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의학박사 학위논문

Radiation-induced esophagitis in vivo

and in vitro reveals that epidermal

growth factor (EGF) is a potential

candidate for therapeutic intervention

strategy

방사선유발 식도염의 in vivo, in vitro

모델에서 표피성장인자(epidermal

growth factor)의 효능 연구

2016 년 8 월

서울대학교 대학원

의학과 방사선종양학과

김경수

i

Abstract

Radiation-induced esophagitis in vivo and in vitro

reveals that epidermal growth factor (EGF) is a

potential candidate for therapeutic intervention

strategy

Kyung Su Kim

Department of Radiation Oncology, School of Medicine

The Graduate School

Seoul National University

Purpose: To establish and characterize radiation-induced esophagitis (RIE) in

vivo and in vitro.

Materials and Methods: Fractionated thoracic irradiation at 0, 8, 12, or 15

Gy was given daily for 5 days to Balb/c or C57Bl/6 mice. Changes in the

body weight gain and daily food intake were assessed. At the end of the study,

we harvested the esophagus and examined: (i) histology by H&E staining; (ii)

immune cell infiltration and apoptosis by Fluorescent activated cell sorting

(FACS); (iii) gene expression changes by qRT-PCR. Het-1A human

esophageal epithelial cells were irradiated at 6 Gy, treated with recombinant

human growth factors, and examined for gene expression changes, apoptosis,

proliferation, and signal transduction pathways.

ii

Results: We observed that irradiation at 12 Gy or 15 Gy per fraction

produced a significant body weight reduction and decreased food intake in

Balb/c mice, but not so much in C57Bl/6 mice. Further analyses of Balb/c

mice irradiated at 12 Gy/fraction revealed an attenuated epithelium, inflamed

mucosa, and increased number of infiltrating CD4+ helper T cells and

apoptotic cells. Moreover we found that expressions of tissue inhibitor for

metalloproteinase-1, plasminogen activator inhibitor-1, granulocyte

macrophage-colony stimulating factor, vascular endothelial growth factor,

and stromal-derived factor-1 were increased while epidermal growth factor

(Egf) was decreased. Irradiated Het-1A cells similarly showed a significant

decrease in EGF and connective tissue growth factor (CTGF) expression.

Treatment of EGF but not CTGF partially protected Het-1A cells from

radiation-induced apoptosis and revealed phosphorylation of EGFR, AKT and

ERK signaling pathways.

Conclusions: We established a mouse model of RIE in Balb/c mice with 12

Gy × 5 fractions, which exhibited reduced body weight gain, food intake, and

histopathologic features similar to human esophagitis. Decreased EGF

expression in the irradiated esophagus suggests that EGF may be a potential

therapeutic intervention strategy to treat RIE.

----------------------------------------------------------------------------------------------

Keywords: Radiation-induced esophagitis, mouse model, epidermal growth

factor

Student Number: 2013-21664

iii

Table of Contents

Abstract …………………………………………………… i

Table of contents …………………………………………. iii

List of figures …………………………………………….. iv

Introduction ……………………………………………….. 1

Materials and Methods ……………………………………. 4

Results ……………………………………………..………10

Discussion ………………………………………..………. 24

References ………………………………………..………. 30

iv

List of Figures

Figure 1. Thoracic irradiation of mice with lead block ….…….. 4

Figure 2. Radiation-induced esophagitis (RIE) in Balb/c or

C57bl/6 mice…………………………………………11

Figure 3. Histological examination of the esophagus from Balb/c

mice………………………………………………….. 12

Figure 4. Examination of infiltrated leukocytes and apoptotic

cells in RIE in Balb/c mice………………………….. 13

Figure 5. Gene expression changes in RIE in Balb/c mice ……15

Figure 6. Epidermal growth factor (EGF) treatment can partially

protect Het-1A human esophageal cell line from

radiation- induced apoptosis ………………………... 17

Figure 7. Fluorescent activated cell sorting (FACS) (A, B) and

cell cycle analysis (C,D,E,F) of Het-1A with or without

irradiation treated with EGF or keratinocyte growth

factor (KGF) or connective tissue growth factor

(CTFG)………………………………………………. 21

Figure 8. FACS and cell cycle analysis of Het-1A with or without

irradiation treated with EGF or KGF or CTFG

v

(A,B,C,D) and western blot of Het-1A cells treated with

EGF (E)………………………………………………22

１

Introduction

Concurrent chemo-radiation has been established as a standard of care in

the management of locally advanced lung cancer [1]. However, severe

toxicity such as radiation-induced esophagitis (RIE) often cause a significant

problem in the clinic limiting dose escalation protocols due to dehydration,

esophageal ulceration, and requirement for treatment interruptions [2]. RIE

peaks by week 3 or 4 in the course of radiotherapy and current therapeutic

options are limited to conservative management with analgesics to manage the

pain [3,4].

There are several agents that have been reported to ameliorate RIE in

clinical and preclinical settings. For example, amifostine, a phosphorothioate

is a radio-protector currently in clinical use whose mechanism of action

involves in free radical scavenging through sulfhydryl moiety [5].

