the importance of dose response for the biology of...
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The importance of dose response for the biology of synthetic triterpenoids
Karen T. Liby Geisel School of Medicine at Dartmouth
Dose-Response 2013 Amherst, MA April 24, 2013
Finkel, Nat Rev Mol Cell Biol, 2005
Disease-specific death rates as a function of time
Age Range (years)
Dis
ease
Dea
th R
ate
(per
100
,000
pop
ulat
ion)
Alzheimer’s Cardiovascular Cancer
Non-resolving inflammation
Nathan Cell 140, 2010
Exist widely in nature, especially in higher plants > 6,000 triterpenoids reported Medicinal uses in Asian countries Anti-inflammatory Anti-tumorigenic Poor potency Resemble steroids Biosynthesis Pleiotropic activities Synthesized by cyclization of squalene
Triterpenoids
O
COOH
H
O
NC
Oleanolic acid
H
HHO
CO2H
Oleanolic Acid
• Weak anti-inflammatory agent • Weak anti-carcinogenic agent • Readily, cheaply available in kilogram quantities from natural sources (e.g., olives, olive leaves)
CDDO Phase I trial terminated
CDDO-Me CDDO-Methyl ester
Phase III trial terminated
CDDO-Im CDDO-Imidazolide
Triterpenoids
CDDO-EA CDDO-Ethyl amide
O
CONHCH2CH3
H
O
N C
O H
O
N C
N
O
N
O
C O O H
H
O
N C
O
C O 2 M e
H
O
N C
Synthetic triterpenoids are multifunctional molecules
.
Triterpenoids
hematopoietic adipogenic neuronal
Ant
i-inf
lam
mat
ion
G 1
G 2
S M
Grow
th Suppression Differentiation
iNOS COX-2 MMPs
Apoptosis
Final Outcome:
Biological response:
Targeted Protein/ Network:
Increasing Concentration of Triterpenoid
Oxi
dativ
e St
ress
Keap/Nrf2/ARE
Induce phase 2 enzymes; Prevent
release of cytokines
Cytoprotection; Anti-inflammatory
Biological responses to triterpenoids are strongly dependent on dose
nM µM
PPARγ, Arp3 & cytoskeleton
IKK and NF-ĸB, JAK/STAT, ErbB2
Block DNA synthesis/ cell cycle progression
Activate caspases
Induce differentiation; Inhibit cell proliferation
Apoptosis
Low nM concentrations
of triterpenoids activate the
Nrf2 cytoprotective pathway
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
IC-50, Suppression of Induction of iNOS
TP-46
O
CO2H
H
H
TP-46
TP-82
O
CO2H
H
O
TP-82
TP-151
O
CO2H
H
O
NC
TP-151 (CDDO)
TP-155
O
CO2Me
H
O
NC
TP-155
TP-225
O
CN
H
O
NC
TP-225
CDDO-Im increases expression of genes regulated by Nrf2
Fold Increase CDDO CDDO-Im
Gene 4 hrs 12 hrs 4 hrs 12 hrs heme oxygenase (decycling) 1 19.70 17.15 90.51 111.43 ferritin, heavy polypeptide 1 2.64 6.06 2.64 6.96 NAD(P)H dehydrogenase, quinone 1 2.00 4.59 1.87 4.59 gamma-glutamylcysteine synthetase, regulatory 2.83 4.00 2.30 4.29 epoxide hydrolase 1, microsomal (xenobiotic) 1.41 2.64 1.87 4.00 NAD(P)H dehydrogenase, quinone 2 1.15 1.41 1.52 4.00 thioredoxin reductase 1 2.46 3.48 2.30 3.48 UDP-glucose dehydrogenase 1.23 1.62 1.41 2.30 glutathione reductase 1.41 2.30 1.52 2.14 crystallin, zeta (quinone reductase) 1.00 1.15 1.32 2.14 gamma-glutamylcysteine synthetase, catalytic 1.32 1.32 1.52 2.00 glutathione S-transferase A4 1.15 1.23 1.62 2.00
Keap1
The transcription factor Nrf2 activates a network of genes that protect against oxidative and electrophilic stress
PKC PI3K
Nrf2 P
Nrf2 P
Nrf2 P
Small Mafs X ARE
MAPK
Keap1
ROS Electrophiles
X=Transcription of anti-oxidative and
cytoprotective genes
Phase 2
Enzymes
• Direct antioxidants • Free radical metabolism • Electrophile detoxification • Glutathione homeostasis • Generation of reducing equivalents • Solute transport • Inhibition of inflammation • DNA damage recognition • Proteasome function
