the environmental stress response as a target for …...the environmental stress response as a...
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The environmental stress response as a target for therapeutic intervention
Jennifer Harbottle
Supervisor: Dr Andreas Kolb
MRP Inter-institutional Post-Graduate Competition 2016
27-28 June 2016
Age-related disease
Office for National Statistics, 2012
Aging and health span
A common denominator
Normal diet Calorie restriction
Stress resistance
Upregulation of the environmental stress response
Nrf2 drives the ESR
↓ Nrf2 signaling with age
↓ antioxidant defence ↓ stress resistance
↑ disease susceptibility
Nrf2, the “guardian of health span and the gate-keeper of species longevity” (Lewis et al. 2010)
Lewis et al. (2010). Integr Comp Biol 50(5):829–843
Keap1
NUCLEUS
Electrophilic phytochemicalse.g. Sulforaphane
Nrf2 Maf
ARE
CYTOPLASM
Transcription of cytoprotective genes
NQO1HMOX-1AKRGST, etc.
↑ cellular defence ↑ stress resistance
↑ longevity and health span
Keap1Nrf2
SHSH
SHSH
Phytochemicals activate the ESR
• Aim 1: Identify and characterise novel, natural compounds that induce the ESR.
A cell based assay system for the analysis of phytochemicals activating the ESR
Stable transfection of HepG2 cells & selection of HepG2 C1 clone
Validation of bioluminescent reporter system using sulforaphane
High throughput screening of natural chemical libraries
Identification and characterisation of compounds that activate the ESR
neoR neoR luciferase
NQO1 promoter Stable transfection
Validation with sulforaphane (1)
pNQO1-luc reporter induction
PAR
T 1
NQO1 gene
pNQO1-luc reporter induction
Validation with sulforaphane (2) AKR1B10 gene
AKR1B10 protein NQO1 protein
0
2
4
6
8
10
12
Untreated 24 48 72 96
Re
lati
ve lu
cife
rase
act
ivit
y co
rre
cte
d f
or
cell
viab
ility
Length of SF (7.5 μM) treatment (hours)
***
***
***
***
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Untreated 24 48 72 96
Re
lati
ve N
QO
1/β
-act
inm
RN
A
Length of SF (7.5 μM) treatment (hours)
NQO1
*
******
***
0
5
10
15
20
25
30
35
Untreated 24 48 72 96
Re
lati
ve A
KR
1B
10
/β-a
ctin
mR
NA
Length of SF (7.5 μM) treatment (hours)
AKR1B10
***
***
***
***
0
1
2
3
4
5
Untreated 24 48 72 96
Re
lati
ve N
QO
1/β
-act
in p
rote
in
Length of SF treatment (hours)
NQO1
0
20
40
60
80
100
120
Untreated 24 48 72 96
Re
lati
ve A
KR
1B
10
/β-a
ctin
pro
tein
Length of SF treatment (hours)
AKR1B10
Untreated 24 h 48 h 72 h 96 h
β-actin
AKR1B10
NQO1
42 kDa
37 kDa
30 kDa
Sulforaphane (7.5 μM) treatment
***
*
**
***
***
***
**
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0 7.5 11 0 7.5 11
HepG2 WT HepG2 C1
NQ
O1
/b-a
ctin
mR
NA
(p
g/p
g)
SF treatment (μM)
0
1
2
3
4
5
6
7
8
9
0 7.5 11 0 7.5 11
HepG2 WT HepG2 C1
HM
OX
1/b
-act
in m
RN
A (
pg
/pg)
SF treatment (μM)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0 7.5 11 0 7.5 11
HepG2 WT HepG2 C1
AK
R1
B1
0/b
-act
in m
RN
A (
pg
/pg)
SF treatment (μM)
Validation with sulforaphane (3)
Endogenous ESR gene expression
in HepG2 WT ‘vs’ HepG2 C1
NQO1 gene
HMOX1 gene AKR1B10 gene
MBC library screening
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12
Re
lati
ve lu
cife
rase
act
ivit
y (c
orr
ect
ed
fo
r ce
ll vi
abili
ty)
MBC compound treatment (20 µM)
MBC37
MBC20
MBC20 and MBC37
• No apparent cytotoxic effect
• Dose-dependent reporter activation
Cell viability (0-200 μM) Reporter induction (0-200 μM)
0
1
2
3
4
5
6
7
Vehicle MBC20 MBC37
Re
lati
ve N
QO
1/b
-act
in m
RN
A
Cell treatment
NQO1
***
***
0
1
2
3
4
5
6
7
Vehicle MBC20 MBC37
Re
lati
ve H
MO
X1
/b-a
ctin
mR
NA
Cell treatment
HMOX1
**
†
0
5
10
15
20
25
Vehicle MBC20 MBC37
Re
lati
ve A
KR
1B
10
/b-a
ctin
mR
NA
Cell treatment
AKR1B10
** **
NQO1 gene HMOX1 gene AKR1B10 gene
0
20
40
60
80
100
120
140
Untreated 1.5 3 6.25 12.5 25 50 100 200
Ce
ll vi
abili
ty (
% v
eh
icle
co
ntr
ol)
MBC treatment (μM)
MBC20
MBC37
0
1
2
3
4
5
6
7
Untreated 1.5 3 6.25 12.5 25 50 100 200
Luci
fera
se a
ctiv
ity
(fo
ld in
du
ctio
n r
ela
tive
to
VC
)
MBC treatment (μM)
MBC20
MBC37
0
20
40
60
80
100
120
140
Untreated 1.5 3 6.25 12.5 25 50 100 200
Ce
ll vi
abili
ty (
% v
eh
icle
co
ntr
ol)
MBC treatment (μM)
MBC20
MBC37
0
1
2
3
4
5
6
7
Untreated 1.5 3 6.25 12.5 25 50 100 200
Luci
fera
se a
ctiv
ity
(fo
ld in
du
ctio
n r
ela
tive
to
VC
)
MBC treatment (μM)
MBC20
MBC37
MBC37 (0-72 h)
• MBC37 ↑ ESR protein expression over time
• ↑ biopotency and ↓ cytotoxicity compared with SF
Successful screening
AKR1B10 protein NQO1 protein Reporter induction
Untreated control
Vehicle control 24 h 48 h 72 h
β-actinAKR1B10NQO1
MBC37 treatment (20 μM)
42 kDa37 kDa30 kDa
0
1
2
3
4
5
6
Untreated 24 48 72
Re
lati
ve N
QO
1/β
-act
in p
rote
in
Length of MBC37 treatment (hours)
0
2
4
6
8
10
12
14
16
Untreated 24 48 72
Re
lati
ve A
KR
1B
10
/β-a
ctin
pro
tein
)
Length of MBC37 treatment (hours)
†
*****
What now?
