diesel exhaust particles and serum modulate airway epithelial cell viability by affecting...
TRANSCRIPT
DIESEL EXHAUST PARTICLES and SERUM
MODULATE AIRWAY EPITHELIAL CELL VIABILITY
by AFFECTING INTRACELLULAR CELL SIGNALING
PATHWAYS, WHICH ARE SENSITIVE to OXIDATIVE
STRESS
Füsun FAKILI1,2, Bülent GÖĞEBAKAN2, Recep BAYRAKTAR2,
Serdar ÖZTUZCU2 ,Hasan BAYRAM1,2
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.
Thank you…