Download (COX-2)?

Phytochemicals in Cancer
Chemoprevention
MJ Wargovich
[email protected]
What Is
Chemoprevention?
 The science of trying to apply
natural and synthetic compounds to
interfere with the earliest stages of
carcinogenesis, before invasive
cancer appears
The Ideal Chemopreventive
Agent
 Is effective
 Easily administered
 Preferably once/twice day
 Little or ideally no toxicity
 Affordable
Mechanisms of Chemoprevention
 Antioxidants: defense against radicals
 Phase 1 enzyme inducers
 Phase 2 enzyme inducers
 Anti-proliferative agents
 Anti-hormonal compounds
 Disruption of mutational gain or loss of
function
 Epigenetic effects
Antioxidants
 Oxygen is actually a very toxic substance
 Antioxidants are the first line defense
mechanism against oxidative damage in
plants
 Fruits and vegetables are loaded with
antioxidant chemicals
Total Daily Intake of
Antioxidants
Polyphemols
Vitamin C
Carotenoids
Antioxidant Potential of Fruits and
Vegetables
1.4
mmol Trolox Eq/d
1.2
1
0.8
0.6
0.4
0.2
0
er
a
rr
be
w
ra
St
le
pp
A a
n
na
Ba e
ng
ra
O
on
ni
O
to
ta
Po o
at
m
To
e
in
W
Be
Te
y
Flavonoids: naturally occurring low molecular wt
phenols consisting of 2 benzene rings linked via a
heterocyclic pyrone or pyran ring
 Anthocyanin - berries
 Flavanone
- citrus
 Flavanol
- red wine
teas
chocolate
fruit
 Flavonol
- fruit
vegetables
 Hydroxycinnamates most fruit & some vegetables
STRUCTURAL REQUIREMENTS FOR
H-DONATING ANTIOXIDANT ACTIVITY:
ortho-dihydroxy substitution in B-ring
2,3-unsaturation in C-ring
4-carbonyl group
OH
OH
HO
O
A
B
C
OH
OH
Bors et al. 1990; Rice-Evans et al. 1996;
O
QUERCETIN
Flavonol
Flavanol
e.g. quercetin
e.g. epicatechin
onion, cranberry, red apple
many fruit and vegetables
red wine, green tea,
as procyanidins in apple, chocolate
OH
OH
Flavanone
OH
OH
HO
O
e.g. hesperetin
HO
Citrus fruit, orange
O
OH
OH
OH
O
OH
OCH3
Anthocyanidin
HO
O
e.g. cyanidin
OH
Hydroxycinnamate
e.g. caffeic acid
major constituents of dark
red fruit berries e.g. raspberries
OH
OH
O
most fruit especially tomato, apple
some vegetables e.g. egg plant
grains
OH
COOH
+
HO
O
OH
OH
OH
OH
Green Tea
Camellia sinensis
 Epidemiologic studies support a protective
effect for green tea but not black tea in
prevention of certain cancers
 Animal studies are highly supportive of a
preventive effect of green tea, GTP, and
purified polyphenols, especially EGCG
against certain cancers
9
OH
3'
HO
B
O
A
OH
4'
O
OH OH
C
5
OH
3
O
H3C
OH
O
O
OH
(-)-Epigallocatechin-3-gallate (EGCG)
(-)-Epigallocatechin (EGC)
(-)-Epicatechin-3-gallate (ECG)
(-)-Epicatechin (EC)
N
CH3
N
N
N
CH3
Caffeine
(1,3,7-trimethylxanthine)
Evidence for Tea Drinking and
Prevention of Human Cancer
 Ecological Studies
 Cohort Studies
 Case-Control Studies
Bioflavonoids
 Naturally occurring chemicals present in
many fruits and vegetables
 Major flavonoids in onion are quercetin
and its glycoside, rutin
 Can reach appreciable levels in onions
but tea also is a major source
Bioflavonoids and
Reduced Risk for Heart
Disease
 Quercetin inhibits oxidation of LDL
cholesterol
 Inhibits development of fatty streaks in
animals
Agents That Influence Drug
Metabolism
Surh et al Nat Reviews 3:2003
Garlic
Allium sativum
 Consumption in China and Italy linked
to lower gastric cancer risk
 Sulfur compounds inhibit cancers of
colon, breast, esophagus, lung, skin
 Primarily acts through modulation of
Phase 1 ( activation) and Phase 2
(detoxification) enzymes
Organosulfur Compounds in Garlic
 Member of the plant genus
Alliin
Allinase
Allicin
Water soluble
compounds
Lipophilic compounds
L-cysteine
diallyl disulfide
S-allyl-l-cysteine
methyl propyl sulfide
S-methyl-l-cysteine
dipropyl disulfide
L-methionine
dimethyl trisulfide
diallyl sulfide
methyl propyl trisulfide
methyl propyl
thiosulfinate
Allium
 Over 20 different compounds
in garlic are being tested for
cancer inhibition.
