Discussion Paper 2017 - E001

DISCUSSION PAPER SERIESOsaka University Graduate School of Medicine
Department of Health Economics and Management

Title：The changes in metastatic potential of radioresistant cancer cell line generated byfrequent X-ray

Name：Kazumasa Minami, Yoshinosuke Hamada, Naomasa Kawaguchi, Seiji Mori,
Masashi Manabe, Kana Nakatani, Kento Tsubouchi, Naoki Hayashi, Yuhki Yokoyama,
Ayako Uchinaka, Hirofumi Yamamoto and Masahiko Koizumi

OHEM Secretariat
Graduate School of Medicine Department of Health Economics and Management
OSAKA UNIVERSITY
1-7 Yamadaoka, Suita, Osaka, 565-0871, Japan

March, 2017

Abstract
In general, cancer cells which survived from X-ray irradiation have acquired radioresistance.
These survived cells are cause of recurrence and/or metastasis.

The purpose of this study is toevaluate metastatic potential of the cancer cells survived from frequent X-ray irradiation.

Theadhesiveness in A549R was remarkably increased than in A549. The motility of A549R was
significantly suppressed than A549.

There was no significant difference in invasiveness.

Integrin β1 expression of A549R was more increased than A549.

In comparison with

non-irradiated each cell line, adhesiveness in both cell lines were most increased in 2 hours or 24hours.

The motility of A549R was most increased in 2 hours compared with non-irradiated celllines whereas motility of A549 was most increased in 24 hours.

As compared with

non-irradiated cell lines, invasiveness in both cell lines was significantly suppressed.
there was no prominent difference in temporal change in both cell lines.

However,

The adhesion ability of

A549R was more increased than A549 consistent with increased integrin β1 expression.

Our

study demonstrates that radio-resistant cancer cell line increase the adhesion capability consistent
with enhance the expression integrin β1

andsuppress the cell migration.

The change inradio-resistant cancer cell line takes place at the time adhesive capacity and cell migration was
most increased compared with parental cancer cell line after irradiation. When radio-resistantmechanism becomes clear, a new treatment policy is established. This leads shortening of thetreatment period, and, as a result, medical expenses are reduced.

In future, it is necessary toinvestigate the effective radiotherapy that consider the effect for health economics.

Keyword：X-ray, radioresistant cell, adhesion, motility, invasion, reduce the medical expense

The changes in metastatic potential of radioresistantcancer cell line generated by frequent X-ray
Kazumasa Minami1, 2, Yoshinosuke Hamada1, 3, 4,
Naomasa Kawaguchi1, Seiji Mori1, Masashi Manabe5, Kana Nakatani2,
Kento Tsubouchi2, Naoki Hayashi2, Yuhki Yokoyama1, Ayako Uchinaka6,
Hirofumi Yamamoto1 and Masahiko Koizumi2
1

Department of Functional Diagnostic Science, 2Department of Medical Physics and Engineering,

3

Department of Health Economics and Management, Osaka University Graduate School of Medicine

4

Department of Pediatric Dentistry, Osaka Dental University

5

The Faculty of Business Innovation, Kaetsu University

6

Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine

Abstract
In general, cancer cells which survived from X-ray irradiation have acquired radioresistance. These
survived cells are cause of recurrence and/or metastasis. The purpose of this study is to evaluate
metastatic potential of the cancer cells survived from frequent X-ray irradiation. The adhesiveness in
A549R was remarkably increased than in A549. The motility of A549R was significantly suppressedthan A549. There was no significant difference in invasiveness. Integrin β1 expression of A549R
was more increased than A549. In comparison with non-irradiated each cell line, adhesiveness inboth cell lines were most increased in 2 hours or 24 hours. The motility of A549R was mostincreased in 2 hours compared with non-irradiated cell lines whereas motility of A549 was mostincreased in 24 hours.