Preferential normal tissue radioprotection of amifostine occurs by: cellular

alkaline phosphatase (which dephosphorylates the compound thereby making

it freely diffusible into cells) concentrations often being higher in normal

tissues than tumors; an increased uptake in certain normal tissues such as the

salivary glands and kidneys [6]; or inducing normal tissue hypoxia through an

increased oxygen use [7]. However, a recent RTOG 98-01 trial has reported

rather disappointing results such that amifostine lacked the efficacy in

reducing RIE [8,9]. Other agents that have been shown some benefits in RIE

include: manganese superoxide dismutase-plasmid liposome (MnSOD-PL), a

gene therapy resulting in MnSOD acting to decrease the availability of

２

superoxide thereby the production of a lethal radical peroxynitrite in tissues

[10-12]; glutamine [13], which supplements glutathione, a radical scavenger

in cells hence exerting radioprotection; and recombinant human granulocyte

macrophage colony-stimulating factor (rhGM-CSF) [14], which has shown to

exert a potent angiogenic effect on the damaged epithelium of irradiated

esophagus.

Recently epidermal growth factor (EGF), a potent mitogen for epithelial

cells maintaining the tissue homeostasis by regulating epithelial cell

proliferation and growth [15] has shown a potential benefit for radiation-

induced oral mucositis in head and neck cancer patients receiving

radiotherapy [16] and for radiation-induced intestinal mucositis in mice [17].

In this study, we hypothesized that EGF would exert a protective effect

towards RIE thereby offering a possibility as a therapeutic intervention. In

order to test our hypothesis, we felt the need of animal models that can

faithfully mimic patients’ symptoms of RIE. Although several preclinical

studies have previously reported RIE in mice [11,18,19], these studies widely

differed in the radiation doses and fractionation schemes. For example, Kim

and colleagues used either 29 Gy × 1 fraction or 11.5 Gy × 4 fractions [18]

while other studies have used 10 ~ 37 Gy single fraction [19-21] of thoracic

irradiation in mice. End-points examining RIE also differed widely such that

some studies examined the histology of irradiated esophagus [20] while other

studies reported the survival of animals [18,19]. Given that RIE peaks by

week 3 or 4 during the course of ‘fractionated’ radiotherapy and that affected

patients’ symptoms include the weight loss, difficulty in swallowing, pain, and

３

inflammation, we hereby established a mouse model for RIE reflecting more

clinically relevant radiotherapy regimen and affected patients’ symptoms.

Hence we hereby report esophagitis model in vivo and in vitro and that EGF

can partially protect cells from radiation-induced apoptosis.

４

Materials and Methods

Mice and in vivo irradiation

Thoracic irradiation at 0, 8, 12, or 15 Gy was given daily for 5 days by

using 320 kV X-ray irradiator X-RAD 320 (320 keV, 12.5 mA at a dose rate

of 1.1 Gy/min using 4 mm Cu filter; Precision X-ray, North Brandford,

Connecticut) to 6 week-old male Balb/c or C57Bl/6 mice that had been

anesthetized mice by ketamine and xylazine cocktail (Fig 1). All animal

experiments were conducted under the guidelines approved by the institution

of Animal Care and Use Committee at Seoul National University College of

Medicine and Pohang University of Science and Technology.

Figure 1. Thoracic irradiation of mice with lead block.

Food intake and weight gain measurements

Body weight was measured every day from the first day of irradiation

(d1) up to day 12 (d12) for Balb/c mice and day 14 (d14) for C57Bl/6 mice.

Once mice reached 20 % body weight reduction, they were sacrificed by CO2

５

asphyxiation. Daily food intake was measured by harvesting the food on the

grill of a cage in every 2~3 days over 12 days thereby calculating daily food

intake per mouse. In some experiments, food intake was measured daily as

indicated in the graph.

Flow cytometry analysis

Immune cell infiltration and cell apoptosis of the esophagus were

determined by flow cytometry analysis (BD LSR II; BD Biosciences, CA).

Esophagus harvested from animals were digested mechanically with surgical

scissors, followed by enzymatic digestion using an enzyme cocktail

containing the mixture of collagenase (Collagenase Type 1; Worthington, NJ),

protease (Pronase; Calbiochem, CA), and DNase (Sigma, Missouri). Digested

esophagus in single cell suspensions were centrifuged for 5 min in 441 g at

4°C. Cells were then resuspended with lysis buffer (Pharm lyse; BD

Biosciences, CA), and filtered through a filter-top tube, followed by an

incubation for 10 min at 4°C to lyse the red blood cells. Cells were finally

washed in PBS containing 2 % fetal bovine serum, and applied with following

antibodies: biotin-CD4 (GK 1.5; ebioscience, CA, USA), FITC-conjugated

CD45 (30-F11; ebioscience), APC-conjugated F4/80 (BM 8; ebioscience),

PE-Cy7-conjugated anti-CD11b (M1/70; ebioscience). Secondary antibodies

used were streptavidin-pacific blue (ebioscience). To detect apoptotic cells,

cells were incubated with FITC Annexin V and propidium iodide provided by

the manufacturer (Apoptosis Detection Kit with PI; BioLegend, CA).

