CHEMOPREVENTION: PRINCIPLES
AND PROSPECTS
Gary D. Stoner, Ph.D.
Department of Internal Medicine
College of Medicine and Public Health
The Ohio State University
Strategies for Cancer Prevention
• Reduce Exposure to Environmental
•
Carcinogens
Identify Individuals at High Risk
• Genetic factors
• Biochemical factors
• Chemoprevention
• Dietary factors
• Synthetic agents
Chemoprevention
The prevention of cancer development
by dietary or synthetic chemicals
Chemoprevention Drug
Development
Preclinical:
Natural Products
Synthetic Agents
In vitro
Assays
Antimutagenic
Cell Transformation
etc.
In vivo
assays
Clinical:
Phase I
Phase II
Phase III
Tumors
Toxicity
Efficacy
Efficacy
Biomarkers
Metabolism
Biomarkers
Cancer
Toxicology
Endpoint
Qualities of an Ideal Chemopreventive Agent
Chemopreventive Agents
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•
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•
•
•
•
Allyl sulfides
Calcium
Coumarins
Dithiolethiones
Indoles
Isoflavones
Isothiocyanates
•
•
•
•
•
•
•
Monoterpenes
NSAIDs
Polyphenols
Protease inhibitors
Retinoids
Selenium
Vitamins (A, C, D3,E)
Classes of Chemopreventive Agents
“Blocking” agents
• Inhibitors of tumor initiation
“Suppressing” agents
• Inhibitors of tumor promotion/
progression
Blocking (Anti-Initiating) Agents
• Influence metabolic activation of
carcinogens
• Deactivate/detoxify carcinogens
• Scavenge electrophiles and oxygen
radicals
• Increase level of fidelity of DNA repair
Carcinogen Metabolism
Carcinogen
Activation
Genetic (DNA)
Damage
Detoxification
Conjugates
Excreted
Cancer
Agents That Influence Carcinogen
Activation
Mechanism
Examples
Inhibition of cytochrome
dithiocarbamates
P450
ellagic acid
diallyl sulfide
isothiocyanates
Induction of cytochrome
P450
M.A. Morse and G.D. Stoner, 1993
indole-3-carbinol
-naphthoflavone
Isothiocyanates
N
N
CH3
N=C=S
O
N-Nitrosomethylbenzylamine (NMBA)
3-Phenylpropyl isothiocyanate (PPITC)
N=C=S
N=C=S
Benzyl isothiocyanate (BITC)
N=C=S
Phenethyl isothiocyanate (PEITC)
4-Phenylbutyl isothiocyanate (PBITC)
N=C=S
6-Phenylhexyl isothiocyanate (PHITC)
NMBA Metabolism
O
(NMBA) H3C N CH2
cytochrome p450
O
N OH
-hydroxynitrosamine
H3C N CH
H
C O
methyldiazohydroxide [ CH3N = NOH ]
benzaldehyde
methyldiazonium ion [ CH3+N  N ]
carbonium ion [ CH3+]
O
NH
H2N
N
OCH3
CH3
N
N
R
N7-Methylguanine
N
N
H2N
N
N
R
O6-Methylguanine
Protocol For Identification of
Blocking Agents in Rat Esophagus
NMBA (1x/wk/15 wks)
0
2
4
19
Inhibitor
25
Effect of Isothiocyanates on DNA Methylation
and Tumorigenesis in Rat Esophagus
O6-Methylguanine
Treatment
(mol/mol guanine)
Tumors / Rat
NMBA
19.6  0.6
7.2  0.7
NMBA + BITC
16.0  0.4
6.5  0.6
NMBA + PEITC
2.1  0.2a
0.1  0.1a
NMBA + PPITC
3.0  0.3a
0.0  0.0a
NMBA + PBITC
7.0  0.6a
4.0  0.4
NMBA + PHITC
20.7  1.0
11.9  0.7a
ap
< 0.05
Agents That Deactivate/Detoxify
Carcinogens
Mechanism
Introduction of phase II
enzymes
• glutathione-Stransferases
• UDP-glucuronyltransferases
• glutathione peroxidase
Examples
• allyl sulfides,
dithioethiones,
isothiocyanates
• polyphenols
• selenium
O
S
H3C
N
C
S
Sulforaphane
Y. Zhang, P. Talalay, C.G. Cho, and G.H. Posner, A major inducer
of anticarcinogenic protective enzymes from broccoli: isolation
and elucidation of structure. Proc Natl Acad Sci USA 89 2399 (1992)
Induction of QR and GST in Mouse
Tissues by Sulforaphane*
(15 mol / mouse / day)
Liver
Forestomach
Small
Intestine
Lung
QR
2.45  0.07
1.70  0.18
2.34  0.19
1.37  0.14