Target gene functions of the Nrf2-ARE pathway
0
20
40
60
80
100
120
0 0.3 1 3 10 30 0 0.3 1 3 10 30
CDDO-Im inhibits NO production in Nrf2 WT cells but not in Nrf2 KO cells
NO
Pro
duce
d (P
erce
nt s
timul
ated
con
trol
)
CDDO-Im (nM)
Nrf2 Wild-type cells Nrf2 Knockout cells
0
30
60
90
120
150
DMSO tBHP 250 µM
+ 0.01 nM Im
+ 0.1 nM Im
+ 1 nM Im
+ 10 nM Im
+ 100 nM Im
Mea
n Fl
uore
scen
ce In
tens
ity
CDDO-Imidazolide protects against ROS in Nrf2 wild-type cells challenged with tBHP
0
30
60
90
120
DMSO tBHP 250 µM
+ 0.01 nM Im
+ 0.1 nM Im
+ 1 nM Im
+ 10 nM Im
+ 100 nM Im
Mea
n Fl
uore
scen
ce In
tens
ity
CDDO-Imidazolide does NOT protect against ROS in Nrf2 knockout cells
10-10
10-9
10-8
10-7
10-6
10-5
CD
, Qui
none
Red
ucta
se In
duct
ion
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
IC-50, Suppression of Induction of iNOS
TP-46 TP-82 TP-151 TP-155 TP-225 TP-46
O
CO2H
H
H
TP-46 TP-82 O
CO2H
H
O
TP-82
TP-151
O
CO2H
H
O
NC
TP-151 (CDDO)
TP-155
O
CO2Me
H
O
NC
TP-155
TP-225
O
CN
H
O
NC
TP-225
Activation of the phase 2 cytoprotective enzyme NQO1 and inhibition of the inflammatory enzyme iNOS are tightly correlated
TP-218 TP-235
TP-224
TP-241
TP-222
TP-226
TP-230 TP-190
TP-233
TP-62 TP-156
TP-162
, -223
TP-225
TP-155
TP-151
TP-82
TP-46
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
IC-50, Suppression of Induction of iNOS
10-10
10-9
10-8
10-7
10-6
10-5 CD
, Qui
none
Red
ucta
se In
duct
ion
Activation of the Keap/Nrf2/ARE pathway by the triterpenoids is cytoprotective
Consequences of Nrf2 activation in cancer
Higher concentrations (low µM) of
triterpenoids increase ROS and
induce apoptosis in cancer cells
0
10
20
30
40
50
60
70
0
100
200
300
400
500
600
Mea
n Fl
uore
scen
ce In
tens
ity
Mea
n Fl
uore
scen
ce In
tens
ity
100 10 1 0.1
nM CDDO-Imidazolide
0 0
+ 250 µM tBHP
nM CDDO-Imidazolide
200 300 400 0 0
+ 250 µM tBHP
Low concentrations of triterpenoids suppress formation of ROS, but high concentrations increase ROS
Targeting the altered redox status of cancer cells to preferentially kill
malignant cells
- Cancer Cell 10:241, 2006
- Nature Reviews Drug Discovery 8:579, 2009
Trachootham, Nat Rev Drug Discovery, 2009
Targeting cancer cells via ROS
NIH3T3-control NIH3T3-Im 1 μM
W780-1control W780-Im 1 μM
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
cont Im-1
NIH3T3
W780
Fold
cha
nge
mea
n flu
ores
cenc
e CDDO-Im, 1 h treatment
CDDO-Im induces higher levels of ROS in Brca-1 defective breast cancer cells
than in normal 3T3 cells
MCF10 model of progressive breast disease
A Immortal
AT1 Ha-ras
transfected
CA1a Malignant
Non-tumorigenic
Premalignant lesions
Poorly differentiated carcinomas
with metastatic potential
Malignancy
In vivo
Cells transformed with activated Ras generate more ROS when
challenged with tBHP
0
10
20
30
40
50
60
No Rx 250 uM t-BHP 500 uM t-BHP
MCF-10 A MCF-10 AT-1
Mea
n Fl
uore
scen
ce In
tens
ity
The production of ROS is dose-dependent
ROS levels in response to tBHP challenge is dependent on triterpenoid dose and cell type
MCF10A cells MCF10 AT-1 cells
RO
S Fl
uore
scen
ce
Triterpenoids increase ROS
MCF10 AT-1 cells MCF10 CA1a cells
RO
S Fl
uore
scen
ce
MCF10 AT-1 cells MCF10 CA1a cells
% a
popt
otic
cel
ls
µM CDDO-Im µM CDDO-Im
Triterpenoids induce apoptosis
Prevention and treatment
of lung carcinogenesis
by triterpenoids
8 weeks of age
1 week after carcinogen
14-20 weeks treatment
Latency period
