phase progression to the G2/M phase.
Reduced expressions of these regulators indicate cell cycle arrest in
the S phase [46,47]. Real-time RT-PCR results showed that TA
treatment decreased mRNA expressions of cyclin A, cyclin E, and
CDK2 in both CT26/5-FU and HCT116/5-FU cells (Fig. 6C). Protein
levels of cyclin A, cyclin E, and CDK2 were also reduced following
treatment with TA (Fig. 6D).

- 33 -
Fig. 6. TA induces S phase arrest in 5-FU-resistant CRC cells by
regulating CDK/cyclin complexes. (A) Cell cycle phase distribution of
CT26/5-FU and HCT116/5-FU with TA treatment for 48 h. The
images are representative of three independent experiments. (B) The
relative cell population in S phase of CT26/5-FU and HCT116/5-FU
cells.

- 34 -
(C and D) mRNA (C) and protein (D) expression levels of cyclin A,
cyclin E, and CDK2 in CT26/5-FU and HCT116/5-FU cells with TA
treatment for 48 h. Results are expressed as the mean ± SD of three
independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

- 35 -
3.7 Effect of TA on EMT of 5-FU-resistant CRC cells

To confirm TA suppresses EMT in 5-FU-resistant CRC cells,

EMT markers were measured by real time RT-PCR and western

blotting. Treatment with non-cytotoxic concentrations of TA (1.25, 2.5,
and 5 μM) down-regulated the mRNA level of mesenchymal phenotypic
markers, including N-cadherin, vimentin, snail, and twist in CT26/5-FU
cells (Fig. 7A). Consistent with the mRNA expression, TA
down-regulated the protein levels of N-cadherin, vimentin, snail, and
twist (Fig. 7B). During the EMT process, cancer cells produce MMPs
to degrade extracellular matrix (ECM), which act as a physical barrier
that prevents the cancer from spreading [48]. The TA treatment
reduced the mRNA and protein expression levels of MMP-2 and
MMP-9 in CT26/5-FU cells (Figs. 7C and 7D). Additionally, the
activity of MMP-2 and MMP-9 was inhibited by TA (Fig. 7E).

- 36 -
Fig. 7. TA inhibits expression of EMT markers in CT26/5-FU cells. (A
and B) mRNA (A) and protein (B) expression of N-cadherin, snail,
vimentin, and twist in CT26/5-FU cells treated with TA for 24 h. (C
and D) mRNA (C) and protein (D) expression of MMP-2 and MMP-9 in
CT26/5-FU cells treated with TA for 24 h. (E) Activity of MMP-2 and
MMP-9 was detected by gelatin zymography. Results are expressed as
the mean ± SD of three independent experiments. *p < 0.05, **p <
0.01, and ***p < 0.001.

- 37 -
3.8 Effect of TA on the migratory and invasive ability of

5-FU-resistant CRC cells

EMT allows stationary tumor cells to acquire the ability of

migration and invasion [49]. To explore the effect of TA on the

migraion of CT26/5-FU cells, the wound healing assay and tumor

spheroid migration assay were performed. Treatment with

non-cytotoxic concentrations of TA (1.25, 2.5, and 5 μM) for 24 h
inhibited the migratory ability of CT26/5-FU cells (Figs. 8A and 8B).
Matrigel invasion assay was performed to further investigate the effect
of TA on the invasive ability of CT26/5-FU cells. The infiltration of
CT26/5-FU cells in the matrigel-coated transwell membrane was

decreased following treatment of TA in a dose-dependent manner (Fig.

8C).

- 38 -
Fig. 8. TA suppresses migratory and invasive abilities of CT26/5-FU
cells. (A) Wound healing assay was performed to investigate the
motility of CT26/5-FU cells following treatment with TA for 24 h at
the indicated concentrations. (B) Tumor spheroid migration assay was
conducted to observe the cells migrating from spheroids after
treatment with TA for 48 h. (C) The invasive ability of CT26/5-FU
cells was determined by matrigel invasion assay following treatment
with the indicated concentrations of TA for 24 h.

