成蹊大学理工学研究報告
J. Fac. Sci. Tech., Seikei Univ.
Vol.43 No.２ (2006) pp.15-19（新任者論文）5-fluorouracil-inducible proteins in a colorectal cancer cell line
Hisashi HISATOMI*1, Shoichi TADO*2, Satsuki EGASHIRA*3
ABSTRACT : 5-fluorouracil (5-FU) is the most common chemotherapeutic agent used in the treatmentof colorectal cancer. To understand the intracellular changes induced by 5-FU, we attempted to identify
the specific proteins affected using a proteomics analysis. A human colorectal carcinoma cell line, WiDr,
was treated with 1.0 µM 5-FU. After 24-72 h, the cellular proteins were analyzed by two-dimensional
gel-electrophoresis in combination with ESI Q-TOF mass spectrometry. A differential display of theproteins using a computer-aided image analysis revealed several protein spots that varied in quantity after
5-FU exposure. The up-regulated proteins were heat shock protein (hsp) 70 protein 5, hsp60 protein 1
and thyroid hormone-binding protein precursor, while the down-regulated proteins were lactate
dehydrogenase (LDH)-A and gamma-synuclein. The distributions of these five protein varied widely, andincluded the cytoplasm, mitochondria, endoplasmic reticulum and plasma membrane. The results of thisstudy demonstrate that hsp70, hsp60, thyroid hormone-binding protein precursor, LDH-A andgamma-synuclein may represent candidates for diagnostic and prognostic markers. Furthermore, theresults demonstrate that the protein expression profile of colon cancer cells can be used to establish thepotential of this methodology as a means by which rational decisions regarding the choice of therapy canbe approached.
Keywords : Proteomics, 5-FU, colorectal cancer, ESI Q-TOF mass spectrometry（Received September 27, 2006）

1. Introduction

activation of DNA damage response pathways, also play key

Various types of malignant tumors, including colorectal

Identification of such pathways would greatly facilitate thecancer, have been treated by multiple chemotherapy, such as

development of new therapeutic strategies to improve thefluoropyrimidine 5-fluorouracil (5-FU) treatment. 5-FU is theefficacy of 5-FU-based chemotherapy. Recently, Maxwell et

most common chemotherapeutic agent used in the treatment

al. identified 5-FU-inducible target mRNAs, such as annexinof colorectal cancer. It is anabolized to the respective

II,

deoxynucleotide monophosphate, FdUMP, which competes

thymosin-beta-10, chaperonin-10 and MAT-8 [7]. However,with the normal metabolite, dUTP, as a potent inhibitor of

the changes in the intracellular environment after 5-FU

thymidylate synthase (TS) [1,2]. This enzyme governs the detreatment may not only involve the expression of mRNAs butroles in determining the cellular response to 5-FU.

spermine/spermidine

acetyltransferase(SSAT),

novo synthesis of thymidylate, and its inhibition results inalso the amounts of certain proteins. To identify thespecific depletion of dTTP. This depletion is associated withdownstream mediators of the tumor cell response to 5-FU, we

inhibition of DNA replication and the ribonucleosideused proteomics technology to identify affected proteins in

triphosphate of 5-FU, FUTP, may be incorporated into RNA,the WiDr colorectal cancer cell line after 5-FU treatment.

and affect the function of the transcripts [3]. In vitro and in

2. Methods

vivo studies have demonstrated that increased TS expression
is correlated with increased resistance to 5-FU [3-5]. It is

2.1. Cell Culture

likely that events downstream of TS inhibition, such as
1* : Associate Professor, Department of Materials and Life Science,
hisatomi@st.seikei.ac.jp*2 : Undergraduate Student, Department of Materials and Life Science*3 : Research Assistant, Department of Materials and Life Science−15−

The WiDr cell line was purchased from the Japan Health
Science Foundation (HSRRB, Osaka, Japan), and maintained

inDulbecco’s

modified

Eagle’smedium

2.3. Identification of proteins(D-MEM)

supplemented with heat-inactivated fetal bovine serum. After

Protein spots on stained 2-D electrophoresis gels wereculture for 48 h, the cells were treated with 1.0 µM 5-FU

excised and digested with 0.4% (w/v) trypsin overnight. The(Kyowa, Tokyo, Japan) for 0, 24, 48 or 72 h. All cells weredigested peptides were desalted and cleaned with ZipTipC18

grown in 5% CO2 at 37°C. Apoptosis was assessed using an

pipette tips (Millipore). Briefly, the ZipTipC18 pipette tips werein situ cell death detection kit (Roche Molecular Biochemicals

equilibrated in 0.1% trifluoroacetic acid (TFA), and the

Diagnostic, IN, USA) (Fig.1). Milli-Q water (Millipore, MA,digested peptides were then bound to the tips by pipetting tenUSA) was used for all solutions.