６

Cell culture and in vitro irradiation

Human esophageal Het-1A cells were purchased from American Type

Culture Collection (ATCC, Rockville, MD), grown in bronchial epithelial cell

growth medium (BEGM BulletKit, Lonza, MD), and harvested by using

triPLE (Gibco, Auckland, NZ) for passaging or preparation of cells for further

analyses. For in vitro irradiation, Het-1A cells were irradiated at 6 Gy by

using Cs irradiator, dose rate at 3.6 Gy/min. Het-1A cells were incubated with

recombinant human EGF (Peprotech, Rocky hill, CT), recombinant human

connective tissue growth factor (CTGF; Peprotech), or recombinant human

Keratinocyte Growth Factor (KGF; Peprotech) at 100 ng/ml for 48 hr.

Western blot

Cells were lysed in modified radio-immunoprecipitation assay (RIPA)

buffer containing 1 mM EDTA, 0.1% SDS, 10 mM Tris, 100 mM sodium

chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, and

protease & phosphatase inhibitor cocktail (Halt™; Thermo, Rockford, IL).

Protein lysates were quantified using BCA protein assay reagent (Pierce™;

Thermo) and 100 μg of the proteins were loaded onto 12% Bis-Tris SDS

NuPAGE gel (Novex; Life technology, Carlsbad, CA). Gel was subjected to

electrophoresis and blotted onto PVDF membrane (0.2 μm; Bio-rad, Hercules,

CA). Primary antibodies were: monoclonal antibodies against Akt (Abcam,

Cambridge Science Park, UK), phosphorylated Akt (Abcam), EGFR (Cell

Signaling Technology, Danvers, MA), ERK1 (BD, San Joes, CA),

phosphorylated ERK1/2 (Cell Signaling Technology), and β-actin (MP

７

biomedicals, Santa Ana, CA); polyclonal antibodies against phosphorylated

EGFR (Thermo).

qRT-PCR

Esophagus were harvested from mice, snap frozen, pulverized to powder

form by using liquid nitrogen. Het-1A cells were harvested and prepared as

pellets by centrifugation. Powdered esophagus or cell pellets were dissolved

in Trizol (Invitrogen) to extract mRNA, followed by synthesis of cDNA using

the following reagents: RNase-free DNase I (Promega, Madison, WI),

SUPERasein (Ambion, Waltham, MA), EDTA (Bioneer, Alameda, CA), dNTP

(Bioneer), random primers (Invitrogen), and GoScriptTM

Reverse

Transcriptase (Promega). Synthesized cDNA was then subjected to PCR

amplification using SYBR GREEN (Applied Biosystems) with the primers

shown in supplemental data. mRNA levels were calculated by relative

quantification methods using comparative threshold cycle (CT) values based

on those of beta-actin according to the manufacturer’s instructions (Applied

Biosystems).

qRT-PCR primer sequences:

Primer sequences used for qRT-PCR analysis were: β-actin Fwd 5‘- CGA

GCG TGG CTA CAG CTT CA, β-actin Rev 3‘- AGG AAG AGG ATG CGG

CAG TG, Timp 1 Fwd 5‘- TAT CCG GTA CGC CTA CAC CC, Timp 1 Rev

3‘- TGG GCA TAT CCA CAG AGG CT, Tgf-β Fwd 5‘- TGG AGC AAC ATG

TGG AAC TC, Tgf-β Rev 3‘- CAG CCG GTT ACC AAG, Pai-1 Fwd 5‘-

８

CTC TCT CTG CCC TCA CCA AC, Pai-1 Rev 3‘- GTG GAG AGG CTC

TTG GTC TG, Gm-csf Fwd 5‘- AGA TAT TCG AGC AGG GTC TAC, Gm-csf

Rev 3‘- GGG ATA TCA GTC AGA AAG GTT, Egf Fwd 5‘- AGC ATC TCT

CGG ATT GAC CCA, Egf Rev 3‘- CCT GTC CCG TTA AGG AAA ACT CT,

Vegf Fwd 5‘- CCA CGT CAG AGA GCA ACA TCA, Vegf Rev 3‘- TCA TTC

TCT CTA TGT GCT GGC TTT, Sdf-1 Fwd 5‘- GCT CTG CAT CAG TGA

CGG TA, Sdf-1 Rev 3‘- TAA TTT CGG GTC AAT GCA CA, Il-1β Fwd 5‘-

GCC TCG TGC TGT CGG ACC, Il-1β Rev 3‘- TGT CGT TGC TTG GTT

CTC CTT G, Egfr Fwd 5‘- TGG CCT TTA AGG GGG ATT CT, Egfr Rev 3‘-

CAG CCC CAG TGA TGT GAT GT, Ctgf Fwd 5‘- AGC CTC AAA CTC

CAA ACA CC, and Ctgf Rev 3‘- CAA CAG GGA TTT GAC CAC. β-ACTIN

Fwd 5‘-GGA CTT CGA GCA AGA GAT GG, β-ACTIN Rev 3‘-AGC ACT

GTG TTG GCG TAC AG, TIMP 1 Fwd 5‘-AAT CGT GGG CTT GTT TCT

TG, TIMP 1 Rev 3‘-CAC ACG GTA TTG GAC ACA GG, TGF-β Fwd 5‘-

GGG ACT ATC CAC CTG CAA GA, TGF-β Rev 3‘-CCT CCT TGG CGT

AGT AGT CG, PAI-1 Fwd 5‘-CTC TCT CTG CCC TCA CCA AC, PAI-1 Rev

3‘-GTG GAG AGG CTC TTG GTC TG, EGF Fwd 5‘-CAG GGA AGA TGA

CCA CCA CT, EGF Rev 3‘-CAG TTC CCA CCA CTT CAG GT, EGFR Fwd

5‘-CAG CGC TAC CTT GTC ATT CA, EGFR Rev 3‘-AGC TTT GCA GCC

CAT TTC TA, CTGF Fwd 5‘-GTT CCA AGA CCT GTG GGA TG, and

CTGF Rev 3‘-TGG AGA TTT TGG GAG TAC GG.

Statistical analysis

Statistical comparisons of the datasets were performed by unpaired

９

unpaired, two-tailed Student’s t-test or ANOVA with Tukey’s post-test using

Prism software (version 4.00; GraphPad Inc). The data were considered to be

statistically significant when P < 0.05.

１０

Results

Balb/c mice are more susceptible to radiation-induced esophagitis (RIE)

To establish a mouse model for radiation-induced esophagitis (RIE), we

irradiated the whole chest of animals by delivering 5 fractionated doses of 0, 8,

12, or 15 Gy/fraction. Upon dissection of mice, we observed that the

esophagus was thickened in Balb/c mice by d12 (Fig. 2A) and C57Bl/6 mice

by d14 (Fig. 2B) irradiate at 12 or 15 Gy/fraction. The thickening of the

esophagus occurred in 4 out of 4 (4/4) Balb/c and 3/3 C57Bl/6 mice irradiated

at 15 Gy/fraction; 3/4 Balb/c and 2/3 C57Bl/6 mice irradiated at 12

Gy/fraction. Changes in the body weight (Fig. 2C & 2D) and food intake (Fig.

2E & 2F) following irradiation were more pronounced and dose-dependent for

Balb/c mice (Fig. 2A, 2C, and 2E) than those for C57Bl/6 mice (Fig. 2B, 2D,

and 2F). Overall, these results suggest that Balb/c mice are more susceptible

to RIE and that 12 Gy/fraction seems to be the best regimen to induce RIE in

this strain. Hence we further examined this RIE animal model in more details

in the following studies.

Histological examination of the esophagus from Balb/c mice irradiated at

12 Gy/fraction at d12 post-radiation revealed an attenuated epithelium and

increased inflammatory cell infiltration in the mucosa and muscle layers (Fig.

3). We also observed vacuole formation in the irradiated esophagus (Fig. 3),

the result consistent with previous studies by other investigators, which have

shown to reflect individual cell degeneration [10,22].

１１

Figure 2. Radiation-induced esophagitis (RIE) in Balb/c or C57bl/6 mice.

Gross morphology of irradiated esophagus (A and B), body weight change (C

１２

and D), and food intake (E and F) of Balb/c (A, C, and E) and C57Bl/6 (B, D,

and F) mice. Arrows in C and D indicate irradiation regimen. * and ** in E

and F indicate P < 0.05 and 0.01, respectively as determined by one-way

ANOVA. The data are the mean ± s.e.m. (n ≥ 5 per group).

Figure 3. Histological examination of the esophagus from Balb/c mice. Non-

irradiated (A) or irradiated at fractionated dose of 12 Gy (B and C) esophagus

in Balb/c mice. Note that irradiated esophagus exhibits an attenuated

epithelium (B), infiltrative leukocytes (indicated with red arrows in C), and

vacuole formation (indicated with black arrows in B).

１３

Increased number of CD4 T cell infiltration and apoptotic cells in RIE

We next examined infiltrated immune cells in the irradiated esophagus

by FACS analysis. We observed that CD45+ leukocyte numbers were similar

between unirradiated (0 Gy) and irradiated (12 Gy) esophagus (Fig. 4A),

while CD4+ helper T cells were significantly increased in the irradiated

esophagus (Fig. 4A). F4/80+CD11b+ macrophages were significantly

decreased in the irradiated esophagus (Fig. 4B) while Gr-1+CD11b+

inflammatory granulocytes were similar between the two groups (Fig. 4C).

Propidium iodide and Annexin V-double positive apoptotic cells were

significantly higher in the irradiated esophagus compared to those in

unirradiated esophagus (Fig. 4D).

１４

Figure 4. Examination of infiltrated leukocytes and apoptotic cells in RIE in

Balb/c mice. FACS analyses of irradiated esophagus at 0 Gy or 12 Gy per

fraction harvested at d12 post-radiation. A, CD45+ leukocytes (gated with

red boxes in the FACS plots on the right) or CD45+CD4+ helper T cells

(gated with blue boxes in the FACS plots on the right); B, F4/80+CD11b+

macrophages (gated with red boxes in the FACS plots on the right); C, Gr-

1+CD11b+ inflammatory granulocytes (gated with red boxes in the FACS

plots on the right); D, Annexin V+ Propidium Iodide+ apoptotic cells. Data

are the mean ± s.e.m. (n ≥ 5 per group). * indicates P < 0.05 analyzed by two-

tailed, unpaired student’s t-test.