- 39 -
3.9 Effect of TA on ABC transporters of 5-FU-resistant CRC cells

To determine whether the sensitivity of 5-FU-resistant CRC

cells to 5-FU by TA is increased due to ABC transporter regulation,

expression of ABC transporters were measured in TA-treated
5-FU-resistant CRC cells. The mRNA and protein expression of
ABCB1, ABCC1, and ABCG2 were down-regulated in both CT26/5-FU
and HCT116/5-FU cells following treatment with TA (Figs. 9A and
9B).

- 40 -
Fig. 9. TA increases sensitivity of 5-FU-resistant CRC cells to 5-FU
by regulating ABC transporters. (A) mRNA expression levels of
ABCB1, ABCC1, and ABCG2 in CT26/5-FU and HCT116/5-FU cells
with TA treatment for 48 h were evaluated by real-time RT-PCR
analysis. (B) Protein expression levels of ABCB1, ABCC1, and ABCG2
in CT26/5-FU and HCT116/5-FU cells with TA treatment for 48 h.
Results are expressed as the mean ± SD of three independent
experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

- 41 -
3.10 Effect of TA and 5-FU combination on the proliferation of

5-FU-resistant CRC cells

To examine whether TA exhibits increased cytotoxic effect in

combination with 5-FU, CT26/5-FU and HCT116/5-FU cells were

co-treated with non-cycotoxic concentrations of 5-FU and TA. The

cell viability of CT26/5-FU was 95.5±6.9% and 90.8±4.1% with 5-FU

or TA alone treatment, respectively, whereas the cell viability of 5-FU
and TA co-treatment decreased to 78.4±5.5% (Fig. 10A). The
apoptosis of CT26/5-FU cells treated with 5-FU and TA increased
from 19.1±2.3% to 30.6±4.1% compared to cells treated with 5-FU
alone (Figs. 10C and 10D). Similarly, the cell viability of HCT116/5-FU

was 94.4±4.1% and 90.1±1.5% with 5-FU or TA alone treatment,

respectively, whereas the cell viability of 5-FU and TA co-treatment
decreased to 68.5±1.6% (Fig. 10B). The apoptosis of HCT116/5-FU

cells treated with 5-FU and TA increased from 13.9±1.4% to

22.2±0.8% compared to cells treated with 5-FU alone (Figs. 10C and
10D).
The expression of apoptosis-related proteins was also detected.
Combined treatment with 5-FU and TA induced cleavage of caspase-3,
caspase-8, caspase-9, and PARP and reduced the expression levels of
Bcl-2 and Bcl-xL, but treatment with 5-FU or TA alone had no effect
(Fig. 10E).

- 42 -
Fig. 10. Combination of 5-FU and TA more decreases cell viability
through apoptosis than treatment with 5-FU or TA alone. (A and B)
CT26/5-FU and HCT116/5-FU cells were treated with 5-FU (10 μM)
and TA (5 μM) alone or in combination for 48 h, and cell viability was
determined.

- 43 -
(C) CT26/5-FU and HCT116/5-FU cells were incubated with 5-FU (10
μM) and TA (5 μM) alone or in combination and stained with Annexin
V and 7-AAD. The images are representative of three independent
experiments. (D) The statistical graph of apoptotic cells.

- 44 -
(E) Protein bands representing caspase-3, caspase-8, caspase-9,
PARP, cleaved PARP, Bcl-2, and Bcl-xL in CT26/5-FU and
HCT116/5-FU cells. Results are expressed as the mean ± SD of three
independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

- 45 -
3.11 Effect of TA on the growth of CT26/5-FU cells implanted in mice

After subcutaneous injection of CT26/5-FU cells, inhibitory

effect of TA on tumor growth was investigated in in vivo. Body weight

remained unaffected following treatment with TA (Fig. 11A). Oral
administration of TA for 4 weeks suppressed growth of tumor volume

in mice (Fig. 11B). Tumors resected from TA-treated mice also

weighed less compared to those of control mice (Figs. 11C and 11D).

- 46 -
Fig. 11. TA suppresses the tumor growth of CT26/5-FU cells in mouse
model. The 5-FU-resistant CRC tumors in BALB/c mice (n=6) were
monitored for 4 weeks of TA (25 or 50 mg/kg) administration. (A) The
body weight of mice during in vivo experiment. (B) Tumor growth
curve for CT26/5-FU carcinoma in the presence or absence of TA
administration. (C) Representative images of subcutaneous tumor mass.
(D) Tumor weight.