As compared with non-irradiated cell lines, invasiveness in both cell lines was

significantly suppressed. However, there was no prominent difference in temporal change in bothcell lines. The adhesion ability of A549R was more increased than A549 consistent with increased
integrin β1 expression. Our study demonstrates that radio-resistant cancer cell line increase theadhesion capability consistent with enhance the expression integrin β1

andsuppress the cell

migration. The change in radio-resistant cancer cell line takes place at the time adhesive capacity
and cell migration was most increased compared with parental cancer cell line after irradiation. Whenradio-resistant mechanism becomes clear, a new treatment policy is established. This leads shorteningof the treatment period, and, as a result, medical expenses are reduced.

In future, it is necessary toinvestigate the effective radiotherapy that consider the effect for health economics.
Key words: X-ray, radioresistant cell, adhesion, motility, invasion, reduce the medical expense

line is compared with parental cell line.

Introduction
Radiotherapy is an effective approach fortreatment of many types of cancers.

RecentMaterials and Methods

developments in radiotherapy technology, such1.

as intensity-modulated radiation therapy (IMRT)

A549 human lung adenocarcinoma cell andand three-dimensional (3D) radiotherapy, allow

A549R radio-resistant cancer cell line wereprecise dose escalation to the tumor.

maintained in DMEM medium (Wako, Osaka,

It hasCell culture

improved local control rates [1, 2].

Japan) with 10 % fetal bovine serum (FBS)

Non-small cell lung cancer (NSCLC) is one of(Biowest,

the most common causes of mortality in Japan,

penicillin/streptomycin (Nacalai Tesque, Kyoto,accounting for approximately 80 % of all lungJapan) at 37 °C in a humidified atmosphere ofcancer patients.

Radiotherapy is the most

5 % CO2 and 95 % air. The establish methods

commonly used treatment option for medicallyof radiation resistant cell line were described

inoperable NSCLC.

Many reviews havepreviously. Briefly, A549 was irradiated with 2

summarized report on early stage NSCLC afterGy/ fraction, 30 fractions with 4 MV X-ray overfractionated

Sibley6 months.

Therefore total dose 60 Gy wasreported that approximately 30 % of patientsdelivered.

After the treatment, the radio

died from distant metastasis [3].

-resistant clone A549R is isolated and cloned

radiotherapy[3-5].

This poor

France,Nuaillé)andprognosis is due to survive from radiotherapy.

from the irradiated subclone.

In addition, some studies have reported that

confirmed that the radiation sensitivity iscancersignificantly lower than that of A549.

cells

after

radioresistance [6, 7].

irradiationacquired

We have

There is a possibility

that the cancer cell survived from X-ray

2.

irradiation

radioresistance

A549 and A549R were cultured with 10 % fetal

promotes metastasis. Thus, poor prognosis inbovine serum and 0.025 % penicillin /NSCLC after treatment will be associated withstreptomycin containing DMEM in 5 % CO2 andthat cell. In the clinic, most of facilities adopt

95% air. The day before radiation treatment,

the

is

each cell was prepared to cell culture dishes. X

approximately 2 Gy × 30 fractions (total 60 Gy).

ray irradiation was performed with medical

Clarification of features of radioresistant cancer

linear accelerator (LINAC) at Osaka University

cell due to survive from radiation therapy is

Graduate School of Medicine with delivering

important to improve survival rate of cancer

dose rate of 1.0 Gy/min. For X-ray irradiation,

patients. We has established the radio-resistant

FBS free DMEM was added to form build-up

cell line named A549R which was irradiatedbefore irradiation.

andconventional

acquiredplanning,

which

Irradiation

with 2 Gy/fraction, total 60 Gy with 4 MV X-ray
over 6 months, and radioresistant cell line was3.

cloned.

A 96-well plastic plates were coated with 10

In this study, we investigate metastatic potential

μg/ml of collagen IV (BD, 354233, Mississauga,of radio-resistant cancer cell line and this cell

Canada), laminin (BD, 354232, San Jose, CA)

Cell Adhesion Assayfibronectin (BD, 354008, San Jose, CA) or

hematoxylin.