 Diallyl sulfide was the first
organosulfur compound
known to inhibit
experimentally-induced
cancer.
 Proposed mechanisms of
chemoprotection lie in
modification of carcinogen
metabolism and/or
detoxification.
Cyp2e1
 One subset of many cytochromes
P450
 Main task is to activate compounds;
eliminate them from the body
 This enzymes metabolizes many small
molecular weight compounds
 Ethanol, nitrosamines, hydrazines
CYP2E1 and Colon Cancer
Dimethylhydrazine (DMH) Model
CH3-NH-NH-CH3
LIVER
COLON
1,2-Dimethylhydrazine
CH3-N=N-CH3
azomethane
CYP2E1
CH3-N=N-CH3
CH3-N N
methyldiazonium
O
Spontaneous
breakdown
H3C
+
azoxymethane
CYP2E1
CH3-N=N-CH2OH
methylazoxymethanol
N2
Typical Chemoprevention Assays
in Rodents
Chemopreventive activity of oilsoluble OSCs in ACF assay
(M. Wargovich, 1997)
Garlic Compounds and Phase II
Enzymes
UDP-Glucoronosyltransferase
activity
Glutathione-S-transferase activity
2500
80
2000
60
1500
40
1000
20
500
0
control
DADS
DPDS
liver
intestine
0
control
DAS
DADS
DPS
DPDS
Antiproliferative or Growth
Suppressing Agents:
Modulation of Cell Signaling
COX Inhibitors Reduce
Colorectal Carcinogenesis
– Observational Data
Polyp/Adenoma
Polyp/Adenoma Incidence
Incidence
Aspirin
0.52
Greenberg ‘93
0.61
Suh ‘93
•
•
Prospective
Retrospective
0.65
Giovannucci ‘94
0.6
Rodriguez ‘00
NSAIDs
Logan ‘93
Martinez ‘95
Peleg ‘96
Sandler ‘98
BreuerBreuer-Katchinski ‘00
0
Cancer
Cancer Incidence
Incidence
Kune ‘88
Rosenberg
‘91
0.49
Suh ‘93 – Males
0.36
Suh ‘93 – Females
Peleg ‘94
0.31
Schreinemachers ‘94
Giovannucci ‘94
0.56
Giovannucci ‘95
PaganiniPaganini-Hill ‘89, ‘91, ‘95
0.21
LaVecchia ‘97
Sturmer ‘98
Neugut ‘98
1 Bucher ‘99
Estimated
Relative
Garcia
Rodriguez Risk
‘01
Peleg ‘96
Muscat ‘94 – Females
Muscat ‘94 – Males
Müller ‘94
Pinczowski ‘94
Bansal ‘96
Reeves ‘96
Rosenberg ‘98
Smalley ‘99
Collett ‘99
Langham ‘00
0.6
0.5
•
•
0.24
0.54
0.08
Prospective
Retrospective
0.74
0.68
0.56
1.5
0.7
1.07
Cancer
-Associated Mortality
Cancer-Associated
Mortality
0.32
0.45
2
0.50
0.58
0.25
Thun0.32‘91 – Females
0.64
0.38
• Prospective
• Retrospective
0.6
0.84
Thun ‘91
0.65 – Males
0.70
0.49
0.57
Giovannucci ‘94
0.76
0
1
0.51
2
Estimated Relative Risk
Bansal ‘96
0
0.68
1
Estimated Relative Risk
2
How Do NSAIDs Work?