- 47 -
Table 3. Effect of TA on parameters of liver and kidney function in

CT26/5-FU-injected mice.

Creatinine
TA (mg/kg) AST (IU/L) ALT (IU/L) BUN (mg/dL)
(mg/dL)
0 161 ± 20.9 29.6 ± 2.4 0.13 ± 0.049 20.8 ± 6.5
25 154 ± 12.3 35.4 ± 13.3 0.11 ± 0.022 22.4 ± 6.9
50 146.6 ± 31.8 25.8 ± 6.1 0.13 ± 0.02 21 ±2.3

- 48 -
 4. Discussion

This study elucidated the reversal effect of TA on the 5-FU

resistance in 5-FU-resistant CRC cells. Insensitivity to apoptosis plays
a crucial role in acquiring resistance of chemotherapy [50]. As shown

in the results of comparing 5-FU-resistant CRC cells and parental CRC

cells, 5-FU-resistant CRC cells evaded 5-FU-induced apoptosis, but
parental CRC cells were occurred apoptosis by 5-FU treatment. On the
other hand, TA reduced cell viability by inducing apoptosis of
5-FU-resistant CRC cells. Apoptosis is mediated by two main
pathways, the extrinsic and intrinsic pathways. The extrinsic pathway
is triggered when death ligands bind to their receptors. Their binding
activates caspase-8 and subsequently leads to the cleavage of
caspase-3. Activation of caspase-3 is followed by the cleavage of
essential substrates for cell survival, such as PARP, which can repair
damaged DNA, and consequently induces apoptosis. The intrinsic
pathway, also known as mitochondrial pathway, is activated by several
stimuli such as DNA damage. Maintenance of mitochondrial membrane
potential is essential for physiological cellular function. Protons
gradient across the inner mitochondrial membrane by proton pumps

creates highly negative membrane potential, which is attributed to ATP

synthesis by the respiratory chain. Therefore, loss of mitochondrial
membrane potential indicates impairment of mitochondria and is a
important event in mitochondrial-dependent apoptosis [51-53]. The
mitochondrial outer membrane permeabilization leads to disruption of

- 49 -
mitochondrial membrane potential and release of intramembrane space

proteins, resulting in the activation of caspases [54,55]. Bax,

pro-apoptotic protein, can cause collapse of mitochondrial membrane

potential by rupture of the mitochondrial membrane through pore

formation and trigger an increase in mitochondiral permeability [56].

On the contrary, Bcl-2 and Bcl-xL which are anti-apoptotic proteins

prevent Bax-induced pore formation as well as mitochondrial

membrane potential loss [57-59]. Previous studies have demonstrated
that TA is able to induce apoptosis by decreasing the mitochondrial

membrane potential in lung, breast, prostate, and gingival cancer cells

[30,31,37,38]. Particularly, TA augments the anticancer efficacy of
cisplatin against liver cancer cells through mitochondria-mediated

apoptosis [34]. In this study, TA-induced apoptosis through extrinsic

and intrinsic pathways by regulating apoptosis-related proteins, and
mitochondrial depolarization by regulating Bcl-2 family.
Deregulation of cell cycle progression underlies uncontrolled
proliferation that is hallmark of cancer cells. The induction of cell
death by cell cycle arrest is one of the effective mechanisms
regulating cell proliferation for cancer treatment [60]. Several studies
have reported that TA induced cell cycle arrest in various cancer cells
including lung, breast, prostate and gingival cancer cells [32]. In

particular, TA leads to arrest in S phase at low doses and sub-G1

phase at high doses in human CRC cell lines HT29 and HCT116 cells,
and S phase accumulation in metastatic CRC cell lines CT26, SW620,

and HCT116 cells [19,41]. The progression through cell cycle phases

- 50 -
is promoted when the CDKs is activated by binding to specific cyclins.

In the S phase, CDK2/cyclin E complex is inactivated due to

degradation of cyclin E to prevent DNA re-replication. Cyclin A is

combined with the CDK2, resulting in activation of CDK2/cyclin A

complex, which induces the transition of S phase to G2/M phase

[46,61]. In the present study, TA induced arrest in S phase via the

inactivation of CDK2/cyclin A complex.