• Further characterisation of MBC20 and MBC37 Bioavailability, absorption, metabolism
• PhytoQuest library screening (MRCT) Stereochemistry and ASR.
Limitations…
• … Due to the nature of the transfection method. - Random integration site; - Variable transgene copy number.
• … Due to the complexity of mammalian transcription . - Regulatory elements (cis-acting enhancers/silencers).
Simply inserting a promoter cannot capture the whole picture.
AAV-mediated integration
Aim 2: Site-specific integration of the luciferase reporter gene into the locus of the HMOX1 gene.
The NanoLuc reporter will reflect the expression pattern of endogenous HMOX1
rAAV dilution range
1x105 copies 5x104 copies 1x104 copies
5x103 copies 1x103 copies Not infected
Screening
gDNA
A1 A2 A3 A4 A5 A7 A8 A9 A10 A11 A12 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 C2 C3
C5 C6 C7 C9 C10 C11 C12 D1 D2 D3 D5 D4 D6 D8 D9 D10 D11 D12 E1 E2 E5 E6 E7 E8
E9 E11 E12 F1 F3 F5 F6 F8 F10 F12 G1 G2 G3 G5 G11 H1 H5 H7 H9 H10 H12
gDNA
gDNA
Integration
Integration
Integration
Site-specific
Site-specific
Site-specific
The ESR – A long term effect?
• Does SF evoke an antioxidant response that persists?
• Are epigenetic mechanisms involved?
Gao et al., 2001. Proc Natl Acad Sci U S A. 98(26):15221-6; Ye et al., 2002. Clin Chim Acta. 316(1-2):43-53
NQO1 mRNA
Plasma SF Prolonged protection against menadione
(QR = NQO1)
0
0.5
1
1.5
2
Untreated 24
Re
lati
ve N
QO
1/b
-act
in m
RN
A
Length of 7.5 μM SF treatment (hours)
No txremoval
24 h txremoval
Phytochemicals and epigenetics
• Epigenetic silencing of Nrf2 and NQO1 with ↑ age.
• Phytochemicals upregulate gene promoter demethylation Facilitate gene transcription.
20-day SF treatment
Does SF (2.5 μM) treatment demethylate ESR gene promoter?
If so, how stable is this epigenetic modification?
0
0.5
1
1.5
2
Untreated 2.5 μM 7.5 μM
Re
lati
ve lu
cife
rase
act
ivit
y
SF treatment (20 days)
0
0.5
1
1.5
2
Untreated 2.5 μM 7.5 μM
Re
lati
ve N
QO
1/b
-act
in m
RN
A
SF treatment (20 days)
0
1
2
3
4
5
Untreated 2.5 μM 7.5 μM
Re
lati
ve N
QO
1/β
-acti
np
rote
in
SF treatment (20 days)
Reporter induction NQO1 gene NQO1 protein
The big picture
• Identification and characterisation of compounds that elicit cytoprotection Food fortification Production of plants with enhanced nutritional quality Optimise health span
• A better understanding of how food provides a conditioning environment that shapes the activity of the (epi)genome and determines the stress adaptive response.
Lab group supervision and support Dr Andreas Kolb Linda Petrie Christopher Knowles Patrikas Pultinevicius
Marine Biodiscovery Centre
Professor Marcel Jaspars
Dr Wael Houssen
Genomics Pauline Shiach
Acknowledgements
Thank you for listening
RAW264.7 NQO1-luc D2
Nitrite production NQO1-luc reporter induction NQO1 gene expression
PCR with a selection of Nrf2-BSP and NQO1-BSP primers that span a CpG island within the respective gene promoters
Obtain 335 bp NQO1-BSP amplicon from nested PCR
gDNA isolation from HepG2 C1 cells after 20 days treatment with SF (n=3)
Bisulfite conversion of gDNA
Unable to obtain Nrf2-BSP amplicon
Purify and clone into pGEMT vector
PCR & Sequence positive clones
Assess methylation status of 24 CpG dinucleotides (> 10 clones)
To be continued
Health span
↑ Health span
Lifespan
Morbidity
‘Normal’ aging process
Optimal longevity