ARTICLE OPEN Clinical Research Exercise in advanced prostate cancer elevates myokine levels and suppresses in-vitro cell growth

Abstract

Background
Altering the systemic milieu through exercise has been proposed as a potential mechanism underlying exercise-driven tumour suppression. It is not yet known whether men with advanced prostate cancer can elicit such adaptations following a program of exercise. The purpose is to examine myokine levels of serum acquired from metastatic castrate-resistant prostate cancer (mCRPC) patients recruited to the INTERVAL-GAP4 trial before and after 6 months of exercise and its tumour-suppressive effect.

Methods
Twenty-five men with mCRPC (age = 74.7 ± 7.1 yrs) were randomised to supervised multimodal (aerobic and resistance) exercise (EX) or self-directed exercise control group (CON). Body composition was assessed using dual-energy x-ray absorptiometry (DXA), and fasting blood in a rested state was collected at baseline and at 6 months. Serum levels of myokines (SPARC, OSM, decorin, IGF-1, and IGFBP-3) were measured. Serum was applied to the prostate cancer cell line DU145, and growth was assessed for 72 h.

Results
No significant change in body composition was observed. Adjusted serum OSM (P = 0.050) and relative OSM (P = 0.083), serum SPARC (P = 0.022) and relative SPARC (P = 0.025) increased in EX compared to CON. The area under curve (AUC) over 72 h showed a significant reduction in DU145 growth after applying post-intervention serum from the EX vs CON (P = 0.029).

Conclusion
Elevated myokine expressions and greater tumour-suppressive effects of serum after 6 months of periodised and autoregulated supervised exercise was observed in men with mCRPC. Exercise-induced systemic changes may slow disease progression in men with advanced prostate cancer.

