Exercise and Prostate Cancer

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Exercise and Prostate Cancer

• Jamie McClellan
• Department of Exercise Science

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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

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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 96450-454, 2004.
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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.

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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

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Definitions

• Physical Fitness
• Measure of a persons 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

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ACLS Cohort and Prostate Cancer Risk
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IGF Axis

• Consists of
• IGF-I
• IGF-II
• IGF receptors
• Six different IGFBPs

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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

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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

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IGF1 and p53 Degredation
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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Previous Research Results




n 10 12
BMI 31.51.6 26.51
Insulin, pmol/1 11038 416
IGF-I, ng/ml 33641 12812

Table 1. Comparison of body mass index and serum
IGF axis for control and exercise subjects
Control
Exercise
IGFBP-1, ng/ml
226
428

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 lt 0.05. These data were previously
published (1).
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Effect of Exercise on Growth

• Figure 1

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Effect of Exercise on PCNA

• Figure 2

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Effect of Exercise on Apoptosis

• Figure 3

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Effect of Exercise on p53 Production

• Figure 4

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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

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p38 Pathway

• IGF-1 suppresses apoptosis and aids proliferation
  via activation of the p38 mitogen-activated
  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

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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

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Insulin Receptors
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Alternate Pathway to Apoptosis
? ?
? ?
Increased p53 with exercise could increase the
ratio of Bax to Bcl-2 which activates the caspase
cascade and leads to apoptosis
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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)

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These data support epidemiological reports that
there is a reduction in prostate cancer risk in
men who are physically active