GST
1.86  0.08
1.98  0.08
2.13  0.20
1.17  0.09
Enzyme
QR = Quinone reductase
GST = Glutathione-S-transferase
Protective Effects of Sulforaphane
on DMBA-Induced Mammary
Tumors in Rats*
Treatment
Control
Tumor Incidence
(% of control)
Tumor Muliplicity
Tumor Weight
(% of control)
(gm) (% of control)
65.2 (100)
1.43 (100)
2.79 (100)
75 mol
35.0 (53.8)
0.45 (31.4)
1.24 (44.4)
150 mol
26.3 (40.3)
0.26 (18.2)
0.68 (24.4)
Sulforaphane
*Y.Zhang, PNAS 91: 3147, 1994
Suppressing Agents
• Inhibit cell growth
• Stimulate cell function
• Stimulate apoptosis
• Inhibit blood vessel formation
(angiogenesis)
POLYAMINE SYNTHESIS
Ornithine
ODC
Putrescine
Spermidine
Carbon dioxide
Chemopreventive Agents:
a. difluromethylornithine (DFMO)
b. curcumin
Spermine
DFMO as an Anti-Tumor Agent
Azoxymethane-induced rat colon tumor model
•
•
•
inhibits tumor incidence and multiplicity
decreases cell proliferation
decreases activated ras expression
NMBA-induced rat esophagus model
• inhibits tumorigenesis when given before,
during and after NMBA
•
decreases cell proliferation and increases apoptosis
Human Neoplasias
•
Barrett’s esophagus; colonic polyps; oral
leukoplakia; uterine cervix (CIN 3)
Action of COX Enzymes
COOH
Arachidonic Acid
Cyclooxygenase Activity
COX-1
COX-2
PGG2
Peroxidase Activity
PGH2
Prostacyclin
PCI2
Prostaglandins
PGE2, PGF2, PGD2
Thromboxane
TxA2
Inhibitors of COX Activity
NSAIDs
•
Aspirin, Indomethacin, Ibuprofen, Naproxen
• Effective chemoprevention agents in rodent
skin, breast and colon tumor models
• Aspirin may be effective as a prevention
agent in human esophageal cancer
Selective COX-2 inhibitors
•
Celecoxib, Rofecoxib and others
• Animal studies indicate that these agents
are as effective
and better tolerated than NSAIDs
COX Activity and Human
Cancers
Human Breast
• Elevated COX-2 activity is seen in tumors
• NSAID use is associated with reduction in
susceptibility to breast cancer (Robertson, Harris)
Human Colon
• Colon polyps have elevated COX-2 activity
• Treatment of FAP patients with Celecoxib leads to
polyp regression
Transcription factors
Growth factor receptor
Other stimuli
Elevated ROS levels
ras
ceramide
Elevated NFkB activity
raf
MEK-1
Erks
Elevated AP-1 activity
Altered gene expression
Increased cell proliferation, resistance to apoptosis
Transcription Factors and
Tumorigenicity
AP-1
• Regulates transcription of genes involved
•
•
•
in cell proliferation, differentiation and
modulation of extracellular matrix
Elevated AP-1 activity is causally related
to tumor promotion and progression in the
mouse skin model
Agents that inhibit AP-1 activity, such as
retinoids, are effective chemoprevention
agents in this model
The role of altered AP-1 activity in other
epithelial tumors is unknown at present
Transcription Factors and
Tumorigenicity
NFkB
• Elevated activity seen in mouse skin
•
•
•
model
Elevated in human Barrett’s
esophagus and esophageal cancers
In cell culture models, elevated NFkB
activity correlates with increased
resistance to apoptosis
Selective inhibitors of NFkB activity
are unavailable at present
Tumor Angiogenesis
• Neovascularization is essential for
sustained tumor growth as well as
establishment of metastases.
• Anti-angiogenesis factors include
endostatin, a product of plasminogen,
that inhibits new vessel growth in
tumor xenografts in mice. Endostatin is
presently being evaluated in human
clinical trials for its efficacy against
metastatic disease.