Full text

ARTICLE OPEN
Clinical Research
Exercise in advanced prostate cancer elevates myokine levels
and suppresses in-vitro cell growth
Jin-Soo Kim
1,2
, Dennis R. Taaffe
1,2
, Daniel A. Galvão
1,2
, Nicolas H. Hart
1,2,3
, Elin Gray
2,4
, Charles J. Ryan
5
, Stacey A. Kenfield
6
,
Fred Saad
7,8
and Robert U. Newton
1,2,8
✉
© The Author(s) 2022
BACKGROUND: Altering the systemic milieu through exercise has been proposed as a potential mechanism underlying exercise-
driven tumour suppression. It is not yet known whether men with advanced prostate cancer can elicit such adaptations following a
program of exercise. The purpose is to examine myokine levels of serum acquired from metastatic castrate-resistant prostate cancer
(mCRPC) patients recruited to the INTERVAL-GAP4 trial before and after 6 months of exercise and its tumour-suppressive effect.
METHODS: Twenty-five men with mCRPC (age =74.7 ± 7.1 yrs) were randomised to supervised multimodal (aerobic and resistance)
exercise (EX) or self-directed exercise control group (CON). Body composition was assessed using dual-energy x-ray absorptiometry
(DXA), and fasting blood in a rested state was collected at baseline and at 6 months. Serum levels of myokines (SPARC, OSM,
decorin, IGF-1, and IGFBP-3) were measured. Serum was applied to the prostate cancer cell line DU145, and growth was assessed
for 72 h.
RESULTS: No significant change in body composition was observed. Adjusted serum OSM (P=0.050) and relative OSM (P=0.083),
serum SPARC (P=0.022) and relative SPARC (P=0.025) increased in EX compared to CON. The area under curve (AUC) over 72 h
showed a significant reduction in DU145 growth after applying post-intervention serum from the EX vs CON (P=0.029).
CONCLUSION: Elevated myokine expressions and greater tumour-suppressive effects of serum after 6 months of periodised and
autoregulated supervised exercise was observed in men with mCRPC. Exercise-induced systemic changes may slow disease
progression in men with advanced prostate cancer.
Prostate Cancer and Prostatic Diseases (2022) 25:86–92; https://doi.org/10.1038/s41391-022-00504-x
INTRODUCTION
Exercise has been established as effective in improving physical
function and supportive care outcomes for cancer patients,
including those with advanced disease [1]. Furthermore, epide-
miological studies of prostate cancer patients consistently report a
positive association between increased physical activity levels and
reduced risk of prostate cancer-specific mortality [2] and disease
progression [3]. However, the causality of exercise-induced
reduction of patient mortality and mechanisms of tumour
suppression has not been thoroughly investigated in men with
advanced prostate cancer [4,5]. Accordingly, a global multi-
centred Phase III randomised controlled exercise trial, INTERVAL-
GAP4 [6], recruiting men with advanced prostate cancer is
currently ongoing to examine the effect of exercise medicine on
clinical outcomes, principally overall survival and disease progres-
sion, and the potential mechanisms by which exercise influences
tumour biology.
Exercise induces multiple physiological changes, including altera-
tion in cell-free and soluble molecules in the circulatory system
known to have tumour-suppressive effects [5]. This has been further
demonstrated in studies in which resting serum acquired after long-
term exercise programs or exercise-conditioned serum obtained
after a single bout of exercise applied to cancer cell lines produces
substantial suppression of growth [7–13], with evidence supporting
the involvement of exercise-induced serological insulin-like growth
factor-1 (IGF-1) axis alteration in prostate cancer cell growth
suppression [7,11–13]. In our recent report [14], we showed
suppression of androgen-independent prostate cancer cell line
DU145 growth by applying serum acquired from patients with
localised prostate cancer undergoing androgen deprivation therapy
(ADT) following a 3-month exercise intervention and observed
alterations in circulating cell-free/soluble factors compared to a pre-
trained state, suggesting a potential of exercise in prostate cancer
suppression [5].
Skeletal muscle has been identified as an endocrine organ and
elicits health-related benefits by producing cytokines termed
myokines, especially during exercise [15]. Furthermore, in vivo and
in vitro application of myokines, such as irisin [16–19], decorin
Received: 20 October 2021 Accepted: 20 January 2022
Published online: 12 February 2022
1
Exercise Medicine Research Institute, Edith Cowan University, Joondalup, WA 6027, Australia.
2
School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA
6027, Australia.
3
Caring Futures Institute, College of Nursing and Health Sciences, Adelaide, SA 5042, Australia.
4
Centre of Precision Health, Edith Cowan University, Joondalup, WA
6027, Australia.
5
Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
6
Departments of Urology and Epidemiology & Biostatistics, University of California San
Francisco, San Francisco, CA 94143, USA.
7
Department of Urology, Centre Hospitalier de l’Université de Montréal, Montréal, QC H2X 3E4, Canada.
8
These authors contributed
equally: Fred Saad, Robert U. Newton. ✉email: r.newton@ecu.edu.au
www.nature.com/pcan Prostate Cancer and Prostatic Diseases
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[20–24], interleukin-6 (IL-6) [25,26], interleukin-15 (IL-15) [27,28],
secreted protein acidic and rich in cysteine (SPARC) [29–32], and
oncostatin M (OSM) [33–35], have reduced the growth and
migration of various types of cancer cell lines, including prostate
[19,20,24–27,30]. In addition, reduced myostatin expression
counters the development of cachexia and may also contribute to
tumour suppression by increasing irisin production [5]. However,
despite the preclinical evidence, myokines are only considered as
apotential molecular player for exercise-induced cancer suppres-
sion [5].
Although myokine expression in the non-cancer population is
well documented [15], there has been no investigation of myokine
expression and tumour-suppressive effects of exercise-
conditioned serum in advanced prostate cancer patients who
have undergone, and continue to receive, a range of cancer
therapies inclusive of androgen blockade. Given the high disease