１５

Reduced EGF gene expression in the irradiated esophagus in vivo and in

vitro

In order to seek for a molecular target for RIE intervention, we decided

to examine gene expression changes in the irradiated esophagus. To do this,

we first repeated an experiment similar to Fig. 2 and measured the daily food

intake. We observed a significant reduction in the body weight and daily food

intake in animals irradiated at 12 Gy per fraction (Fig. 5A), consistent with

our earlier results (Fig. 2). We then harvested the esophagus at d5 (i.e.,

immediately following the last dose of fractionated irradiation) or d11 post-

radiation (i.e., at the lethal level of body weight reduction), and performed

qRT-PCR analyses against genes involved in extracellular matrix remodeling

(tissue inhibitor of metalloproteinase 1 (Timp1); plasminogen activator

inhibitor-1 (Pai-1); transforming growth factor-beta (Tgf-β), growth factor

signaling (granulocyte macrophage-colony stimulating factor (Gm-csf);

vascular endothelial growth factor (Vegf); epidermal growth factor (Egf);

epidermal growth factor receptor (Egfr); connective tissue growth factor

(Ctgf)), and inflammatory cytokines (stromal-derived factor-1 (Sdf-1);

interleukin-1 beta (Il-1β)). We observed that at d5, Timp1 and Egf gene

expression was significantly upregulated while Gm-csf gene expression was

decreased in the irradiated esophagus (Fig. 5B). At d12, we observed that

Timp-1 was increased by approximately 130 times and other genes including

Pai-1, Gm-csf, Vegf, Sdf-1, and Il-1β were also increased in the irradiated

esophagus (Fig. 5C). Egf, Egfr, and Ctgf gene expressions were decreased in

the irradiated esophagus compared to unirradiated control esophagus (Fig. 5C).

１６

Gene expression changes in the irradiated esophagus at d12 were not

significantly different compared to those of unirradiated esophagus, perhaps

due to high inter-individual variability in mRNA levels observed among 5

mice per group that had been analyzed.

Figure 5. Gene expression changes in RIE in Balb/c mice. A, Body weight

changes (left) and food intake measured every day (right) in Balb/c mice

irradiated at 0 Gy or 12 Gy/fraction. Arrows indicate irradiation time. # and

## indicate the time at which the esophagus was harvested for gene expression

analyses in B (#) and C (##). *** on the left and * on the right graphs in A

indicate P < 0.001 and 0.05, analyzed by two-way ANOVA and Student’s t-

１７

test, respectively. B and C, Gene expression changes of irradiated esophagus

by qRT-PCR analysis at d5 (B) and d12 (C) post-radiation. Gene expression

was expressed as relative quantification (RQ) methods by dividing mRNA

levels of the esophagus irradiated at 12 Gy/fraction by those of the esophagus

irradiated at 0 Gy/fraction. Data are the mean ± s.e.m. for n ≥ 5 per group. *

in B indicates P < 0.05, analyzed by one-way ANOVA.

We then examined whether the reduced expression in Egf and Ctgf would

also occur in irradiated epithelial cell line in vitro. To do this, we utilized Het-

1A human esophageal epithelial cell line and irradiate these cells at 6 Gy,

followed by gene expression analysis by qRT-PCR at 48 hr post-radiation. We

observed that EGF and CTGF expression was indeed significantly reduced in

irradiated Het-1A cells (Fig. 6A). Consistent with in vivo data, we also

observed that TIMP-1 expression was increased by approximately 2-fold in

irradiated Het-1A (Fig. 6A).

１８

Figure 6. Epidermal growth factor (EGF) treatment can partially protect Het-

1A human esophageal cell line from radiation-induced apoptosis. A, Gene

expression changes in Het-1A cells, expressed as relative quantification of

mRNA of irradiated cells (6 Gy) compared to that of unirradiated cells (0 Gy).

B, FACS analysis for Annexin V+ Propidium Iodide+ apoptotic cells

irradiated at 0 Gy or 6 Gy, treated with or without EGF. EGF was treated for

48 hr prior to FACS analysis. Red boxes show gated populations used for

quantification in C. C, Quantification of apoptotic cells in B. Treatment of

EGF or KGF was for 48 hr. D, Clonogenic survival of Het-1A cells irradiated

at 0 Gy or 6 Gy with or without EGF. Bar graph shows quantification of

１９

colonies survived over 2 weeks shown in a picture above. Data in A, C, and

D are the mean ± s.e.m. for triplicate determinations. *, **, and *** indicate

P < 0.05, 0.01, and 0.001, respectively, as analyzed by one-way ANOVA. E,

Western blot analysis of Het-1A cells irradiated at 0 Gy or 6 Gy with or

without EGF, KGF or CTGF for EGFR, AKT, and ERK signaling pathways.

