DIESEL EXHAUST PARTICLES and SERUM MODULATE AIRWAY EPITHELIAL CELL VIABILITY by AFFECTING INTRACELLULAR CELL SIGNALING PATHWAYS, WHICH ARE SENSITIVE to OXIDATIVE STRESS Füsun FAKILI 1,2 , Bülent GÖĞEBAKAN 2 , Recep BAYRAKTAR 2 , Serdar ÖZTUZCU 2 ,Hasan BAYRAM 1,2 Gaziantep University , School of Medicine, 1 Department of Respiratory Medicine 2 Cell Culture Laboratory The 13 th Annual Congress of Turkish Thoracic Society 201
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DIESEL EXHAUST PARTICLES and SERUM MODULATE AIRWAY EPITHELIAL CELL VIABILITY by AFFECTING INTRACELLULAR CELL SIGNALING PATHWAYS, WHICH ARE SENSITIVE to.
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Gaziantep University , School of Medicine, 1 Department of Respiratory Medicine 2 Cell Culture Laboratory
The 13th Annual Congress of Turkish Thoracic Society 2010
Introduction-1
• There is a close association between increases in
particulate matter 10µm (PM10), and respiratory
morbidity and cardiopulmonary mortality
- McConnell R et al, AJRCCM 2003,
- Pope CA et al, NEJM 2004)
• Diesel exhaust particles (DEP) increase release of inflammatory mediators from airway epithelial cells
- Bayram H et al, AJRCMB 1998
Introduction-2
• DEP induce A549 cell proliferation, and inhibit
apoptosis through oxidative stress, inhibition of
p21CIP1/WAF1 and stimulation of JNK and NF-B pathways
under serum free condition
- Bayram H et al, Eur Respir J 2006
Introduction-3
• c-Jun N-Terminal Kinaz (JNK), ‘Extra Cellular
Regulated Kinase’ (ERK) and Nuclear Factor
(NF)-kB pathways are activated in inflammatory
airway diseases such as asthma and chronic
obstructive pulmonary diseases (COPD)
- Hoshino S et al, Biochemical and Biophysical Research Com. 2005- Rahman I. Journal of Biochemistry and Molecular Biology 2003- Lee YC at al, Am J Physiol Lung Cell Mol Physiol 2008- Edwards MR et al, Pharmacology & Therapeutics 2009
Objectives
• To create a model of inflamed airways by adding serum
to cell culture media in vitro
• To investigate the role of oxidative stress and cell
signalling pathways including c-jun N Terminal Kinase
(JNK), Extra Regulated Kinase (ERK) and Nuclear
Factor (NF)-κB
• To investigate the effects of DEP on airway epithelial cell
viability, apoptosis and p21, p27 and p53 expression
Methods-1• A549, BEAS-2B and primary bronchial epithelial
cell cultures were incubated with 0, 50, 100, 200,
400 ve 1000 g/ml DEP in the presence and
absence of N-acetylcysteine (NAC), an inhibitor
of JNK (SP600125), an inhibitor of ERK (PD
98059) and NF-B inhibitor (SN50) under 0%, 1,
3.3 ve 10 FCS condition for 48 hours.
• Cells viability was evaluated by MTT assay
Methods-2
• Cells were double stained by Annexin V-PE and
7AAD dyes and analyzed by flow cytometry to
assess apoptotic cells and necrotic cells
• p21, P27 and p53 mRNA expression was
studied by means of real-time (PCR)
Effect of DEP on A549 Cell Viability of in the Presence of Different Concentrations of FCS
0.0
0.5
1.0
1.5
2.0
DEP (g/ml) + FCS (0%)
*** ******
**
Op
tica
l De
nsi
ty
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
***
DEP (g/ml) + FCS (1%)
Opt
ical
Den
sity
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
2.5
* **** ***
***
DEP (g/ml) + FCS (3.3%)
Op
tical
Den
sity
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
2.5
***
DEP (g/ml) + FCS (10%)
Opt
ical
Den
sity
*p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP
Effects of DEP on BEAS-2B Cell Viability in the Presence of Different Concentrations of FCS
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0**
***
*** ***
DEP (g/ml) + FCS (1%)
Opt
ical
Den
sity
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
***
*** *** ***
*
DEP (g/ml) + FCS (0%)
Opt
ical
Den
sity
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
2.5
*** ***
*
*
DEP (g/ml) + FCS (3.3%)
Opt
ical
Den
sity
0 50 100
200
400
1000
0.0
0.5
1.0
1.5
2.0
2.5
***
***
DEP (g/ml) + FCS (10%)
Opt
ical
Den
sity
*p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP
Effects of DEP on Primary Bronchial Epithelial Cell Viability in the Presence of Different Concentrations of FCS
0 50 100 2000.0
0.5
1.0
1.5
*
***
DEP (g/ml) + FCS (0%)
Opt
ical
Den
sity
0 50 100 2000.0
0.5
1.0
1.5
2.0
****
DEP (g/ml) + FCS (3.3%)
Opt
ical
Den
sity
0 50 100 2000.0
0.5
1.0
1.5
2.0
2.5
*
***
DEP (g/ml) + FCS (1%)
Opt
ical
Den
sity
0 50 100 D2000.0
0.5
1.0
1.5
*** ***
***
DEP (g/ml) + FCS (10%)
Opt
ical
Den
sity
*p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP
Effect of NAC and JNK inh. on A549 Cell Viability Modified by Serum (3.3%) and DEP (200µg/ml)
0 3.3 10 33 0 3.3 10 330.0
0.5
1.0
1.5
2.0
2.5
3.3% FCS + NAC(mM)
DEP (200g/ml)
******
***
***
Opt
ical
Den
sity
0
DMSO 3.