Human Clinical Trials
Phase I
• Toxicity, metabolism
Phase II
• Biomarkers
Phase III
• Long term prospective studies
Target Populations for
Chemoprevention
High Risk Individuals
• Hereditary predisposition
• High exposure to carcinogens
• precancerous lesions
• previous treatment for cancer
General Population ?
Chemoprevention Trials
Evaluation
• Modulation of Intermediate Endpoints
(Biomarkers)
• Prevention and/or Reversal of
Precancerous Lesions
• Extension of Latency Period for Cancer
Development
• Reduction of Cancer Incidence and
Mortality
Phase I Trials
Population
• Usually high risk
• 18-24 subjects
Escalating Dose
Objectives
• Determine pharmacokinetic parameters
• Minimum and maximum tolerated dose
• Time course of the reversibility of side effects
• Dose selection for Phase II trials
Phase I Clinical Trial of Freeze-Dried
Black Raspberries
• 10 normal volunteers, > 18 years of
age
• “Phenol-free” diet
• 90 g BRB / day, 14 days
• Blood and urine
• Clinical signs of toxicity
Results of Phase I Clinical Trial of
Freeze-Dried Black Raspberries
• Well tolerated
•  blood levels of ellagic acid and
several anthocyanins
• Reduced levels of 8-OH-dG in urine
Phase II Trials
Randomized placebo controlled
Population
• High risk
• 100-200 subjects
Objectives
• Further evaluation of toxicity
• Dose response vs. biomarker modulation
• Dose selection for Phase III trials
BIOMARKERS
Anti-Initiation
1. Phase I enzyme activities
2. Phase II enzyme activities
3. DNA repair enzyme activities
4. DNA adducts (carcinogen and free radicals)
5. Hemoglobin adducts
6. Urinary metabolites
•
•
Detoxification products
Mutagenicity assays
7. Micronuclei, binucleated cells
8. Chromosome damage (FISH)
BIOMARKERS
Anti-Promotion/Progression
1.
Proliferation Index: PCNA, Ki67, BrdU, 3H-thymidine
2.
ODC activity; polyamine levels
3.
Growth factor and receptor expression
4.
Prostaglandin levels
5.
Cell differentiation
• Blood group antigens
• Keratins
• Retinoid receptors
• Squamous metaplasia mucociliary differentiation
6.
Apoptosis
7.
Morphometric
1. DNA ploidy
2. Nuclear/nucleolar-morphology
Barrett’s Esophagus
Normal
Barrett’s Esophagus
Esophageal Cancer
Multi-Center Clinical Trial Study Schema
Barrett’s Esophagus
Intestinal Type Metaplasia
Low Grade Dysplasia
Biopsies for Surrogate
Endpoint for Biomarkers
Randomize
DFMO 900mg, Once Daily
x 26 Weeks
Placebo, Once Daily
x 26 Weeks
Endscopy with Biopsies for
Endpoint Biomarkers
Endoscopy with Biopsies for
Endpoint Biomarkers
26 Weeks, No Treatment
Endoscopy with Biopsies for
Endpoint Biomarkers
26 Weeks, No Treatment
Endoscopy with Biopsies for
Endpoint Biomarkers
Overall Study Objectives
Determine if oral DFMO given in a randomized, placebo-controlled,
double-blinded study will significantly alter:
Primary Endpoint:
Ki-67 labeling index
Secondary Endpoints:
p53 protein accumulation
Levels of Cyclin D1, EGFR, or TGF-alpha
DNA ploidy, nuclear and nucleolar morphometry
Cellular apoptosis
Pathology & morphology of Barrett’s
Phase III Strategies
• Target Populations
• Length of Study
• Conclusiveness of Study
• Statistical Methods
Combination Strategies
• Synergistic Activity
• Lower Doses of Each  Lower Toxicity +
Fewer Adverse Effects
• Mechanistic Combination Strategy
(Anti- Initiator/ Anti-Proliferator)
Collaborators
OSU
OSU - Former Students
Robeena Aziz
Rajaram Gopalakrishnan
Peter Carlton
Leena Khare
Tong Chen
Laura Kresty
Ashok Gupta
Dian Wang
Keith Harris
Qian-Shu Wang
Tamaro Hudson
American Health Foundation
Beth Liston
Fung-Lung Chung
Mark Morse
Univ. of Minnesota
Ron Nines
Stephen Hecht
Haiyan Qin
Sharon Murphy