EGF partially protects Het-1A epithelial cells from radiation-induced

apoptosis

Above results demonstrating decreased EGF and CTGF expression in

irradiated esophagus indicate that these molecules may have potentials as a

therapeutic intervention strategy for RIE. To determine whether EGF or

CTGF protects esophagus from radiation-induced damage, we irradiated Het-

1A at 6 Gy and treated them with either recombinant human EGF (EGF),

recombinant human keratinocyte growth factor (KGF), or recombinant human

connective tissue growth factor (CTGF) for 48 hr. By analyzing Annexin V-

positive apoptotic cells using FACS, we observed that irradiation alone

produced approximately 10 % of apoptotic cells and that EGF treatment

partially decreased the level of radiation-induced apoptosis (Fig. 6B & 6C).

On the other hand, neither KGF (Fig. 6C & Fig. 7A) nor CTGF (Fig. 7B)

exerted similar effects as to EGF (i.e., partially protected Het-1A cells from

the radiation-induced apoptosis).

To investigate how EGF protects cells from radiation-induced apoptosis,

we examined cell cycle distribution by FACS, survival by clonogenic assay,

and signal transduction pathways by western blot analysis. We observed that

２０

irradiation alone caused a decrease in the number of cells in G1 phase while

those in G2/M phase were increased (Fig. 7D, 7F, 8B, and 8D), consistent

with other studies demonstrating radiation-induced G2/M arrest [23,24]. We

further found that addition of EGF, KGF, or CTGF in irradiated Het-1A cells

did not significantly alter the proportion of cells in each cell cycle phase (Fig.

7D, 7F, 8B, and 8D) although EGF seemed to have a small effect in increasing

the number of cells in G2/M phase (Fig. 8D). By clonogenic cell survival

assay, we observed that EGF alone increased survival of Het-1A cells by

approximately 1.3-fold (Fig. 6D) and that irradiation alone at 6 Gy caused

approximately 95% cell kills (Fig. 6D). Cell survival of irradiated Het-1A

cells treated with EGF was not significantly different to that of irradiation

alone (Fig. 6D), suggesting that EGF was not able to protect cells from

radiation-induced cell kill. Western blot analysis revealed that treatment of

EGF to Het-1A cells resulted in a rapid internalization of EGFR by 48 hr (Fig.

8E) accompanied by phosphorylation of EGFR, AKT, and ERK1/2 signaling

(Fig. 8E). Irradiation at 6 Gy alone did not result in such effect, as

demonstrated by intact presence of EGFR (Fig. 6E) and the basal level of

phosphorylated AKT (Fig. 6E). We found that treatment of EGF but not with

KGF nor CTGF in irradiated Het-1A cells lead to a rapid internalization of

EGFR and an increase in phosphorylated EGFR, AKT, and ERK1/2 protein

(Fig. 6E), effects similar to unirradiated Het-1A cells treated with EGF. These

results therefore suggest that EGF could partially protect radiation-induced

apoptosis in Het-1A cells via activation of EGFR signaling pathway involving

in AKT and ERK.

２１

Figure 7. A, FACS plot of apoptotic cells in irradiated Het-1A cells treated

２２

with KGF for 48 hr. B, Quantification of Het-1A cells with or without

irradiation treated with EGF or CTGF. C, Cell cycle analysis of Het-1A cells

with or without irradiation treated with EGF or KGF for 48 hr by FACS.

Quantification of each phase of cell cycle is shown in D (G1 phase), E (S

phase), and F (G2/M phase). Data are the mean ± s.e.m. for triplicate

determinations. *, **, and *** indicate P < 0.05, 0.01, and 0.001,

respectively determined by one-way ANOVA.

２３

Figure 8. A, Cell cycle analysis of Het-1A cells with or without irradiation

treated with EGF or CTGF for 48 hr by FACS. Quantification of each phase

of cell cycle is shown in B (G1 phase), C (S phase), and D (G2/M phase).

Data are the mean ± s.e.m. for triplicate determinations. *, **, and ***

indicate P < 0.05, 0.01, and 0.001, respectively determined by one-way

ANOVA. E, Western blot of Het-1A cells treated with EGF demonstrating a

rapid internalization of EGFR accompanied by phosphorylation of EGFR,

AKT, and ERK.

２４

Discussion

In the present study, we established a mouse model of radiation-induced

esophagitis (RIE), reflecting symptoms of RIE patients including reduced

food intake and the body weight loss. Previously, other studies have utilized

various mouse strains such as C3H/HeNsd and C57BL/6 mice irradiated

either at single fractionation of 28~37 Gy or fractionated daily doses of

11.5Gy for 4 days to induce RIE and examined the histology, weight loss, and

the overall survival as the end point [10,11,18,19,25,26]. Although C57Bl/6

mice have also been utilized by other investigators to induce RIE [18,19], we

found that this strain of mice was very resistant to single high dose irradiation

up to 30 Gy (data not shown) as well as to fractionated irradiation investigated

in this study. It has been well established that C57Bl/6 and Balb/c mice are

prototypical Th1- and Th2-type mouse strains, respectively [27]. Studies have

shown that Balb/c mice are much more susceptible towards bacterial infection

[27] and methacholine-induced allergic asthma [28]. Mechanistically, these

mice have been reported to possess more CD4+CD25+ T regulatory cells,

which can suppress immune responses [29]. However, this is not always the

case. For example, C57Bl/6 mice have been shown to be more sensitive

towards ischemia-reperfusion lung injury [30], diet-induced obesity [31], and

dextran sulfate sodium-induced acute colitis [32] than Balb/c mice.