3 1
0 33 0
DMSO
3.3 10 33
0.0
0.5
1.0
1.5
2.0
2.5
***
***
DEP (200g/ml)
3.3% FCS+JNK inh.(M)
Opt
ical
Den
sity
NAC JNKinh.
***p<0.0001 vs 0µg/ml DEP♦p<0.0001 vs 200µg/ml DEPΦp<0.01 vs 200µg/ml DEP + DMSO
Effect of Inhibitors of ERK and NF-κB on A549 Cell Viability Modified by Serum (3.3%) and DEP (200µg/ml)
0
DM
SO 3.3 10 33 0
DM
SO 3.
3 10 330.0
0.5
1.0
1.5
2.0
***
*** ***
DEP (200g/ml)
***
3.3% FCS+ ERK inh.(M)
Opt
ical
Den
sity
03.
3 10 33 03.
3 10 330
1
2
3
******
***
DEP (200g/ml)
3.3% FCS+ NFKB inh.(M)
Opt
ical
Den
sity
***p<0.0001 vs 0µg/ml DEP
ERKinh. NF-kBinh.
Effect of DEP(200µg/ml) on A549 Cell Apoptosis in the Presence of FCS (3.3%)
LL(A-/P-) LR(A+/P-) UR(A+/P+) UL(A-/P+)0
5
10
15
20
60
90
120FCS (% 3.3)
FCS (%3.3) +DEP (200g/ml)
Kadranlar
Hü
crel
er %
SF 0 50 2000.00
0.05
0.10
0.15
0.20
3.3% FCS + DEP(g/ml)
** **p<0.05; **p<0.005 vs0g/ml DEP
p53
mR
NA
% o
f G
AP
DH
SF 0 50 2000.0
0.2
0.4
0.6
0.8
3.3% FCS + DEP(g/ml)
p21
mR
NA
% o
f G
AP
DH
SF 0 50 2000.00
0.05
0.10
0.15
%3.3 FCS + DEP (g/ml)
p27
mR
NA
% o
f G
AP
DH
Effect of DEP on mRNA
Expression of p21, P27 and
p53 by A549 Cells in the
Presence of FCS(3.3%)
p21 p27
p53
Summary-1• Although DEP induced A549 cell viability under serum free
condition, they reduced cell viability in the presence of
3.3%FCS
• The role of oxidative stress and the oxidative stress pathways
(JNK and ERK) and NF-κB in this process looks limited.
• NAC and inhibitors of JNK, ERK and NF-κB inhibit A549 cell
viability in the presence of serum
• DEP do not affect A549 cell apoptosis/necrosis in the
presence of serum
• DEP induce mRNA expression of p53 in the presence of 3.3%
serum
Summary-2
• In BEAS-2B cells, although lower concentrations of DEP
induced cell viability under 0-3.3 % FCS condition,
relatively higher doses decreased cell viability. In the
presence of 10% FCS, higher concentrations
suppressed cell viability
• In primary bronchial epithelial cells, higher DEP
concentrations reduced cell viability under 0-10% FCS
condition.
Conclusion
• The activated status of JNK, ERK, and NF-kB in inflamed
airways that can be seen in respiratory disorders such as
asthma and COPD may, at least in part, be due to the
leakage of serum to airway mucosa
• Under such conditions, the toxic effects of pollutants such as
DEP may be enhanced at cellular level.
• Airway epithelial cells from different origins may be affected
by the toxic effects of DEP at different levels
• The underlying mechanisms need to be investigated further
• This study funded by the Research Fund of Gaziantep University.