Pro-inflammatory cytokines play a key role in the development of radiotherapy-induced gastrointestinal mucositis

Ong Zhi, Gibson, Bowen, Stringer, Darby, Logan, Yeoh, Keefe

Description

Mucositis is a toxic side effect of anti-cancer treatments and is a major focus in cancer research. Pro-inflammatory cytokines have previously been implicated in the pathophysiology of chemotherapy-induced gastrointestinal mucositis. However, whether they play a key role in the development of radiotherapy-induced gastrointestinal mucositis is still unknown. Therefore, the aim of the present study was to characterise the expression of pro-inflammatory cytokines in the gastrointestinal tract using a rat model of fractionated radiotherapy-induced toxicity. Methods Thirty six female Dark Agouti rats were randomly assigned into groups and received 2.5 Gys abdominal radiotherapy three times a week over six weeks. Real time PCR was conducted to determine the relative change in mRNA expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF in the jejunum and colon. Protein expression of IL-1β, IL-6 and TNF in the intestinal epithelium was investigated using qualitative immunohistochemistry. Results Radiotherapy-induced sub-acute damage was associated with significantly upregulated IL-1β, IL-6 and TNF mRNA levels in the jejunum and colon. The majority of pro-inflammatory cytokine protein expression in the jejunum and colon exhibited minimal change following fractionated radiotherapy. Conclusions Pro-inflammatory cytokines play a key role in radiotherapy-induced gastrointestinal mucositis in the sub-acute onset setting.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 50
• Langue: English