Our irradiated esophagus in vivo (Fig. 5) and irradiated Het-1A human

esophageal cell line in vitro (Fig. 6) consistently showed reduced EGF gene

２５

expression, indicating that EGF may be a potential therapeutic strategy to treat

RIE. EGF is a polypeptide that regulates epithelial cell proliferation, growth,

and migration [15]. Several preclinical studies exploiting EGF as a protective

agent against chemotherapy or radiotherapy have demonstrated that EGF

accelerated recovery of small intestinal mucosa after radiation damage in mice

[33] and decreased radiation-induced oral mucositis in rats [34]. Moreover,

clinical studies have also shown that EGF ameliorates oral mucositis of

patients treated with radiotherapy of head and neck [16,35]. Suggested

mechanism for EGF in treatment of oral mucositis is via enhancing mucosal

wound healing and tissue generation [36] or protects epithelial cells against

apoptosis [37]. Consistent with the latter, we also observed that the exposure

of EGF to irradiated cells partially protected cells from radiation-induced

apoptosis, possibly via an increased AKT and ERK signaling pathway (Fig. 6).

Activation of AKT and ERK signaling pathway resulted from EGF-mediated

EGFR activation may offer protection of epithelium against RIE via not only

their well-known cellular survival pathway (for example AKT inhibiting

apoptosis by inactivating various pro-apoptotic proteins such as BAD, BAX,

and caspase-9 [38] or by upregulation of several antiapoptotic proteins such as

FLIP and XIAPs [39-41]) but also enhancing DNA repair capacity. Several

studies have demonstrated that AKT activation promotes DNA-PKcs

accumulation at the DNA double strand break sites and enhances its kinase

activity necessary to mediate DNA repair process [42]. AKT activation has

also shown to upregulate MRE11 expression, a central protein binding to

RAD50 and NBS1 thereby forming MRN (MRE11, RAD50, and NBS1)

２６

complex, which senses the DNA breaks and recruits ATM necessary for

further DNA repair process [43]. Moreover, ERK activation resulting from

EGF activation has also shown to activate DNA repair genes ERCC1 and

XRCC1 in cancer cells [44].

In the gastrointestinal tract, ulceration of the gastrointestinal epithelium

induces development of ulcer associated cell lineage from gastrointestinal

stem cells, which have been shown to secrete EGF to stimulate cell

proliferation and ulcer repair [45]. In this study, we observed that at d5, Egf

gene expression was significantly upregulated in the irradiated esophagus

with 12 Gy/fraction (Fig. 5B). However, EGF gene expression was reduced at

d12 (Fig. 5C), which justifies exogenous EGF application. Another study

previously reported that macrophages express EGF, thereby signaling in a

paracrine manner towards cells expressing EGFR [46]. Since we observed

decreased F4/80+ CD11b+ macrophage infiltration in the irradiated esophagus

(Fig. 3) and that EGF expression was decreased in this setting (Fig. 5), it

would be interesting if macrophage infiltration would correlate with EGF

levels in RIE.

For the wide use of the in vivo model in the development of new

therapeutic agents for RIE, we should easily evaluate the degree of RIE.

Changes in body weight and food intake might be an easy outcome to

evaluate, but these could not be sensitive outcomes when using an animal

model. Instead, evaluation of apoptotic cells via FACS analysis would provide

the possible endpoint for a therapeutic agent like EGF. Moreover, in the

present study, we observed that TIMP-1 gene expression was increased in the

２７

irradiated esophagus in vivo (Fig. 5). Although irradiated epithelial cells

themselves can express TIMP-1 (Fig. 6) possibly via a compensatory

mechanism resisting against radiation-induced cell death [47], studies have

reported that a major cellular source of TIMP-1 is CD4 T helper cells [48].

This is in a good agreement with our results where we observed an increase in

infiltrating CD4 helper T cells in the irradiated esophagus in vivo (Fig. 4).

Therefore, for the therapeutic agent targeting immune modulation, Timp-1

expression could be a sensitive marker for the RIE in vivo.

Although EGF protects epithelial cells from radiation-induced apoptosis,

clinical study is needed to test whether EGF could alleviate RIE-associated

pain or not. In the phase II clinical study that showed EGF reduced radiation

induced oral mucositis in patients treated with radiotherapy, the grade of

mucositis was evaluated using the Radiation Therapy Oncology Group

(RTOG) scoring criteria based an evaluation of the patients’ pain [16]. By

inferring this study, it is likely that EGF could prove helpful in alleviating

pain associated with RIE.

