Screening for Anti-Cancer Agents in Herbal Medicines:

1
Screening for Anti-Cancer Agents in Herbal
Medicines Prostate whats in it for the
tomato ?
Emma Guns, Ph.D. Natural Health Products
Pharmacological Research Program
2
Screening for Anti-Cancer Agents in Herbal
Medicines Prostate whats in it for the
tomato ?
Emma Guns, Ph.D. Natural Health Products
Pharmacological Research Program
3
Anti-Cancer Activity should we focus on lycopene
?

• Both epidemiologic and case-control studies have
  associated increased consumption of tomato
  products and greater blood concentrations of
  lycopene with a reduced risk of mortality from
  several cancers, including prostate cancer.
• In prostate cancer a reduction in risk of 33 was
  found among men who consumed gt 10 servings of
  tomato products per week (30-50mg per day)
  (Giovanucci et al., 1995, JNCI 871767).
• Levels of serum PSA were found to decline in
  patients who received lycopene tomato extract
  (Kucuk et al., 2001, Cancer Epidemiol Biomarkers
  Prev 10861).
• Lycopene has now been implicated in epidemiologic
  studies to be preventive against a broad range of
  epithelial cancers (prostate, lung)

4
Lycopene

• Lycopene is abundant in several fruits including
  guava, watermelon, pink grapefruit, rosehips and
  tomatoes.
• Tomato products (such as tomato sauce, tomato
  paste and ketchup) are the major dietary source
  of lycopene.
• Lycopene is a carotenoid in the same family as
  ß-carotene.
• It is a powerful antioxidant that has shown
  beneficial activity in several diseases
  (cardiovascular, cancer, Alzheimers disease,
  multiple sclerosis).

5
Anti-Cancer Activity

• In vitro data conclusively supports an
  anti-proliferative effect of lycopene and other
  tomato derived carotenoids on prostate and other
  cancer cells (data from Guns lab to follow).
• Preclinical data in animal models suggests a
  preventive role for lycopene/tomato extracts
  (most evident for prostate and lung).
• Preventive effects have recently been shown to be
  enhanced in animals treated with whole tomato
  extracts vs lycopene (prostate cancer).
• Lycopene serum levels have recently been
  associated with more rapid clearance of an
  oncogenic HPV infection prevention of cervical
  cancer. Lycopene or carotenoids ?
• Results supporting therapeutic effects of
  lycopene in animal models are inconclusive.
  Lycopene vs whole tomato extract ?

6
Lycopene Quantitation in Lycomato
Lycomato 3 lycopene plus other carotenoids
Standard curve from HPLC for pure lycopene
obtained from LKT laboratories. HPLC chromatogram
for Lycomato shown below (lycopene RT 12.04
min)
0.30
0.20
Absorbance Units
0.10
0.00
-0.10
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Elution Time, Minutes
7
Lycomato? on Cytotoxicity LNCaP Cells
LYCOPENE CYTOTOXICITY AT 24 HOURS
(Crystal Violet Assay)
120.0
100.0
80.0
ABSORBANCE AT 592 nm
60.0
40.0
20.0
0.0
0
10
09
08
07
06
05
1.00E-
1.00E-
1.00E-
1.00E-
1.00E-
1.00E-
LYCOPENE CONCENTRATION (M)
The ability of lycopene to kill LNCaP cells in
culture was confirmed using a crystal violet
assay. Lycomato is cytotoxic to LNCaP cells at
concentrations of 1µM and above. The arrow marks
the hypothetical pharmacologically achievable
concentration.
8
Lycomato? on proliferation in LNCaP Cells
1µM
Nikita data
3.3µM
5µM
9
Lycomato? on Protein Kinase Phosphorylation
LNCaP cells were treated for 10 minutes with 10-8
M lycopene and protein was screened using Western
blot by Kinexus (www.kinexus.ca) for changes in
the phosphorylation states of an array of
proteins.
10
Lycomato? on Protein Kinase Phosphorylation
LNCaP cells were treated for 10 minutes with 10-8
M lycopene in lycomato and protein was screened
using Western blot by Kinexus (www.kinexus.ca)
for changes in the phosphorylation states of an
array of proteins.
11
Cell signaling made simple
Soluble growth factors
Extracellular matrix
Integrin
Surface receptor
Cytoskeletal tension
Ras
Rho
ERK
Cyclin D1 CDK 4/6
P21/p27
Cyclin E CDK 2
G0
pRb E2F
G1
M
S
G2
12
Cell signaling made simple
Soluble growth factors
Extracellular matrix
Integrin
Surface receptor
Cytoskeletal tension
Ras
Rho
ERK
Cyclin D1 CDK 4/6
P21/p27
Cyclin E CDK 2
G0
pRb E2F
G1
M
S
G2
13
Lycomato??on Retinoblastoma Phosphorylation
The Kinexus data indicated that lycomato altered
the phosphorylation state of the tumour
suppressor gene Retinoblastoma. This finding was
validated by using Western blot with lycomato
treated LNCaP cells
LNCaP cells were treated with lycopene in
lycomato and and a Western blot of cell lysates
was probed for changes in the phosphorylation
state of Retinoblastoma at several sites.
Phosphorylation at tyrosine 780 was markedly
decreased by lycomato treatment.
14
Cell signaling made simple
Soluble growth factors
Extracellular matrix
Integrin
Surface receptor
Cytoskeletal tension
Ras
Rho
ERK
Cyclin D1 CDK 4/6
P21/p27
Cyclin E CDK 2
G0
pRb E2F
G1
M
S
G2
15
Lycomato? on cdk or p27 protein levels
10 minutes 30 minutes 8 hours 24
hours
Lycopene uM
0
0
16
Cell signaling made simple
Extracellular matrix
IGF-1
Integrin
IGF-1 receptor
Cytoskeletal tension
Ras
Rho
ERK
Cyclin D1 CDK 4/6
p21/p27
Cyclin E CDK 2
G0
pRb E2F
G1
M
S
G2
17
Insulin-Like Growth Factor Axis Components