EMT is involved in cancer progression and metastasis as well
as acquired resistance to 5-FU [16]. Several studies have reported

that many kinds of cancer cells with acquired resistance to

chemotherapy undergo EMT. For example, gefitinib-resistant non-small
cell lung cancer cells [62], paclitaxel-resistant ovarian cancer cells

[63], tamoxifen-resistant breast cancer cells [64] and 5-FU-resistant

CRC cells [65] showed not only phenotypic changes consistent with
EMT, but also enhancement of migration and invasion ability.
Collectively, EMT is an important phenotype that drives
chemoresistance in cancer cells, and inhibition of EMT might increase
sensitivity of cancer cells to chemotherapy [66]. EMT is regulated by
transcriptional factors such as snail and twist. It has been reported
that deletion of snail or twist in genetically engineered mouse model of
pancreatic ductal adenocarcinoma enhances the expression of

nucleoside transporters in tumors, contributing to increased sensitivity

to gemcitabine [67]. EMT in cancer cells contributes to production of
MMPs, and increased MMPs promote the pathogenic EMT process

[48]. TA inhibits EMT by down-regulating MMP-2 and MMP-9,

- 51 -
resulting in inhibition of migration and invasion of metastatic prostate

cancer cells [36]. In this study, TA suppressed MMP-2 and MMP-9 as

well as mesenchymal markers such as N-cadherin, snail, vimentin, and

twist in CT26/5-FU cells. Consequently, TA inhibited the migratory and

invasive ability of 5-FU-resistant CRC cells.

Molecular events underlying chemoresistance include EMT,

down-regulation of apoptosis and failure of drug delivery by facilitating

drug efflux through a variety of membrane transporters [68].
Overexpression of ABC transporters is one of the major causes of

chemoresistance, which can mediate the efflux of drugs from cancer

cells, thereby reducing intracellular accumulation of anti-cancer drugs
[69]. TA has a synergistic effect with 5-FU and mitomycin C by

decreasing the expression of ABCB1, ABCC1, and ABCC2 membrane

pumps involved in drug efflux and chemoresistance [70]. ABCB1,
ABCC1, and ABCG2 are clinically significant ABC transporters that
have been investigated with respect to their role in chemoresistance in
cancer cells [17]. In this study, TA down-regulated expressions of
ABC transporters such as ABCB1, ABCC1, and ABCG2. Further
experiments are needed to investigate the inhibitory effect of TA on
efflux activity of ABC transporters.
To improve the efficacy of anti-cancer therapy, TA has been

investigated in combination with chemotherapy with the expectation

that such combinations might enhance the cytotoxicity. Previous studies
have shown that TA sensitizes osteosarcoma [71], ovarian [35], and

liver [34] cancer cells to cisplatin. In addition, TA exhibits

- 52 -
chemo-sensitivity effects with doxorubicin and docetaxel in C4-2 and

PC-3 prostate cancer cells [37]. In this study, TA increased

cytotoxicity and improved sensitivity to 5-FU in 5-FU-resistant CRC

cells by modulating apoptosis compared to 5-FU alone treatment.

The inhibition effect of TA on the growth of the 5-FU-resistant

CRC cells implanted in mice was evaluated. The doses of TA

administered orally to mice were selected based on other studies. Oral

administration of TA (50 mg/kg) resulted in gastroprotective effect
against gastric ulcer in mice [72]. Another study found that TA

treatment at a dose of 75 mg/kg for 15 days did not alter the serum
AST and ALT levels, known markers of hepatocellular injury [73]. In
addition, as a result of analyzing AST, ALT, creatinine, and BUN

levels, it was considered that TA did not induce hepatotoxicity or

nephrotoxicity in mice orally administered 5 days a week for 4 weeks
(Table 3).
In conclusion, TA not only inhibited 5-FU-resistant CRC cells
by inducing apoptosis and cell cycle arrest, but also enhanced
sensitivity by suppressing apoptosis evasion, EMT, and ABC
transporters-mediated drug efflux. Therefore, TA could be used as a

potential therapeutic agent for the treatment of 5-FU-resistant CRC.

- 53 -
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