Prostate cancer cell growth characteristics in serum and prostate-conditioned media from moderate-intensity exercise-trained healthy and tumor-bearing rats

Abstract

Physical activity is associated with diminished risk of several cancers, and preclinical studies suggest exercise training may alter tumor cell growth in certain tissue(s) (e.g., adipose). From moderate-intensity exercise-trained rats versus sedentary controls, we hypothesized 1) there will be a decreased prostate cancer cell viability and migration in vitro and, within the prostate, a reduced 5α-reductase 2 (5αR2) and increased caspase-3 expression, and 2) that exercise training in tumor-bearing (TB) animals will demonstrate a reduced tumor cell viability in prostate-conditioned media. Serum and prostate were harvested from sedentary or exercise-trained (treadmill running, 10-11 weeks) immune-competent (Copenhagen; n = 20) and -deficient (Nude; n = 18) rats. AT-1 and PC-3 prostate cancer cells were grown in one or more of the following: serum-supplemented media (SSM), SSM from TB rats (SSM-TB), prostate-conditioned media (PCM) or PCM from TB rats (PCM-TB) for 24-96 h under normoxic (18.6% O₂) or hypoxic (5% O₂) conditions. Under normoxic condition, there was a decreased AT-1 cell viability in SSM and PCM from the exercise-trained (ET) immune-competent rats, but no difference in PC-3 cell viability in SSM and PCM from ET Nude rats versus the sedentary (SED) group, or in SSM-TB from ET-TB Nude rats versus the SED-TB group. However, there was a decreased PC-3 cell viability in the PCM-TB of the ET-TB group versus SED-TB group. PC-3 cell viability in all conditioned media types was not altered between groups with hypoxia. In the prostate, exercise training did not alter 5αR2 expression levels, but increased caspase-3 expression levels. In conclusion, prior exercise status reduced prostate cancer cell viability in the serum and prostate of trained rats but did not modify several other key prostate tumor cell growth characteristics (e.g., migration, cell cycle except in S phase of PC-3 cells in PCM-TB). Importantly, once the tumor was established, exercise training reduced tumor cell viability in the surrounding prostate, which may help explain the reduced severity of the disease in patients that exercise.

Keywords: 5α-reductase 2; Exercise; caspase-3; growth characteristics; hypoxia; normoxia; prostate cancer.

Figure 1

Effect of 24, 48 and 96 h incubation with serum-supplemented media (SSM) from Copenhagen sedentary (SED; n = 10) rats without FBS (-FBS) and with FBS (+FBS) on viable AT-1 cell number. There was a significant increase in cell number in SSM (+FBS) vs. SED SSM (-FBS) from Copenhagen SED rats at 48 and 96 h (P ≤ 0.05). Values are expressed as mean cell number (%) ± SEM. *P ≤ 0.05 vs. SSM (-FBS).

Figure 2

Effect of 24, 48 and 96 h incubation with serum-supplemented media (SSM; A) and prostate-conditioned media (PCM; B) from sedentary (SED; n = 6-10) and exercise-trained (ET; n = 9-10) Copenhagen rats on viable AT-1 cell number. There was a significant decrease in cell number in ET vs. SED for SSM and PCM at 24-96 h (P ≤ 0.05). Values are expressed as mean cell number (%) ± SEM. *P ≤ 0.05 vs. SED, within SSM or PCM treatment group.

Figure 3

Effect of 24, 48 and 96 h incubation with serum-supplemented media (SSM; A) and prostate-conditioned media (PCM; B) from sedentary (SED; n = 6-7) and exercise-trained (ET; n = 8-9) Copenhagen rats on migration of AT-1 cells. A representative image of cell migration utilizing a cell exclusion zone technique in a 96-well tissue culture plate is illustrated to the right of each bar graph. No significant difference in cell migration between SED and ET groups was observed in SSM or PCM from 24-96 h (P > 0.05). Bars with different letters are statistically different from one another within SED group over time. Bars with different numbers are statistically different from one another within ET group over time. Values are expressed as mean cell number ± SEM. P ≤ 0.05 for 24 vs. 48, 24 vs. 96, and 48 vs. 96 h within SED or ET group.

Figure 4

Effect of 24, 48 and 96 h incubation with serum-supplemented media (SSM; A) and prostate-conditioned media (PCM; B) from sedentary (SED; n = 4) and exercise-trained (ET; n = 4-6) Nude rats on viable PC-3 cell number in normoxia. There was no significant difference in cell number in ET vs. SED group for SSM or PCM at 24-96 h (P > 0.05). Values are expressed as mean cell number (%) ± SEM.