Control diet
Chemopreventive agents in diet
Heavy tumor burden
Reduced tumor burden
Prevention of lung carcinogenesis - A/J mouse model
Administer Carcinogen
(vinyl carbamate)
Vinyl carbamate induces highly invasive carcinomas
High grade carcinoma invading into a bronchus
CDDO-methyl ester and CDDO-ethyl amide prevent lung carcinogenesis
Control CDDO-ME 60 mg/kg diet
CDDO-EA 400 mg/kg diet
CDDO-methyl ester and CDDO-ethyl amide prevent lung cancer
Control CDDO-Me CDDO-EA
Ave tumor size, mm3 (% control)
2.2 (100%)
0.2 * (9%)
0.2 * (9%)
Ave tumor burden, mm3 (% control)
7.2 (100%)
0.1 * (2%)
0.2 * (2%)
*, P < 0.05 vs. control
8 weeks of age
8 wks
Treat 12 wks
Control diet
Therapeutic agents in diet
Administer Carcinogen
(vinyl carbamate)
Treatment of lung carcinogenesis - A/J mouse model
Heavy tumor burden
Reduced tumor burden
C/P injections
Triterpenoids protect against carboplatin/paclitaxel toxicity – Survivors after 5 C/P injections
Treatment Survivors
C/P WITHOUT triterpenoid 3/8 (38%)
C/P WITH triterpenoid 14/16 (88%)
8 weeks of age
12 wks
Treat 12 wks
Control diet
Therapeutic agents in diet
Administer Carcinogen
(vinyl carbamate)
Treatment of lung carcinogenesis - A/J mouse model
Heavy tumor burden
Reduced tumor burden
C/P injections
Treatment Survivors
Carboplatin/paclitaxel alone 21/23 (91%)
Triterpenoids + C/P 31/32 (97%)
Triterpenoids protect against carboplatin/paclitaxel toxicity – Survivors after 5 C/P injections
0
4
8
12
16
control CDDO-ME CDDO-EA
NQ
O1
mR
NA
(F
old
indu
ctio
n vs
. con
trol
) Triterpenoids increase NQO1 mRNA in PBMCs
Triterpenoids increase NQO1 enzyme activity in A/J mice
NQ
O1
enzy
me
activ
ity
∆ in
Abs
orba
nce/
mg
of P
rote
in
Liver Lung
Control lungs CDDO-Me + C/P
The combination of CDDO-Me and C/P inhibits lung carcinogenesis in A/J mice
Control CDDO-ME 80
mg/kg diet
Carboplatin &
Paclitaxel
CDDO-Me +
C/P
Ave # of tumors/slide (% control)
3.5 (100%)
2.5 (71%)
2.1 * (62%)
1.5 * (43%)
Ave tumor volume (mm3) per tumor (% control)
4.6 (100%)
1.6 * (34%)
1.5 * (33%)
1.0 * (21%)
Ave tumor volume (mm3) per slide (% control)
15.9 (100%)
3.9 * (25%)
3.3 * (21%)
1.4 ** (10%)
* , P < 0.05 vs. control **, P < 0.05 vs. Me and C/P
Histopathology of tumors in A/J Mice
n = 22 slides, 55 tumors
Control
30%
70%
n = 52 slides, 180 tumors
n = 32 slides, 45 tumors
High grade
Low/med grade
*, p < 0.001 vs control
CDDO-Me
45% *
CDDO-Me + C/P
57% * 64% *
36% *
n = 42 slides, 90 tumors
Carboplatin/Paclitaxel
55% *
43% *
100X 400X
High grade tumors in control group
100X 400X
Treatment – Carboplatin/Paclitaxel
Treatment – Triterpenoid + Carboplatin/Paclitaxel
100X 400X
Summary
• Low doses of triterpenoids activate the Nrf2 cytoprotective pathway and reduce inflammation and ROS
• High doses of triterpenoids increase ROS and induce apoptosis in cancer cells
• Triterpenoids also reduce tumor burden and enhance treatment with carboplatin and paclitaxel in experimental lung cancer
Acknowledgements
Dartmouth Michael Sporn Renee Risingsong Darlene Royce Charlotte Williams Ryan Collins Andrew Place Nanjoo Suh
Duke Medical School Thomas Sporn
Dept of Chemistry Gordon Gribble Tadashi Honda
DHMC Ethan Dmitrovsky Candice Black Eric York
Funding Robert E. Gosselin Fellowship Sidney Kimmel Fdn for Can Res American Cancer Society Breast Cancer Res Fdn Komen for the Cure NCI, NIH Reata Pharmaceuticals
Johns Hopkins Paul Talalay Albena Dinkova-Kostova Thomas Kensler Melinda Yates