times. The bound peptides were washed with 0.1% TFA, andeluted with 0.1% TFA and 50% acetonitrile. The eluted

2.2. 2-D electrophoresis

peptides were determined using an electrospray ionization

Two-dimensional (2-D) polyacrylamide gel electrophoresis(ESI)-quadrupole-time of flight (Q-TOF) system (Q-TOF(PAGE) analysis was performed using a previously described

Ultima™ API mass spectrometer; Waters, Milford, MA,

standard protocol [8]. Briefly, proteins from untreated cells

USA). Database searches were carried out using the Mascot(control) or cells after treatment with 1.0 µM 5-FU for 24, 48

Search engine in Matrix Scienceor 72 h were extracted with 8.5 M urea, 2% Triton X-100,(http://www.matrixscience.com/) (Fig. 3).

0.1% sodium dodecyl sulfate, 1.2% DeStreak™ reagent

3. Results(Amersham Biosciences Corp., NJ, USA), 1% phosphataseinhibitor cocktail 1&2 (Sigma, MO, USA), 1/10 tablet/mL
protease inhibitor cocktail tablets, complete mini (Roche

Remarkable apoptosis was not observed at any of the time

Molecular Biochemicals Diagnostic) and 2% pharmalyte™

points (Fig. 1). The protein expression profiles of the treated

pH 3-10 (Amersham Biosciences). The suspension wasand untreated populations are shown in Figure 2. We foundsonicated for approximately 30 sec and then centrifuged atthree proteins that were up-regulated by >2-fold, and two

10000 xg for 30 min to sediment any undissolved material.

proteins that were down-regulated by >2-fold (Table 1). The

The protein concentration was approximately 50 mg/mL after

up-regulated proteins were heat shock protein (hsp) 70 protein

filtration through an Ultrafree UFV5BGC25 filter (Millipore).

5 (BiP), hsp60 protein 1 and thyroid hormone-binding protein

Samples containing approximately 0.5 mg were applied toprecursor, while the down-regulated proteins were lactate

immobilized pH 3-10 linear gel strips (Bio-Rad, Hercules, CA,dehydrogenase (LDH)-A and gamma-synuclein. ThyroidUSA) in a rehydration buffer (6 M urea, 2 M thiourea, 2%hormone-binding protein precursor and LDH-A were

Triton X-100, 1.2% DeStreak™ reagent, 1% pharmalyte™

cytoplasmic proteins, hsp60 protein 1 was a mitochondrial

pH 3-10, 6 mM Orange G). The first dimension of isoelectric

protein, hsp70 protein 5 was distributed in the endoplasmic

focusing (IEF) was carried out using a Protean IEF Cell

reticulum,System (Bio-Rad). Electrofocusing was performed at 250 V

cytoplasm and plasma membrane, indicating that thefor 1 h, followed by a linear increase in voltage from 250 V todistributions of the up- and down-regulated proteins varied

10000 V within 6 h, and then at 10000 V for 6 h. The 2-DAandgamma-synuclein

wasdistributed

theB

separation was performed in vertical 7.5%T polyacrylamidegels (Anatech, Tokyo, Japan) in a Tris/Tricine buffer using a
CoolPhoreStar Tetra-200 vertical slab gel electrophoresis
apparatus (Anatech) under a constant current of 20 mA/gel.
After electrophoresis, the gels were stained using a SYPRO
Ruby protein gel stain kit (Invitrogen Corp., Carlsbad, CA,USA), and scanned in a FluoroPhoreStar 3000 blue diodeFig. 1. Apoptosis in the WiDr cell line.

scanner (Anatech). Noise reduction, background subtraction,

A: Untreated WiDr cells (0 h) and WiDr cells after treatment
with 1.0 µM 5-FU for 24, 48 and 72 h. No differences areobserved among the cells. B: Apoptosis in 5-FU-treated WiDrcells. Remarkable apoptosis is not observed.

spot detection, quantification, gel-to-gel matching anddifferential analysis were carried out using PDQuest softwarever. 7.1 (Bio-Rad) (Fig. 2).

−16−

widely. The possibility that the changes in the identified

since triplicate experiments based on independent cell

proteins could have resulted from artifacts was excluded,

extractions yielded consistent results.

HSP70 protein 5 (BiP)
pH5.03 72071Da

HSP60 protein 1
pH5.70 60986Da

Thyroid hormone binding
protein precursor
pH4.82 57069Da

Lactatedehydrogenase A
pH8.44 36665Da

gamma-Synuclein
pH4.97 13293Da

pH 3

10Fig. 2. Protein expression map of the WiDr cell line.

Spot detection, quantification and gel-to-gel matching were carried out using the PDQuest software. The newly identified spots were
excised, digested with trypsin and subjected to ESI Q-TOF mass spectrometry.