Figure 5

Effect of 24, 48 and 96 h incubation with serum-supplemented media (SSM; A) and prostate-conditioned media (PCM; B) from sedentary (SED; n = 3-4) and exercise-trained (ET; n = 4-6) Nude rats on viable PC-3 cell number in hypoxia. There was no significant difference in cell number in ET vs. SED group for SSM or PCM at 24-96 h (P > 0.05). Values are expressed as mean cell number (%) ± SEM.

Figure 6

Bar graph representing the percentage of cells in G0/G1, S, and G2/M cell cycle phase after treatment of AT-1 cells with serum-supplemented media (SSM; A) and prostate-conditioned media (PCM; B) from sedentary (SED; n = 5-10) and exercise-trained (ET; n = 9-10) Copenhagen rats, or PC-3 cells (in normoxia) with SSM (C) and PCM (D) from SED (n = 4) and ET (n = 6) Nude rats. There was no significant effect of media type on AT-1 or PC-3 cell population in different cell cycle phase, in SED vs. ET rats (P > 0.05). Values are expressed as mean proportion of cells (%) ± SEM.

Figure 7

Representative images of immunohistochemical sections stained for 5αR2 (A and B) and caspase-3 (C and D) in the prostate of SED and ET Copenhagen rats, respectively. The arrows within the tissues are representative stained sections showing 5αR2 and caspase-3 staining. Images were captured at 200X magnification. Bar graph representing prostate 5αR2 (E) and caspase-3 (F) expression between sedentary (SED; n = 10) and exercise-trained (ET; n = 10) Copenhagen rats. No significant difference in 5αR2 between SED and ET groups was observed (P > 0.05). Caspase-3 was significantly increased in ET vs. SED group (*P ≤ 0.05 vs. SED). Values are expressed as mean positive cells per tissue area ± SEM.

Figure 8

Effect of 24, 48 and 96 h incubation with serum-supplemented media from tumor-bearing rats (SSM-TB; A) and prostate-conditioned media from tumor-bearing rats (PCM-TB; B) from sedentary tumor-bearing (SED-TB; n = 4) and exercise-trained tumor-bearing (ET-TB; n = 4) Nude rats on viable PC-3 cell number in normoxia. There was no significant difference in cell number in ET-TB vs. SED-TB group for SSM-TB at 24-96 h (P > 0.05). However, a significant decrease in cell number in ET-TB vs. SED-TB group was observed for PCM-TB at 48 and 96 h (P ≤ 0.05). Values are expressed as mean cell number (%) ± SEM. *P ≤ 0.05 vs. SED-TB, within PCM-TB.

Figure 9

Effect of 24, 48 and 96 h incubation with serum-supplemented media from tumor-bearing rats (SSM-TB; A) and prostate-conditioned media from tumor-bearing rats (PCM-TB; B) from sedentary tumor-bearing (SED-TB; n = 4) and exercise-trained tumor-bearing (ET-TB; n = 4) Nude rats on viable PC-3 cell number in hypoxia. There was no significant difference in cell number in ET-TB vs. SED-TB group for SSM-TB or PCM-TB at 24-96 h (P > 0.05). Values are expressed as mean cell number (%) ± SEM.

Figure 10

Effect of 24 h incubation with serum-supplemented media from tumor-bearing rats (SSM-TB; A) and prostate-conditioned media from tumor-bearing rats (PCM-TB; B) from sedentary tumor-bearing (SED-TB; n = 3-4) and exercise-trained tumor-bearing (ET-TB; n = 4) Nude rats on migration (via Transwell migration assay) of PC-3 cells in normoxia. No significant difference in cell migration between groups was observed (P > 0.05). Values are expressed as mean cell count/total area (pixels²) ± SEM. Bar graph representing the percentage of PC-3 cells in G0/G1, S, and G2/M cell cycle phase after treatment of PC-3 cells with serum-supplemented media from tumor-bearing rats (SSM-TB; C) and prostate-conditioned media from tumor-bearing rats (PCM-TB; D) from sedentary tumor-bearing (SED-TB; n = 4) and exercise-trained tumor-bearing (ET-TB; n = 4) Nude rats in normoxia. There was no significant effect of SSM-TB on cell population in different cell cycle phase in SED-TB vs. ET-TB rats (P > 0.05). PCM-TB however caused a significant decrease of cells in S phase in ET-TB vs. SED-TB (P ≤ 0.05). Values are expressed as mean proportion of cells (%) ± SEM. *P ≤ 0.05 vs. SED-TB, within PCM-TB.