 Aspirin transfers acetyl group to serine
on the cyclooxygenase enzyme,
blocking its affinity for arachidonic acid
 All other NSAIDs are competitive
inhibitors of the COX enzymes
BIOLOGICAL ROLE OF PROSTANOIDS
Inflammation and Cancer
 Chronic, clinically invisible,
unresolved inflammation may
create a very high risk for
common cancers
 Tumors corrupt the
inflammatory pathway to
survive
A model of chronic inflammation
setting a threshold for cancer
Chronic
Inflammation
Esophagitis
Gastritis
Colitis
Pancreatitis
Hepatitis
Initiation
ROS/RNS
Mutation
Growth
advantage
ROS/RNS
Cell damage
Tumor
Replacement
hyperproliferation
Promotion
 apoptosis
 angiogenesis
APC
mutation
K-ras
mutation
Loss of 18q
p53
mutation
COX-2
Overexpression
Multistage Colon Carcinogenesis
NSAIDs Inhibit ACFs in the Colon
Indomethacin
100
A
Control
0.2 g/kg
80
0.4 g/kg diet
*
60
40
20
*
*
*
*
*
0
Total 1 Crypt 2 Crypts >3 Crypts
per Aberrant Crypt Focus
Number of Aberrant Crypts per Focus
Number of Aberrant Crypts per Focus
Ibuprofen
100
A
Control
0.025 g/kg
80
0.5 g/kg diet
*
60
*
40
*
*
20
0
*
Total 1 Crypt 2 Crypts >3 Crypts
per Aberrant Crypt Focus
Sulindac sulfide
Number of Aberrant Crypts per Colon
200
180
160
140
120
100
80
60
40
20
0
Control
0.16 g/kg diet
0.32 g/kg diet
*
*
*
*
*
**
*
200
180
160
140
120
100
80
60
40
20
0
Control
0.5 g/kg diet
1 g/kg diet
*
*
*
Total 1 Crypt 2 Crypts >3 Crypts
per Aberrant Crypt Focus
200
180
160
140
120
100
80
60
40
20
0
Control
1 g/kg diet
2 g/kg diet
*
*
* *
*
*
Total 1 Crypt 2 Crypts >3 Crypts
per Aberrant Crypt Focus
Sulindac sulfone
Number of Aberrant Crypts per Colon
Number of Aberrant Crypts per Colon
Sulindac sulfoxide
*
**
* *
**
Total 1 Crypt 2 Crypts >3 Crypts
per Aberrant Crypt Focus
NSAIDS, COX Specificity and Chemoprevention
COX 1
100%
0%
COX 2
Indomethacin
0%
Sulindac
Piroxicam
Diclofenac
Naproxen
Meclofenamate
Etodolac
Nimsulide
Celecoxib
100%
Toxicity of NSAIDs
Compound
Toxicity*
 Ibuprofen
-
 Diclofenac
 +/-
 Naproxen
 +/-
 Ketoprofen
+
 Indomethacin
 +++
 Piroxicam
 +++
Inhibit ACF?