Extrait

Ong et al. Radiation Oncology 2010, 5:22
http://www.ro-journal.com/content/5/1/22
RESEARCH Open Access
Pro-inflammatory cytokines play a key role
in the development of radiotherapy-induced
gastrointestinal mucositis
1 2* 1 1 1 3Zhi Yi Ong , Rachel J Gibson , Joanne M Bowen , Andrea M Stringer , Jocelyn M Darby , Richard M Logan ,
1 4Ann SJ Yeoh , Dorothy M Keefe
Abstract
Background: Mucositis is a toxic side effect of anti-cancer treatments and is a major focus in cancer research. Pro-
inflammatory cytokines have previously been implicated in the pathophysiology of chemotherapy-induced
gastrointestinal mucositis. However, whether they play a key role in the development of radiotherapy-induced
gasstinal mucositis is still unknown. Therefore, the aim of the present study was to characterise the
expression of pro-inflammatory cytokines in the gastrointestinal tract using a rat model of fractionated
radiotherapy-induced toxicity.
Methods: Thirty six female Dark Agouti rats were randomly assigned into groups and received 2.5 Gys abdominal
radiotherapy three times a week over six weeks. Real time PCR was conducted to determine the relative change in
mRNA expression of pro-inflammatory cytokines IL-1b, IL-6 and TNF in the jejunum and colon. Protein expression
of IL-1b, IL-6 and TNF in the intestinal epithelium was investigated using qualitative immunohistochemistry.
Results: Radiotherapy-induced sub-acute damage was associated with significantly upregulated IL-1b, IL-6 and TNF
mRNA levels in the jejunum and colon. The majority of pro-inflammatory cytokine protein expression in the
jejunum and colon exhibited minimal change following fractionated radiotherapy.
Conclusions: Pro-inflammatory cytokines play a key role in radiotherapy-induced gastrointestinal mucositis in the
sub-acute onset setting.
Introduction (in particular interleukin (IL)-1b (IL-6) and tumour
Mucositis is a debilitating side effect of cytotoxic che- necrosis factor (TNF)) have been suggested to play a
motherapy (CT) and radiotherapy (RT). It involves key role in this 5 phase mucositis model [5].
inflammation and mucosal ulceration of the alimentary Previous research has clearly shown that IL-1b,IL-6
tract, resulting in symptoms including pain, abdominal and TNF are upregulated in the buccal mucosa, jejunum
bloating, nausea, vomiting and diarrhoea [1-3]. The and colon of rats following administration of che-
effects of mucositis often limit the dose of cytotoxic motherapy [6]. Furthermore, elevated levels of IL-1b
agents that can be administered and in some cases, even and TNF have been detected in the buccal mucosa of
prevents patients from undergoing further treatment to hamsters who received combined chemotherapy and
control the malignancy [4]. radiotherapy [7,8]. In addition, various studies have
It has been postulated that mucositis occurs in five attempted to target pro-inflammatory cytokines as a
overlapping phases: initiation, upregulation and message preventive measure for intestinal mucositis [8-11]. For
generation, signalling and amplification, ulceration and example, palifermin and IL-11 have been reported to be
healing [5]. Nuclear factor kappa B (NFB), cyclooxy- successful in lowering the levels of pro-inflammatory
genase-2 (COX-2) as well as pro-inflammatory cytokines cytokines in the development of mucositis [8-11].
Furthermore, they also attenuate mucositis in animal
models [8-12], thus supporting the current view that* Correspondence: [e-mail]
2School of Medical Sciences, University of Adelaide, Adelaide, South Australia
© 2010 Ong et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Ong et al. Radiation Oncology 2010, 5:22 Page 2 of 8
http://www.ro-journal.com/content/5/1/22
pro-inflammatory cytokines play a major role in the Table 1 Experimental Design
development of mucositis. Group Rat Number Treatment Total Radiation
Duration (Weeks) Dose (Gy)Recently, we have developed a fractionated radiother-
1 n = 5 1 7.5apy-induced mucositis model in the Dark Agouti (DA)
2n=5 2 15rat [13]. The model involves rats receiving one to six
3 n = 5 3 22.5weeks of radiotherapy. In the clinical setting, fractio-
4n=5 4 30nated radiotherapy is usually more common than a sin-
5 n = 5 5 37.5gle high dose. Thus, this model provides the ideal
6n=5 6 45opportunity to explore various avenues involved in frac-
tionated radiotherapy-induced mucositis, with rats Control n = 6 6 0
receiving between one and three weeks of radiotherapy Groups of rats (n = 5) were exposed to varying doses of fractionated
radiotherapy over a six week period. Control rats (n = 6) received norepresenting short-term, and those receiving between
fractionated radiotherapy.
four and six weeks representing long-term radiotherapy
in the clinical setting [13]. Damage which occurs in the
short term is an acute event, while damage in the long The small and large intestines were separated and
term is considered sub-acute. Histological damage peaks flushed with chilled saline to remove intestinal contents.
mid treatment and begins to subside towards the com- Sections of jejunum (collected at 33% of the length from
pletion of radiotherapy, despite worsening clinical symp- the pyloric sphincter) and colon (collected at 50% of the
toms of intestinal toxicity [14]. The cause of this is length) were collected and either fixed in 10% neutral
unknown but may be related to inflammatory changes. buffered formalin and embedded in paraffin for histo-
Therefore the aim of the present study was to character- pathology and immunohistochemistry or snap frozen
ise the expression of pro-inflammatory cytokines in the with liquid nitrogen and stored at -70°C for real time
intestines during six weeks of fractionated radiotherapy. PCR.
We hypothesise that pro-inflammatory cytokine levels in
the jejunum and colon will be elevated following radio- Histopathology
therapy and that this increase will correlate with the Routine histopathological examination using standard
increasing duration and total doses of radiotherapy. haematoloxylin and eosin staining was conducted. These
methods have previously been validated and described
Methods elsewhere [15].
Ethics
This study was approved by the Animal Ethics Commit- RNA extractions
tee of the Institute of Medical and Veterinary Sciences, Total RNA was isolated and purified using the NucleoS-
Adelaide and the University of Adelaide. Animal work pin® RNA II kit (Macherey-Nagel, Duren, Germany) fol-
and handling were complied with the National Health lowing manufacturer’s instructions. The integrity of
and Research Council (Australia) Code of Practice for RNA extracted was determined by comparing sharp 28S
Animal Care in Research and Teaching (2004) [13]. and 18S rRNA bands electrophoresed on a 1.5% formal-
dehyde gel and 260/280 ratios.
Irradiation Protocol and Experimental Design
Thirty six female DA rats (150 g - 170 g) were obtained Reverse Transcription
from the University of Adelaide Breeding Facility. All 1 μg RNA was reverse transcribed to generate cDNA
animals were maintained in an environmentally con- using the iScript™ cDNA Synthesis Kit (Bio-Rad Labora-
trolled condition of 12-h light/12-h dark cycles and tories, Hercules, CA) according to manufacturer’s
allowed free access to food and water. Rats were ran- instructions. 100 ng of cDNA from each sample was
domly assigned to groups based on RT dose as follows: subsequently used in real time PCR.
Control (no treatment); 7.5 Gy; 15 Gy; 22.5 Gy; 30 Gy;
37.5 Gy and 45 Gy (Table 1). Detailed radiation proce- Real Time PCR
dures have been described previously [13]. Briefly, rats The amplification reactions were conducted in a volume
were anaesthetised prior to receiving 2.5 Gys of radio- of 10 μL containing 1× Quantitect SYBR Green master
therapy to the abdomen three times a week for up to mix (Qiagen) forward and reverse primers each at a
six weeks. final concentration of 2.5 ng/μL and 100 ng cDNA. Pri-
mer sequences for IL-1b, IL-6, TNF and b-actin are sta-
Tissue Collection ted in Table 2. Real time PCR was carried out using
Rats were killed by exsanguination followed by cervical Rotor-Gene 6000 real time rotary analyser (Corbett Life
dislocation and the entire gastrointestinal tract removed. Science, Sydney, Australia). Taq DNA polymerase wasOng et al. Radiation Oncology 2010, 5:22 Page 3 of 8
http://www.ro-journal.com/content/5/1/22
Table 2 Primer sequences for IL-1b, IL-6, TNF and b actin Statistical Analysis
Statistical analyses were conducted using either one-wayGene Primer Sequence Size Accession
(bp) No ANOVA followed by Tukey’s Post Hoc test, or Kruskal
IL-1b Forward: 5’-CACCTCTCAAGCAGAGCACAGA-3’ 81 NM_031512 Wallis test followed by Dunn’s Post Hoc test. Results
Reverse: 5’-ACGGGTTCCATGGTGAAGTC-3’ were deemed significant should p < 0.05.
IL-6 Forward: 5’-ATATGTTCTCAGGGAGATCTTGGAA-3’ 80 NM_031512
Reverse: 5’-GTGCATCATCGCTGTTCATACA Results
TNF Forward: 5’-GTGATCGGTCCCAACAAG-3’ 71 X66539 Histopathology
Reverse: 5’-AGGGTCTGGGCCATGGAA-3’ Pathological changes over time in the rat intestinal tract
caused by fractionated radiotherapy have previouslyb actin Forward: 5’-AGGCCAAC