1. MECHANISTIC STUDY OF
EBSELEN-INDUCED
NRF2/ARE-MEDIATED
ANTIOXIDANT/ PHASE II
DETOXIFYING SIGNALING
Li Ching Sheng

2. Agenda
 Background/ Introduction
 Objective
 Methods
 Results
 Discussion
 Questions

3. Oxidative Stress and Cellular
Response
Antioxidants & Redox Signaling 8:88, 2006

4. Nrf2/ARE-mediated Defense
Mechanism
ARE genes
 Antioxidant proteins
 Phase II
metabolizing/
 detoxifying
enzymes
 Phase III drug efflux
pumps
A short half-life
protein (13-20 mins)Stress signaling, electrophiles,
ROS, chemopreventive agents,
etc.
5’-(A/G)TGACNNNGC(A/G)-3’)Mol Cell Biol 27: 6334, 2007
Nuclear Factor Erythroid 2 p45-related Factor 2 (Nrf2)
Cytoprotection
to carcinogen-
induced damage
Molecular Target
for Cancer
Chemoprevention

5. Ebselen
 Catalyzes some reactions for cellular
protection against oxidative and free radical
damages
 Under clinical trials for prevention and
treatment of eg. Cardiovascular diseases,
arthritis, stroke, atherosclerosis, and cancer

6. To investigate the role of Ebselen in
activating cytoprotective effects against
oxidative stress through Nrf2 pathway
Objectives

7.  Luciferase Assay: to quantify the expression
levels
 Nrf2 Activation
 Kinase Inhibitors
 MTT Assay: to assess cell viability
 RT-PCR: to visualize and quantify the
expression of selected genes
Methods

8.  Western Blot: to visualize the amount of
proteins
 Cytoprotective proteins
 Nrf2 Fraction
 Glutathione (GSH) Assay: to measure the level
of GSH in the cells
 Oxidative Stress Assay: to assess the level of
intracellular reactive oxygen species (ROS) in
the cells
 Used tert-butyl hydroperoxide (t-BOOH)
Methods

9. Results 1.1: Activation of Nrf2
Pathway
0
0.5
1
1.5
2
2.5
3
3.5
4
DMSO 3.125 6.25 12.5 25 tBHQ
50uM
LuciferaseActivity
(FoldtoControl)
Ebselen
(μM)

10. Results 1.2: Cell Viability (MTT
Assay)
0
20
40
60
80
100
120
140
DMSO 3.125 6.25 12.5 25
CellViability(%)
Ebselen
(μM)

11. Results 2: Nrf2 Fraction (Western
Blot)
0 6.25 12.5 25
Ebselen
(μM)
0 6.25 12.5 25
Ebselen
(μM)
p-Nrf2
p-Nrf2
Lamin
B
Lamin
B
Acti
n
Nucleus
Cytoplasm

12. Results 3: RT-PCR
0 6.25 12.5 25
Ebselen
(uM)
GAPDH
AKR1C1
GCLC
GCLM
-
1.00
2.00
3.00
0 6.25 12.5 25
-
1.00
2.00
3.00
0 6.25 12.5 25
-
0.50
1.00
1.50
0 6.25 12.5 25
-
1.00
2.00
0 6.25 12.5 25

13. Results 3.1: Cytoprotective
Genes
0 6.25 12.5 25
Ebselen
(μM)
AKR1C1
-
1.00
2.00
3.00
0 6.25 12.5 25
AKR1C1
Actin
0 6.25 12.5 25
Ebselen (μM)
Aldo-keto reductase family 1 member C1
(AKR1C1) Quantification
(Fold to control)

14. Glutamate—cysteine ligase, catalytic subunit
(GCLC)
Glutamate-cysteine ligase, modifier subunit
(GCLM)
Results 3.2: Cytoprotective
Genes
GCLC
GCLM
-
1.00
2.00
3.00
0 6.25 12.5 25
-
1.00
2.00
0 6.25 12.5 25
0 6.25 12.5 25
Ebselen
(μM)
Quantification
(Fold to control)

15. Results 3.3: GSH Measurement
0
20
40
60
80
100
120
140
GSH(Arbitrary
Units)
Ebselen 0 6.25 12.5 0 6.25 12.5 0 6.25 12.5
(mM)
t-BOOH - - - +250 +250 +250 +500 +500 +500 (mM)

16. Results 4.1: ROS Measurement
0
1
2
3
4
5
6
RelativeintracellularROS
(FluorescenceIntensity)
Ebselen 0 6.25 12.5 25 (mM)
t-BOOH - +500 +500 +500 (mM)

17. Results 5: Kinase Inhibitors
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
LuciferaseActivity
(FoldtoControl)
DMSO
25μM Ebselen
PI3K MEK P38 PKC

18.  Ebselen activates Nrf2 pathway in a dose-
dependent manner without cytotoxicity
 Cytoprotective genes—AKR1C1, GCLC, and
GCLM—are activated, and their respective
detoxifying enzymes and antioxidant are
produced
 Ebselen-treated cells display protective abilities
when exposed to ROS
 P38 signaling pathway is involved in the
regulation of Ebselen-induced detoxifying
enzymes and antioxidants
Conclusions and Discussion

20. SPECIAL THANKS TO
TAIWAN TECH TREK
NATIONAL HEALTH RESEARCH
INSTITUTES
DR. KUO AND HER LAB
Thank you for listening!

21. Thank you for listening!
Questions?