+
+
+
++
+++
+++
* Clinical manifestations: Colitis, perforation, fistulae, GI bleeding
Peptic and small bowel ulcers, strictures
Multiplicity (tumors/animal)
Celecoxib Inhibits Tumor Multiplicity in
the MIN Mouse Model
35
Early treatment
(days 30-80)
30
35
30
25
25
20
20
15
15
*
10
*
5
0
0
500
1500
50
Celecoxib Piroxicam
(mg/kg diet)
*
*
10
5
Vehicle
Late treatment
(days 55-80)
Vehicle
150
500
Celecoxib
1500
50
Piroxicam
(mg/kg diet)
* P < 0.05; n=12/group
Jacoby et al: Cancer Res 60:5040-4, 2000
Effect of COX-2 Selective Inhibition on Colorectal
Adenomas in Patients with FAP
Steinbach et al, NEJM, 2000
Plant-based
NSAIDS...
Herbal NSAIDS
 Overuse could potentially result in bleeding,
prolonged platelet aggregation, and
ulceration
 Combined use might reduce the need for high
strength NSAIDs
 Could help in long term chemoprevention
trials for colon and breast cancer
Are there plant-based
NSAIDS?
 If so, the source should:
 Have anti-inflammatory effects
 Be available for oral use
 Be safe
 Efficacious
Resveratrol (Red Grapes)
 A phytoalexin in red grapes
 Inhibits cell transformation in cultured
tumor cells
 Inhibits cell proliferation in cells in culture
and in tumors grown in mice
 Inhibits mammary cancer in rats
 Induces apoptosis
Rosemary
 Rosemary is a potent source of
antioxidants
 Three antioxidants have cancer preventive
properties:
rosmarinic acid
ursolic acid
caffeic acid
Gingko biloba
 Widely used in Europe
  cognitive function
 No side effects in > 10,000
patients
 Useful in dementia and
Alzheimer’s
 Anti-inflammatory
 Anti-oxidant
Gingko biloba
Turmeric
 The yellow spice used in
cooking Indian dishes
 Yellow color due to phenolic
compound-curcumin
 Strongly antioxidant
 Suppress many types of
tumors in animal
 Clinical studies underway
Curcuma longa
What is Cyclooxygenase-2 (COX-2)?
Cytokines
growth factors
tumor promoter
Arachidonic acid
Aspirin
NSAIDs
COXIBs
COX-1
COX-2
(Inducible)
Prostaglandin H2
Prostaglandin synthase
Prostaglandins
Increased cell
proliferation
Inhibition of
apoptosis
Stimulation of
angiogenesis
Colon Cancer
Increased
invasiveness
Inhibitory Effect of EGCG on Cell Growth
MTS assay : cell viability
120
140
48h
24h
6h
100
48h
24h
6h
100
Viable Cells (% control)
Viable Cells (% control)
120
80
60
40
20
80
60
40
20
0
-20
0
100
200
300
400
500
0
0
EGCG (uM)
HT-29
50
100
150
EGCG (uM)
HCA-7
200
250
300
350
Inhibitory Effect of EGCG on COX-2 expression
EGCG
COX-2 protein
Western Blot Analysis
EGCG
0
10
50
125
200
250
1.00
0.87
0.93
0.54
0.43
0.86
0
10
50
125
1.00
0.80
0.95
0.70
200
250 (M)
COX-2
(72 kDa)
-actin
(47 kDa)
HT-29
HCA-7
0.56
0.37
Conclusion
EGCG
PGE2
Plasma membrane
PI3K
MAPK
COX-2 protein
ERK1/2
p38
Akt
COX-2 mRNA
Nuclear Membrane
p65 p50
NF-kB
COX-2/ other target gene
Inhibition of cell growth in colon
cells
Effect of EGCG on MAPKs and PI3K pathways
Dose:
0
10
50
125 200
250
(M)
PhosphoERK1/2
Decrease of ERK activation
ERK1/2
44kDa
42kDa
PhosphoAkt
Decrease of Akt activation
Akt
60kDa
Phosphop38
Increase of p38 activation
