Transcript Exercise alters the IGF axis in vivo and increases p53

Exercise and Prostate Cancer
Jamie McClellan
Department of Exercise Science
Those who think they have not time for bodily
exercise will sooner or later have to find time for
illness. ~Edward Stanley
Lack of activity destroys the good condition of
every human being, while movement and
methodical physical exercise save it and
preserve it. ~Plato
Exercise alters the IGF axis in
vivo and increases p53 protein in
prostate tumor cells in vitro
Pak-Shan Leung, William J.
Aronson, Tung H. Ngo, Lawrence
A. Golding and R. James Barnard
Journal of Applied Physiology 96:450-454, 2004.
Epidemiology of Prostate Cancer
Prostate cancer is 2nd most common
cancer among men in US.
Approx. 1 in 11 men will eventually be
diagnosed with prostate cancer.
Twice as common among Black American
males as it is among White American
males.
Found most often in males over 50; more
than 70% of men diagnosed with prostate
cancer are over the age of 65 years.
Environmental Factors
Western High-fat Diet
Physical Inactivity
20% of Asian men have malignancy of the
prostate at autopsy, even though prostate
cancer is rare among Asian men
– Asian men have the same incidence rate of
prostate cancer when they migrate to the US
and adopt our lifestyle of a high-fat diet and
very little physical activity
– It could be the American lifestyle that leads to
the progression of prostate cancer into its later
stages of invasiveness and metastasis
Definitions
Physical Fitness
– Measure of a person’s ability to perform
physical activities that require endurance,
strength, or flexibility
Physical Activity
– Any bodily movement produced by skeletal
muscles that results in an expenditure of
energy (kcals) and includes a broad range of
occupational, leisure-time, and routine daily
activities
Exercise
– Planned physical activity that is done to
improve or maintain one or more components
of physical fitness
ACLS Cohort and
Prostate Cancer Risk
IGF Axis
Consists of:
– IGF-I
– IGF-II
– IGF receptors
– Six different IGFBPs
How IGF-1 Works
Stimulates cell proliferation and
inhibits apoptosis
IGF-1R is activated by ligands (IGF-1
and IGF-2) and provides protection
against pro-apoptotic factors
IGFBP-1 sequesters IGF-1 so that it
cannot interact with IGF-1R
IGF-1 in Prostate Cancer
Positive association between prostate
cancer risk and circulating IGF-1 level
– Particularly strong in younger men indicating
the importance of IGF-1 in the initial
development of prostate cancer
IGF-1 one is used as a predictor of
prostate cancer and increased IGFBP-1 is
associated with a decreased risk of
prostate cancer
IGF-1 is directly correlated with tumor cell
growth and IGFBP-1 is inversely related
IGF1 and p53 Degredation
Model for IGF-I action after 4-NQO-induced DNA damage
Heron-Milhavet, L. et al. J. Biol. Chem. 2002;277:15600-15606
LNCaP vs LN-56
Both are prostate tumor cell lines
LNCaP cells are androgen dependent
LN-56 cells are derived directly from
LNCaP cells so they are genetically
identical except for a non-functional p53 in
LN-56 cell line
– GSE-56 sequence that acts as inhibitor of p53
was taken from retroviral vector and put into a
plasmid that was resistant to noemycin
– Plasmid was transfected into LNCaP cells and
GSE-56 cells were selected by adding
neomycin
Purpose of the Study
To measure the effects of exercise on
apoptosis, proliferation, and p53
production in serum-stimulated prostate
tumor cell lines
Focuses on the ability of IGF-1 to suppress
normal function of p53
p53 protects genome from mutations by
activating genes or factors to cause cell
cycle arrest, DNA repair, or to induce
apoptosis
Experimental Groups
Control Subjects
– At risk for prostate cancer based based on
sedentary lifestyle and diet (Western high-fat
diet)
– Normal PSA levels
Experimental Subjects
– At least 10yr in Adult Fitness Program at UNLV
(14.7yr average)
– 5 days per week for 1 hour, 45-50 minutes of
strenuous cardiovascular exercise
– No reported results of participation in the
program or fitness levels of the subjects
Serum Collection
Fasting blood
samples taken in
the morning were
allowed to clot and
then centrifuged
Samples analyzed
for IGF-1 and
IGFBP-1 by ELISA
Cell Culture
Cells grown in flask
in medium with
food, antibiotics,
and FBS
Maintained at 37
degrees and
supplemented with
CO2
Passaged to fresh
media every 3rd
day at 80%
confluence
Experimental Groups
Dish 1
– LNCaP cells with control serum
Dish 2
– LNCaP cells with exercise serum
Dish 3
– LN-56 cells with control serum