Cell migration was quantitatedvitronectin (BD, 354238, San Jose, CA) inby counting the number of stained withphosphate-buffered saline (PBS) (Gibco, New

hematoxylin in three random fields at 200 ×York, USA) for 2 h at 37°C and then treatedmagnification with a microscope.

with 3 % bovine serum albumin (BSA) for 1 h at
37°C, or were coated with only BSA for
4

5.

Matrigel invasion Assay

negative control. 2.5 × 10 cells in FBS free

Invasion of cancer cells was measured by the

DMEM containing 0.1 % BSA were added andinvasion of cells through transwell inserts withincubated for 2 h at 37°C. After removal of the

8-μm pores that were coated with Matrigel

medium, a 0.04% crystal violet solution was(Bectonadded and incubated for 10 min at room

Irradiated cells were trypsinized, washed twotemperature. The wells were washed three times

times

with PBS and 20 μL of dimethyl sulfoxide were

0.1 %BSA, and 200 μL of cell suspension

added for permeabilization. Distilled water was(5×105 cells/ml) were added to the upper well.

then added to 100 μL, and the number of

DMEM supplement with 700 μL of 10 % FBS

adherent cells was assessed with a microplate

as a chemoattractant was added to the lower well.

reader (measurement wavelength 550 nm and

The number of cells that had invaded to thereference wavelength 630 nm).

lower surface of the Matrigel-coated membrane

Dickinson,
with

DMEM

California,
supplemented

USA).
with

was counted in three random fields under a4.

microscope.

Boydenchamber Assay

The cells were collected by using trypsin-EDTA
in PBS and suspended with serum-free medium
containing 0.1 % BSA after the cells had been
washed with the same medium.

6.

Integrin

expression

analysisusingflowcytometery

The migratory

The cells were collected with Trypsin-EDTA.

activities of A549 and A549R cells throughThe cells were dissolved in 0.1 % FBS and8-μm pores were assessed. A filter was placed

0.03 % NaN3.

on a 24-well plate and coated with fibronectin

were added in the 96-well plate. The following

for 30 min at room temperature.

processes were done at 4℃.

DMEM

Three hundred thousand cells
0.1% FBS andsupplemented with 10 %FBS was used as a0.03 % NaN3 were used as the washing solution.

chemoattractant into the lower wells.

ThirtyAnti integrin β1 antibody (MAR4) (BD, 555442,

thousand of untreated or treated cells were

San Jose, CA) was diluted 1:100 with washingplated onto the filter membrane and the allowed

solution, and used as primary antibody. As thecells to migrate through the membrane at 37°C

control, washing solution of 100 μL was added.

in an atmosphere of 5 % CO2. After 3 hours, not

After 30 minutes, it was washed three times with

migrated cells were scraped off with a cotton

the solution.

swab, and the filter membrane was removed(DAKO, F0261, Copenhagen, Denmark) of thewith a blade. The cells that had migrated to theundiluted solution and 100 μL of washingbottom side of the membrane were fixed withsolution was added.

10

was used as the secondary antibody of β1.

%

formalin

and

then

stainedwith

Secondary anti-mouse IgG

Mouse immune serum

After 30 minutes, it was washed three times.

tendency as collagen IV was observed (Fig. 2B).

Flow Cytometry analysis was performed using

In comparison with non-irradiated each cell line,

FACS Calibur (BECTON DICKNSON Co.) andadhesion ability on laminin in A549 and A549R

integrin expression on the cell surface wascells was most increased by about 220 % andmeasured.

170 % in 2 hours after irradiation whereas on
vitronectin in both cells was most promoted by

7.

about 160 % in 24 hours after irradiation (Fig.

Statistics

The results were expressed as mean values with

2C, D).

standard deviations. The statistical significancewas tested by means of Student’s t-test. A

3.

p-value of less than 0.05 was considered to bestatistically significant.

Effectsofirradiationonmigration

capabilities
The results of the migration of both cells in 2, 6,
24 hours after irradiation are shown in Figure

Results

3A.

1.

Difference in metastatic potential between

significantly increased in 24 hours compared

A549 and A549R

with non-irradiated each cell line.