p38
43kDa
GSK-3
Axin
APC
Cell-cell adhesion
E-cadherin
, catenin
Beta Catenin
Degradation
c-jun
CD1
c-myc
TCF/LEF
Beta Catenin
A 3rd Pathway for Beta Catenin Regulation:
RXR- Epigenetics
J. Biol. Chem., Vol. 278,
Issue 32
, 29954-29962, August 8,
2003
Methylation of RXRα gene
DNA Methyltransferase
RXRα
APC
Protein
Accumulation of β-Catenin
in the cytoplasm then its nuclear
translocation
Activation of down stream targets
Such as cyclin D1 and c-myc
Uncontrolled cell proliferation
Tumor Formation
Intestinal Medium RXR alpha negative Foci
Intestinal Large RXRa negative Foci
The Effects of Green tea on The Formation of
RXRa Down-Regulating Foci
Average Number of Foci/Animal
5
4.5
4
3.5
3
*
Tea- Small RXRa DR
Foci
2.5
Without Tea- Small
RXRa DR Foci
2
1.5
1
0.5
0
1
AOM+GT
AOM+W
S+GT
S+W
Control
RXRα
 actin
Cyclin D1
 actin
AOM+GT
AOM+W
S+GT
S+W
RXR
 Mediate activity of retinoids along with
RARs
 Dysregulated in breast, lung, prostate
cancers
 CpG islands in the RXR promoter
 Hypermethylation  RXR expression
and heterodimer partners (RAR, VITD,
PPAR)
Wnt
Retinoids
EGCG?
frizzled
Dvl
Degradation
Axin
Mutan
t
Apc
Apc

GSK3




iDNMTs?
P


 ?

Lef1/TCF
 RXR ?
Wnt
Responsive
Genes
CpG Islands
In RXR Promoter?
?
= RXR, RAR, PPAR, VITD
Medicinal Plants from West AfricaNew COX Inhibitors?
 New collaboration
between my lab at
the University of
South Carolina and
The University of
Conakry and the
Ministry of Public
Health, Republic of
Guinea, West Africa
West African Traditional Medicinal Plants With
Anti-inflammatory Activity
Common
Name
Botanical
Name
Medicinal
Use
How Used?
Neem tree
Azadirachta
indica
Reduce fever
Decoction of
bark
Baobob tree
Adamsonia
digitata
GI pain; fever;
rheumatic pain
Decoction of
the leaves
African basil
Ocimum
viridae
Common
analgesic for
fever
Syrup made of
water extract of
leaves
Senegal
mahoghany
Khaya
sengalensis
Joint pain and
menstrual pain
Syrup made of
conc. extract of
stem & bark
MS spectrum of K-4 : 3,7 -dideaetylkhivorin
12-Jun-2006 16:34:56
Phytochemical_K-4 2006-6-12 104 (3.106) Sm (SG, 2x3.00); Cm (93:127)
1: TOF MS ES+
476
407.2061
100
Name:
O
Molecular formula: C28H38O8
425.2092
OAc
O
Molecular weight:
443.2285
O
O
3, 7-dideacetylkhivorin
502
Reference: Adesida, G.A.; Adesogan, E.K.; Okorie, D.A.; Taylor, D.A.H.Phytochemistry, 1971, 10, 1845
1005.4966
OH
HO
[2M+H]+
%
1022.5241
1027.4792
503.2484
[M+H]+
520.2820
1028.4617
521.2878
284.2733
1029.4785
382.2860
310.2859
525.2466
267.1077
987.4972
625.3002
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1043.4409
1050
1100
1150
m/z
1200
Bioactivity of the isolates K1-5
Caco2
Cell Viability (%)
120
100
80
60
K-A-4(IC50=35ppm)
40
K-A-5
20
0
0
2
20
50
[Concentration],ppm
100
200
Acknowledgments
 Wargovich Lab






G Peng
S Volate
A Issa
S Hudson
X Androulakis
S Cavitt
 Dr. S Muga
 Dr. T Smith
 Dr. Dan Dixon
 Supported by
NIH CA 96694
NIH CA 107138
 Ministry of Public Health,
Guinea
Dr. Y Koita
 EngenderHealth, Guinea
Dr. B. Toure
 Clemson University
Dr. Feng Chen