Exercise and Prostate Cancer

1. Exercise and Prostate Cancer Jamie McClellan Department of Exercise Science

2. 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

3. 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.

4. 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. Epidemiology of Prostate Cancer

5. 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 Environmental Factors

6. 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

7. ACLS Cohort and Prostate Cancer Risk

8. IGF Axis • Consists of: • IGF-I • IGF-II • IGF receptors • Six different IGFBPs

9. 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

10. 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

11. IGF1 and p53 Degredation

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. Previous Research Results Table 1. Comparison of body mass index and serum IGF axis for control and exercise subjects Control Exercise IGFBP-1, ng/ml 22±6 42±8 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).

24. Figure 1 Effect of Exercise on Growth

25. Figure 2 Effect of Exercise on PCNA

26. Figure 3 Effect of Exercise on Apoptosis

27. Figure 4 Effect of Exercise on p53 Production

28. 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

29. 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

30. 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

31. Insulin Receptors

32. Alternate Pathway to Apoptosis κ κ κ κ Increased p53 with exercise could increase the ratio of Bax to Bcl-2 which activates the caspase cascade and leads to apoptosis

33. 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)

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