Celladhesion,

migration,

and

After

irradiation, both

cells

were

On the otherinvasion

hand, in comparison with non-irradiated eachcapabilities and integrinβ1 expression in A549

cell line, cell migration only in A549R wasand A549R are shown in Figure 1. Adhesion

remarkably promoted in 2 hours after irradiation.

ability to extracellular matrix in A549R was
significantly increased compared with A549 (Fig.

4.

1A). Cell migration in A549R was suppressed

The results of the invasion of both cells in 2, 6,

than A549 (Fig. 1B). There was not difference

24 hours after irradiation are shown in Figure 3B.

in cell invasion capability between both cells

Compared with non-irradiated each cell line,

Integrin β1 expression in A549R

after irradiation, invasiveness in both cells was(Fig. 1C).

Effects of irradiation on cell invasion

was promoted, compared with expression ofsignificantly suppressed.

However, when

integrin β1 in A549 (Fig. 1D).

compare the temporal variation of both cells,
there was no remarkable difference in the feature2.

Effects of irradiation on cell adhesionof the temporal variation of both cells.

capabilities
Cell adhesion ability of A549 and A549R toeach extracellular matrix in 2, 6, 12, 24 hours

5.

Effects of irradiation on expression of
integrin β1

after X-ray irradiated is shown in Figure 2.

Figure 4 has shown expression of integrinβ1 in

Adhesion capability to collagen IV in A549R

both cells in 2 and 24 hours after irradiation.

was most increased by about in 2 hours 160 %Integrinβ1

after X-ray irradiated whereas in A549 was mostincreased in 24 hours after irradiation. On the

promoted by about 210 % in 24 hours after

other hand, in comparison with non-irradiatedirradiation compared with non-irradiated each

each cell line, both cells irradiated in 2 hours

cell line (Fig. 2A). With fibronectin, the same

were slightly promoted.

expression

wasdramatically

: A549: A549R

Figure 1. Metastatic potential in both cells, A549 and A549R is examined by celladhesion assay (A), cell migration assay (B), matrigel invasion assay (C) and integrin β1
expression analysis using flow cytometry (D). Each arrows show below.
:
Negative control in A549.
: Negative control in A549R.
: The expressionof integrin β1 in A549.
: The expression of integrin β1 in A549R. **, p<0.01, *,p < 0.05 (Student’s t test, compared with A549).

Figure 2. Temporal changes of cell adhesion capability to (A) collagen IV, (B)fibronectin, (C) laminin and (D) vitronectin in A549 or A549R cells after 10 Gy
irradiation. The absorbance in each cell after irradiation was averaged and normalized tothe absorbance of non-treated each cell.
: A549R after irradiation.
: A549 after
irradiation.

Figure 3. The evaluation of cell migration (A) and cell invasion (B) capability in A549(
) or A549R () cells after 10 Gy irradiation. **, p < 0.01 (Student’s t test,compared with untreated cell line).

Figure 4. Expression of integrin β1 in A549 cells at 2 hours (A) and 12 hours (B) after 10
Gy irradiation. (C) and (D) show the expression of integrin β1 in A549R cells at 2 hours
and 12 hours after 10 Gy irradiation, respectively. Each arrows show below.
: Non-irradiation.
: 10 Gy irradiation.

Discussion

withThe aim of this study is to examine theRadiation-induced

metastatic potential of the radio-resistant cancer

capability in both cells in 24 hours aftercell line. First in our study, we performed all

irradiation

experiment under non-irradiated condition toradiation-induced

evaluate differences in metastatic potential

However, expression of integrin β1 in both cell

between both cells.

In order to reach distantlines did not change in 2 hours compared withorgans away from primary tumor site, tumor

each non-irradiated cell whereas adhesive

cells have to interact with extracellular matrix,

capability in both cell lines was significantly

and then enter the lymphatic capillary system or

promoted after in 2 hours.

blood flow [6]. Various factors are related toreported that the reason why increased adhesion

metastatic potentials.

Changes in integrin

capability after irradiation even though there

expression level are likely to affect cell

was hardly difference in expression of integrin

functions.