While acute toxicity causing odynophagia is the main problem during

radiotherapy, late radiation change i.e, stricture formation is the critical issue

in RIE. By reducing severe acute toxicity through reduced apoptosis and re-

epithelialization with EGF application, we can prevent late consequential

effect. Several studies reported that EGF prevents stricture formation or

wound contraction. Juhl et al. reported that EGF prevents sclerotherapy-

induced esophageal ulcer and stricture formations in pigs [49], while Inoue et

al. reported that EGF application to the collagen gel prevents wound

２８

contraction by inhibiting unwanted fibroblast action [50]. Further study is

needed to evaluate the relevance of EGF and late stricture change in the

esophagus after radiation.

While growth factors such as EGF [16,35] and KGF [51] have shown to

result in a significant reduction of radiation-induced oral mucositis in humans,

exploiting them as therapeutic intervention strategy for RIE may not be easy.

An intraesophaeal delivery of such treatment may result in only limited

efficacy due to physiological peristalsis of the esophagus leading to a rapid

flushing thereby diminishing the therapeutic efficacy of the delivered agents.

Therefore in order to treat RIE, a careful consideration should be made for

therapeutic agents in their formulation as well as delivery strategy

counteracting such peristaltic condition of the esophagus.

A possible approach to overcome such limitation has been proposed by

Koukourakis and colleagues [14] where they utilized glycol that stabilize

rhGM-CSF, which otherwise would have easily degraded in the aqueous

solution. These investigators have shown by using scintigraphic imaging that

approximately 20 % remained in the esophagus from the orally administrated

radioactive material and that even these small quantities were sufficient to

expose the esophageal mucosa at high concentrations of rhGM-CSF [14]. In

the previous studies, EGF was also administered using various formulations

such as carboxymethylcellulose (bioadhesive carrier matrix) [52] or slow-

release, protease-protected form [53] in the treatment of duodenal ulcer or

necrotizing enteritis. Conjugation with hyaluronic acid is also a potential

strategy in the treatment of RIE currently being investigated.

２９

In summary, we report a model of RIE in vivo and in vitro, and

demonstrate that EGF may be a potential therapeutic intervention strategy to

treat RIE. Further studies are warranted to examine effective delivery

strategies of EGF to the esophagus evaluating the efficacy of EGF in reducing

RIE in vivo.

３０

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３８

국문초록

방사선유발 식도염의 in vivo, in vitro 모델에서

표피성장인자(epidermal growth factor)의

효능 연구

서울대학교 의학과 방사선종양학 김경수

연구목적: 본 연구는 방사선 유발 식도염의 in vivo, in vitro 모델에서

표피성장인자 (epidermal growth factor: EGF) 의 효능에 대해

연구하고자 하였다.

연구방법: Balb/c와 C57Bl/6 쥐들에게 흉부 방사선 조사를 매일

0,8,12,15 Gy씩 5일에 걸쳐 조사 하고 몸무게 변화와 음식물

섭취량의 변화를 측정 하였다. 실험 종료 후 쥐의 식도를 적출 하여

다음과 같은 검사를 하였다. : (i) H&E 염색을 통한 병리조직 검사;

(ii) 형광표지세포분류 분석법 (FACS) 을 이용한 면역 세포 침윤과

세포자멸사 조사; (iii) qRT-PCR 을 이용한 유전자 발현 조사. 또한

Het-1A 인간 식도 상피 세포주를 6Gy 방사선 조사를 하고 성장

인자들을 처리 후 유전자 조사와 세포자멸사, 증식 및 세포내 신호

３９

전달 경로를 조사 하였다.

결과: 회당 12Gy 혹은 15Gy 방사선 량이 Balb/c 쥐에서는 유의한

몸무게 변화와 음식섭취 량의 변화를 가져왔지만, C57Bl/6 쥐에서는

그 정도가 작았다. 회당 12Gy 조사된 Balb/c 쥐의 식도에서는

일그러진 상피세포와, 염증반응을 확인 할 수 있었고, CD4+ helper T

세포의 침윤과 세포자멸사가 증가됨을 확인할 수 있었다. 또한 tissue

inhibitor for metalloproteinase-1, plasminogen activator

inhibitor-1, granulocyte macrophage-colony stimulating factor,

vascular endothelial growth factor, and stromal-derived

factor-1 의 유전자 발현이 증가된 반면 Egf 의 발현은 감소됨을

확인하였다. 방사선 조사된 Het-1A 세포주에서도 이와 비슷하게

EGF 와 connective tissue growth factor (CTGF) 의 발현이

감소되었다. Het-1A 세포주에 EGF 를 처리 한 결과 EGFR의 인산화

및 AKT, ERK 신호전달과정을 통해 방사선 유발 세포자멸사를

일부분 감소시킴을 확인하였다.

Conclusions: 이 연구에서는 Balb/c 쥐 에게 12 Gy × 5 회

방사선이 몸무게 및 음식 섭취량 감소를 일으키고 식도의 병리학적

소견의 변화를 보임을 확인함으로써 방사선 유발 식도염의 동물

모델을 구축하였다. 또한 in vivo, in vitro 모델에서 EGF 발현의

감소는 EGF 가 방사선 방사선유발 식도염의 치료에 잠재적인 역할을

할 수 있을 것임을 시사한다.

４０

-------------------------------------

주요어: 방사선유발 식도염, 동물 모델, 표피성장인자

학번: 2013-21664