4. Discussion

post-transcriptional expression profiles of cancer cell lines andtumors have begun to identify genes that may be associatedProteomics analysis usually employs the separation of awith a response or resistance to these anticancer agents [9,10].

protein mixture by 2-D electrophoresis and the identification ofIn the present study, we used such an approach to identify

the separated proteins by mass spectrometry. The major

proteins up- or down-regulated in WiDr colorectal cancer cells

advantage of 2-D electrophoresis is that it enables theafter 5-FU treatment. We found three proteins that weresimultaneous separation and visualization of thousands of

up-regulated

unknown proteins in different modification states. No otherdown-regulated by>2-fold after 5-FU treatment. Hsp represents

method can achieve this at the present time. The assessment ofa complex family of proteins exerting chaperone-like activities

protein expression profiles by 2-D electrophoresis afterthat are classified according to their molecular weight [11-13].

treatment with chemotherapeutic agents has the potential to

The up-regulated hsp60 protein 1 is a member of the hsp family

identify novel signaling pathways involved in mediating theand has been attributed numerous roles, including regulation ofdownstream responses to these therapies, and could greatly

DNA synthesis, cell proliferation and apoptosis [13,14].

facilitate the discovery of novel potential therapeutic targets

Hsp60 protein 1 binds hsp10 (chaperonin-10) to regulate theand/or markers of chemoresistance. Studies examining thefolding of mitochondrial proteins [15]. Hsp10 mRNA was−17−by>2-fold,andtwoproteins

thatwereA

Galetto et al. also reported that hsp70 was up-regulated in
granulocyte macrophages after gamma-radiation and 5-FU
treatment [18]. Gamma-synuclein was previously identified asa breast cancer-specific gene [19], and Jiang et al.
demonstrated that it had a chaperone activity in the hsp-based
multiprotein chaperone complex for stimulation of estrogen
receptor (ER)-alpha signaling [20,21]. The gamma-synucleinmediated stimulation of ER-alpha transcriptional activity isB

consistent with its stimulation of mammary tumorigenesis inresponse to estrogen. The anti-proliferative effects of tamoxifen
plus

5-FUonKATOIII (poorlydifferentiatedgastricadenocarcinoma) cells were not dependent on ER-alphaexpression [21]. Gamma-synuclein is thought to have many
functions as an hsp besides its participation with ER-alpha. No
previous reports regarding any relationship between thyroid
hormone-binding protein and 5-FU or hsp were found, and wetherefore have no explanation for this at the present time.
Using a cDNA microarray analysis, Maxwell et al. identified
5-FU-inducible target mRNAs, including SSAT, annexin II,Fig. 3. Protein identification results from the mass spectrum
obtained in a Q-TOF Ultima™ API mass spectrometer anda database search on the Mascot Server.
A: Mass spectrum obtained after in-gel digestion and ESI
Q-TOF analysis of one of the identified proteins, hsp60 protein
1. B: Probability-based Mowse Score on the Mascot Server.
The identified peptides and their molecular masses are reported
using a root mean square (RMS) error graph. The amino acidsequence “VTDALNATR” corresponds to amino acids
421–429 of hsp60 protein 1. There are three oxidation peptides
and a carbamidomethyl peptide among the identified peptides.

thymosin-beta-10, chaperonin-10 and MAT-8 [8]. They used 10µM 5-FU for their transcriptional study, which was 10-fold theconcentration used in the current study. Therefore, we areunable to validate the 5-FU-inducible genes they identified.
However, hsp10, which was identified as an up-regulated geneby Maxwell et al. in their cDNA microarray analysis, binds
hsp60 protein 1, which was identified as an up-regulated
protein in this study. It is considered that only a few proteinchanges were observed in the present study because theconsistently up-regulated in the MCF7 breast cancer cell line

sensitivity of WiDr cells to 5-FU is comparatively low.

after treatment with 10 µM 5-FU [7]. In addition, it is known

The results in the current study demonstrate the potential ofthat hsp70 can control the decomposition of LDH [16,17], and2-D electrophoresis to identify novel genes involved inboth the up-regulation of hsp70 protein 5 and down-regulation

mediating the response of tumor cells to chemotherapy. Sinceof LDH-A we identified will be related to this phenomenon.

there are five candidate proteins that appear to be involved inTable 1. Functional grouping of the proteins identified by 2-D electrophoresis as being up- or down-regulated in the WiDr cellline after 5-FU treatment.
Regulation Fold induction
Protein nameUp2.53
HSP 70 protein 5 (BiP)Up
2.22
HSP60 protein 1UpDown

2.00
2.12

Thyroid hormone binding protein precursor
LDH-A

Down

2.06

Gamma-synuclein

Functional classifications
Heat shock protein
Heat shock protein

Structial classifications
Endoplasmic reticulum
Mitochondrial protein

HormoneSimple carbohydrate metabolism

Cytoplasmic protein
Cytoplasmic protein

Heat shock protein

Plasma membrane, cytoplasmic protein−18−

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