β1 in cancer cell after irradiation compared with

Integrin plays an important role tonon-irradiated

each

cell

increase

was

in

in

with

integrin

β1.

Tsutsumi et al.

regulate not only the adhesion to extracellular

non-irradiatedmatrix but also cell motility and invasion byradiation induces the localization of focal

controlling cell adhesion [7].

In comparison

adhesion molecules, including integrin β1 [11].

with A549, expression of integrinβ1 in A549R

Our results that cell adhesion in both cells in 2

was increased.

This result might cause

hours after irradiation was increased while

increase of adhesiveness in A549R. However,expression levels of integrin β1 did not change

cell invasiveness in A549R was not promoted

in both cells after irradiation might denote thecompared with A549. Gliemroth, et al reported

same tendency.

that compared with non-irradiated cell line,

Previous findings have suggested that ionizing

human glioma cell line which irradiated with 2

radiation promotes tumor migration, metastatic

Gy/fraction, total 60 Gy dramatically suppressed

and the invasiveness potential of cancer cells [10,cell12-14].

migration

and

did

not

changethewasadhesive

consistency

increase

cell

of

line.

becauseionizing

The results of present study showedinvasiveness [8]. These results were similar tothat irradiation remarkably promotes migration

ours.

in both cells in 24 hours, and cell migration in

Guo, et al has reported that radiation-induced

A549R alone significantly was increased in 2

increasebehours by irradiation. Jung et al. showed thatmodulated by radiation-induced increase inradiation-induced cell migration is due to be

integrin β1 expression [9]. In addition, Jung etactivated the p38 MAP kinase pathway and JNK

al. reported that irradiation promoted tumor cell

pathway in A549 cells in 30 minutes after

motility and focal adhesion formation, and these

irradiation [10]. These pathways are shown toevents already occur in 30 minutes after

be a prerequisite for promotion of cell migration

irradiation and are mediated by increased[15-17].

phosphorylation of FAK [10].

tendency.

ofadhesion

capacity

could

Our findings

Our study might show the same

showed that adhesiveness in both cell lines were

It is well established that the MMP family plays

most increased in 2 hours or 24 hours compareda key role in metastatic processes, especiallyMMP-2 [18-19].

Some studies showed that

Acknowledgments

increased cell invasion after irradiation is due toThis study was supported by Grants-in Aid forthe enhancement of MMP-2 activity by X-ray

Scientific Research (No.26290055) by the Japan

irradiation

Society for the Promotion of Science.

[11-13,20].

However,

wepreviously reported that cell invasiveness in
A549 after 10 Gy irradiation significantly was

Referencessuppressed [21].

More investigations are1) Onishi H, Araki T, et al. Stereotactic

needed to elucidate the relevant molecularhypofractionated high-dose irradiation formechanism behind the different response tostage I nonsmall cell lung carcinoma:ionizing radiation.

clinical outcomes in 245 subjects in a
Japanese multiinstitutional study. Cancer,
101: 1623–1631, 2004.

Importance of cost-saving aspects
It is a critical problem that medical expenses

2) Palazzi M, Orlandi E, et al. Furthere

increase more and more on cancer patients andimprovementinoutcomesoftheir families. The share of cancer care in thenasopharyngeal carcinoma with optimized

general practice costs is around 11%. Recently,

radiotherapy and induction plus concomitant

growth rate of cancer care cost is going up to

chemotherapy: an update of the Milan

42%, more than three times that of national

experience. Int. J. Radiat. Oncol. 74:

medical expenditure. Performing the radiation

774–780, 2009.

therapy efficiently is likely to suppress the3) Sibley GS. Radiotherapy for patients withcancer care cost growth, and improve the labor

medically inoperable StageI nonsmall cell

productivity:

lung carcinoma: smaller volumes and higher

QOL of patients is improved andtheir rehabilitation goes smoothly.

doses-a review. Cancer. 82: 433–438, 1998.