• Ligands
• Insulin-like Growth Factors I (IGF-I)
• Binding Proteins
• IGFBP 1 6
• BP-2 5 are upregulated in tumours and promote
  the IGF response,
• BP 3 is generally antagonistic to IGF-mediated
  signaling
• Receptors
• IGF-IR Tyrosine kinase

IGF-I
IGF-I
-ss-
-ss-
-ss-
-ss-
-ss-
-ss-
-ss-
-ss-
18
IGF-I and Cancer

• Serum IGF-I levels are correlated with increased
  risk for developing prostate cancer (Chan et al.,
  1998, Sci. 279563).
• Human population studies strongly indicate high
  circulating levels of IGF-1 and low IGF-BP3 with
  lung cancer (Wakai et al., Cancer Res. 2002).
• Serum lycopene levels inversely correlate with
  IGF-I levels (Mucci et al., 2001, BJU Int.
  87814).
• Lycopene treatment in MCF 7 cells (breast cancer
  cells) has been shown to downregulate IGF-IR
  signaling (Levy et al 1995, Nutr. Cancer 24257).
  
• Upregulation of IGF-BP3 has been inversely
  correlated with lung cancer incidence in ferrets
  exposed to smoke carcinogens (Lui et al., Cancer
  Res 2003).

19
IGF-I Receptor Expression
- - - -

Lyc-o-mato 48h
- - - -

- -
- -
- -
- -
R1881 10-9 M 48h
- -
- -
- -
- -
IGF-I 100 ng/ml 10 min
IGF-I Receptor (IGF-IR) was immunoprecipitated
from cells treated with 1?M lycopene in lycomato,
R1881, and/or IGF-I. Lycomato produced a
decrease in the expression of IGF-IR in the LNCaP
cells but had no effect upon the phosphorylation
state of the receptor.
20
Effects of Lycomato? on IGF Signalling
The impacts of lycomato treatment upon components
of the IGF signalling pathway were investigated
in LNCaP cells using Western blot.
IGFBP2 Expression
- - - -

Lyc-o-mato 48h
- - - -

- -
- -
- -
- -
R1881 10-9 M 48h
- -
- -
- -
- -
IGF-I 100 ng/ml 10 min
Protein was prepared from LNCaP cells treated
with 1?M lycopene in lycomato, the synthetic
androgen R1881, and/or IGF-I. Western blot shows
that lycomato induces increased expression of IGF
Binding Protein 2 (IGFBP2)
21
Lycomato Interaction with the Androgen Receptor
To determine whether lycomato was acting through
the androgen receptor a reporter gene assay was
used.
LNCaP cells transfected with AR and an androgen
responsive luciferase reporter were treated with
lycopene. At concentrations gt1?M an agonist
effect was seen in the absence of DHT. Lycomato
behaves as a weak antagonist against the action
of DHT.
22
Summary. whats in it for the tomato ?

• Lycomato does not alter cdk 1, 4, 5 or p27
  expression.
• Lycomato inhibits phosphorylation of the tumour
  suppressor, Rb, thus preserving growth
  suppression properties.
• Lycomato treatment opposes androgens effect on
  IGF-I signaling by decreasing IGF-I receptor
  expression.
• Analogous to androgen suppression, IGF-BP2 levels
  increase upon treatment with lycomato.
• In the presence of androgen, lycomato acts a weak
  antagonist of androgenic response. At higher
  lycomato concentrations stimulation of the
  androgenic pathway occurs in the absence of
  androgen.

23
And. is lycopene all thats in it for the tomato
?

• ?-carotene, ?-carotene, ?-carotene, phytoene,
  phytofluene all shown to accumulate in prostate
  tissue upon dietary inclusion of tomato.
• In Acycloretinoic acid has been shown to be an
  oxidative product of lycopene in pig and human
  liver fractions.
• Bioleau et al. have recently shown whole tomato
  extract had greater efficacy than lycopene
  treatment alone on lethal prostate cancer
  development in a rat model PC induced by
  N-methyl-N-nitrosourea and testosterone
  treatment.

24
And. is lycopene all thats in it for the tomato
? NO

• ?-carotene, ?-carotene, ?-carotene, phytoene,
  phytofluene all shown to accumulate in prostate
  tissue upon dietary inclusion of tomato. Lycopene
  accumulation is most pronounced.
• In Acycloretinoic acid has been shown to be an
  oxidative product of lycopene in pig and human
  liver fractions.
• Bioleau et al. have recently shown whole tomato
  extract had greater efficacy than lycopene
  treatment alone on lethal prostate cancer
  development in a rat model PC induced by
  N-methyl-N-nitrosourea and testosterone
  treatment.
• .likelihood is that lycopene has some
  anti-proliferative activities which are enhanced
  in the context of tomato extract either in an
  additive or synergistic manner.

25
Many Thanks to

• Dr. Simon Cowell
• Nikita Ivanov
• Dr. Michael Cox
• Dr. Paul Rennie
• Catherine Wood
• Venket Rao, U. of Toronto
• Dr. Vasu Venketswaran, Princess Margaret
  Hospital, University of TO
• Prostate Cancer Research Foundation of Canada
  (research grant)
• Canadian Prostate Cancer Research Initiative
  (Bio-NET grant)