Dish 4
– LN-56 cells with exercise serum
Cell Preparation
Trypsan used to detach cells by digesting protein
and centrifuged to get pellet
Cells resuspended in fresh media
Trypan blue used to assess cell viability
Cells were plated and allowed 24 hours to
stabilize
Fresh media and either FBS or human serum (con
or ex) was added to the cells and incubated at 37
degrees for 48 hours
Growth Assay
CellTiter assay was used to measure
proliferation
MTS in the reagent is reduced into
formazan in the presence of proliferating
cells
Amount of formazan produced is directly
related to the amount of proliferation and
it is read on the spec or plate reader
Apoptosis Assay
Cells were plated and FBS or human serum
added (con or ex)
Cell Death Detection ELISA
– Measures mono- and oligo-nucleosomes in the
cytoplasm due to DNA degredation
– Nucleosomes bind to anti-histone anitbody via
their histone component and anti-DNA
peroxidase reacts with DNA part of the
nucleosome
ELISA
Enzyme-Linked ImmunoSorbent Assay
– Monoclonal antibody comes already attached
to the bottom of the plate
– Sample is added and the antibody attaches to
the substance of interest (p53 or PCNA)
– Polyclonal antibody (with enzymes) is added
and it attaches to the substance of interest
creating a sandwich
– The enzymes on the polyclonal antibodies will
generate a color signal proportional to the
amount of target substance present in the
original sample
PCNA and p53 Measurement
Cells were lysed and centrifuged to
obtain supernatant
Bradford protein assay on
supernatant to determine protein
content and concentrations adjusted
to 10 ug/mL
Cell lysate supernatant was used in
PCNA (proliferating cell nuclear
antigen) ELISA and p53 ELISA
Previous Research Results
Table 1. Comparison of body mass index and serum IGF axis for control and exercise
subjects
Control
Exercise
n
10
12
BMI
31.5±1.6
26.5±1
Insulin, pmol/1
110±38
41±6
IGF-I, ng/ml
336±41
128±12
22±6
42±8
IGFBP-1, ng/ml
Values are means ± SE; n, no. of subjects. Serum concentrations reflect
fasting levels. BMI, body mass index; IGFBP-1, IGF binding protein-1. All
exercise values were significantly different from control, P < 0.05. These
data were previously published (1).
Effect of Exercise on Growth
Figure 1
Effect of Exercise on PCNA
Figure 2
Effect of Exercise on Apoptosis
Figure 3
Effect of Exercise on p53
Production
Figure 4
Major Findings
Regular exercise alters serum factors that
allow LNCaP prostate cancer cells to
initiate apoptosis, where there is little
apoptosis when the control serum was
added to the cells
Serum changes due to exercise also
decreased cell proliferation/growth
Increased apoptosis and decreased
proliferation attributed to 100% increase
in p53
They never proved that the increase in
IGFBP-1 and the decrease in IGF-1 caused
this effect; it could have been many other
factors that changed in the exercise serum
p38 Pathway
IGF-1 suppresses
apoptosis and aids
proliferation via activation
of the p38 mitogenactivated protein kinase
pathway
LN-56 cells further suggest
the p38 pathway to
apoptosis
Increased p53 leads to
increased p21 which has
an effect on decreasing cell
cycle activity
With exercise, IGF-1
decreases and IGFBP-1
increases, so p53 is not
targeted for degradation
via p38 MAPK pathway
Model for IGF-I action after 4-NQO-induced DNA damage
Heron-Milhavet, L. et al. J. Biol. Chem. 2002;277:15600-15606
The Role of Insulin
Increased circulating insulin is associated
with increased risk of prostate cancer
Insulin stimulates the liver to produce
IGF-1 and inhibits the production of
IGFBP-1 and SHBG
Exercise increases insulin sensitivity by
increasing insulin receptors on muscle cell
membranes to sequester glucose into the
muscle to be directed into glycogen,
lowering fasting insulin levels
Increases in IGF-1 and decreases in
IGFBP-1 are thought to be the most
important factors, not insulin
Insulin Receptors
Alternate Pathway to Apoptosis
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Increased p53 with exercise could increase the ratio of Bax to
Bcl-2 which activates the caspase cascade and leads to
apoptosis
Previous Studies on Exercise
and Prostate Cancer
When IGF-1 was added to the serum of
the exercise group, the reduction in LNCaP
growth was completely eliminated
After 11 days of diet and exercise, same
serum changes in insulin, tumor growth,
and apoptosis, while the men remained
obese (fitness more important than BMI)
These data support
epidemiological reports that
there is a reduction in prostate
cancer risk in men who are
physically active