In summary, our study demonstrates that

4) Rowell NP, and Williams CJ. Radical

radio-resistant cancer cell line increase theradiotherapy for stage I/II non-small celladhesion capability consistent with enhance thelung cancer in patients not sufficiently fit for

expression integrin β1 and suppress the cellor declining surgery (medically inoperable):

migration. The change in radio-resistant cancera systematic review. Thorax. 56: 628-638,

cell line takes place at the time adhesive

2001.

capacity and cell migration was most increased5) Qiao X, Tullgren O, et al. The role ofcompared with parental cancer cell line afterradiotherapy

irradiation. When radio-resistant mechanism

non-small cell lung cancer. Lung Cancer. 41:

becomes clear, a new treatment policy is

1–11, 2003.

established. This is likely to shorten thetherebytreatment

of

stage

I

6) Benavente S, Huang S, et al. Modification oftreatment

period,medicalradiation and EGFR inhibitor response in

expenses.

In future, it will be necessary tohuman tumor cells following establishment

investigate

theof radiation resistance, Int. J. Radiat. Oncol.

effective

reduceinradiotherapy thatconsiders cost-saving aspects of the treatments.

Biol. Phys. 60: S350, 2004.
7) Xu Q, Gao Y, et al. Identification ofdifferential gene expression profiles ofpotential of human pancreatic cancer cells

radioresistantandestablished

lung
by

cancercell

fractionatedline

ionizing

radiation in vitro. Chin. Med. J. 18:
1830-1837, 2008.

its

blockadebyamatrix

metalloproteinase inhibitor, CGS27023. Clin.
Cancer Res. 8: 1223–1227, 2001.
16) Camphausen K, Moses MA, et al. Radiation

8) Tarin D, and Matsumura Y. Recent advances

therapy to a primary tumor accelerates

in the study of tumour invasion andmetastatic growth in mice. Cancer Res. 61:

metastasis. J. Clin. Pathol. 47: 385–390,

2207–2211, 2001.

1994.

17) Huang C, Jacobson K, and Schaller MD.

9) Guo W, and Giancotti FG. Integrin signaling
during tumor progression. Nat. Rev. Mol.
Cell Biol. 10: 816–826, 2004.
10) GliemrothJ,

MAP kinases and cell migration. J. Cell Sci.
117: 4619–4628, 2004.
18) Xia Y, and Karin M. The control of cell

Feyerabend

T,

et

al.

motility and epithelial morphogenesis by

Proliferation, migration, and invasion of

Jun kinases. Trends Cell Biol. 14: 94–101,

human glioma cells exposed to fractionated2004.

radiotherapy in vitro. Neurosurg Rev. 26:
198–205, 2003.

basal JNK activity differentially affects key11) Cordes N, Blaese M, et al. Ionizing radiationinduces

up-regulationoffunctional

β1-integrin in human lung cancer tumour
cell lines in vitro. Int. J. Radiat. Biol. 78:
347–357, 2002.

fibroblast functions important for woundhealing. J. Biol. Chem. 278: 24624–24628,
2003.
20) Chambers A, and Matrisian L. Changing
views of the role of matrix metalloproteinase12) Jung J. W, Hwang S. et al. Ionising radiationinduces19) Javelaud D, Laboureau J, et al. Disruption ofchanges

associatedwithin metastasis. J. Natl. Cancer Inst. 89:
1260-1270, 1997

epithelial-mesenchymal transdifferentiation

21) Deryugina EI, Luo GX, et al. Tumor celland increased cell motility of A549 lunginvasion through matorigel is regulated byepithelial

activated

cells.

Eur.

J.

Cancer.

43:

1214–1224, 2007.
Motility and Invasiveness in Tumor cells
that survive 10 Gy irradiation. Cell Struct.
Funct. 34: 89–96, 2009.
14) Wild-Bode C, Weller M, et al. Sublethal
promotes

migrationandinvasiveness of glioma cells: implications
for radiotherapy of human glioblastoma.
Cancer Res. 61: 2744-2750, 2001.
15) Qian

LW,

Mizumoto

Radiation-induced

metalloproteinase-2.

Anticancer Res. 17: 3201-3210, 1997.

13) Tsutsumi K, Tsuda M, et al. Increased

irradiation

matrixincrease

K,

et

al.

in

invasive