load of these patients, with metastases and a shortened survival
time, in addition to their castrate-resistant status underpinning an
altered endocrine environment, it is important to determine if
they can respond to exercise with the development of a more
anti-tumour systemic milieu. Thus, in this study, we investigated
resting serum myokine levels (irisin, decorin, IL-6, IL-15, SPARC,
OSM, and myostatin) and growth hormone levels (IGF-1 and
IGFBP-3) over the initial 6-month period of 2-year exercise
intervention vs control group in metastatic castrate-resistant
prostate cancer (mCRPC) patients. We also evaluated the potential
exercise-induced tumour-suppressive effect by comparing serum
acquired at baseline and after 6 months to the prostate cancer cell
line DU145. We hypothesised that despite the heavy disease load
and continuous treatment in this patient group (mCRPC), 6 months
of supervised aerobic and resistance exercise would alter
circulatory myokine levels and that exercise-conditioned serum
obtained from the patients would reduce the growth of the
prostate cancer cell line DU145.
MATERIALS/SUBJECTS AND METHODS
Participants and exercise program
Serum was collected from 25 men with mCRPC (EX, n=13; CON,
n=12) who were recruited for the INTERVAL-GAP4 trial (Clinical
Trials Registry: NCT02730338) [6] from March 2016 to May 2020 at
the Exercise Medicine Research Institute (EMRI; Edith Cowan
University (ECU); WA, Australia) which was used for analysis
(Fig. 1). The recruitment and randomisation of patients were
undertaken as previously described [6]. Briefly, patients who had
been identified as mCRPC (adenocarcinoma of the prostate with
systemic metastatic disease despite castrate levels of testosterone
(<50 ng/dl) due to orchiectomy or luteinising hormone-releasing
hormone (LHRH) agonist, undergoing ADT (gonadotropin-releas-
ing hormone (GnRH) agonist/antagonist or prior bilateral orch-
iectomy)), and capable of performing exercise were recruited by
clinician referrals.
Patients were randomly allocated to supervised exercise (EX) or
a self-directed exercise control group (CON). The current study
examines the initial 6 months of resistance and aerobic training
completed thrice weekly as previously described [6], as the
protocol for INTERVAL GAP4 initially commences with full super-
vision before transitioning to home-based exercise. Briefly, in the
first and third exercise session of the week, structured resistance
exercise (6 exercises, 2–5 sets, 6–12 RM intensity adjusted using
repetition maximum (RM)) with a combination of high-intensity
interval training (HIIT) (aerobic exercise, 6 x 60 s, intensity adjusted
to a rating of perceived exertion (RPE) of 8 on 0–10 Borg scale)
was prescribed, and 30–40 min of moderate-intensity continuous
aerobic training (MICT) (cycling/walking) was undertaken at an
intensity adjusted to RPE 6 in the second exercise session of the
week. The exercise program was periodised and autoregulated
across the week, month and 3-month cycles and autoregulated so
that intensity, volume and exercise selection was adjusted
depending on the patient’s readiness on the day. CON were
provided with the American College of Sports Medicine (ACSM)
guidelines for cancer survivors [36]. The study was funded by the
Movember Foundation and ethically approved by the Human
Research Ethics Committee at Edith Cowan University (ID: 13236
NEWTON). Written informed consent was obtained from all
patients before inclusion.
Body composition
Body composition was assessed at baseline and after the initial
6 months of the study by Dual-energy X-ray Absorptiometry (DXA;
Horizon A, Hologic, Washington, USA). Values derived were whole-
body lean mass (LM, kg), upper-body LM (kg), lower-body LM (kg),
whole-body fat mass (FM, kg), percent LM, percent FM, and
LM index (total lean mass/height squared; kg/m
2
). Body mass
index (BMI) was calculated from weight divided by height squared
(kg/m
2
).
Blood assessment and analysis
Resting blood samples were collected early in the morning for
fasting specimens and at least 48 h post any exercise. The
collected blood samples were processed to serum and stored at
−80 °C until serum myokine analysis. Serum myokine levels for
irisin, IL-6, IL-15, SPARC, OSM, and myostatin, were analysed using
multiplex magnetic bead panels (HMYOMAG-56k-15 Huma,
Millipore, Billerica, MA, USA), and serum decorin (ab99998, Decorin
Human ELISA Kit, Abcam, Cambridge, United Kingdom), IGF-1
(ab211651, Human IGF-1 SimpleStep ELISA Kit, Abcam, Cambridge,
United Kingdom), and IGFBP-3 (ab211652, Human IGFBP3 Simple-
Step ELISA Kit, Abcam, Cambridge, United Kingdom) levels were
analysed using appropriate enzyme-linked immunosorbent assay
(ELISA) kits.
Cell culture and real-time cellular analysis
The human prostate cancer cell line, DU145 (ATCC HTB-81), was
obtained from The Harry Perkins Institute for Medical Research,
Nedlands, WA, Australia. Cells were cultured in RPMI-1640 media
containing 10% fetal bovine serum (FBS), incubated at 37 °C, 5%
CO2, and routinely passaged at ~80% confluence. Growth of
DU145 cells was assessed using a Real-Time Cellular Analysis
(RTCA) system, xCELLigence DP unit and E-plate (ACEA Bioscience,
CA, USA) in the presence of human serum. Each well of E-plate
was seeded with 15,000 DU145 cells with 100 µl of serum-free
RPMI-1640. After 24 h of starvation, 100 µl of growth media (RPMI-
1640) containing 20% human serum (final concentration of 10%)
was added to each well of the E-plate. The plates were incubated
for 72 h while recording the Cell Index every hour.
Fig. 1 Consort diagram. Cycle 6 indicates end of the initial 6-month
phase of the INTERVAL GAP4 trial. Due to COVID-19 restrictions,
patients who could not visit the centre for cycle 6 assessments were
excluded from the analysis.
J.-S. Kim et al.
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Statistical analysis
Based on Cell Index results from our previous research (Pre:
5.829 ± 1.112; Post: 4.566 ± 1.515) [14], 24 participants (12 each
group) is required to achieve 0.80 power at an αlevel 0.05 two-
tailed. As a result, we obtained data and samples for 25
participants (CON, n=12; EX, n=13) from the INTERVAL GAP4
trial investigating the effect of exercise treatment on patients
with mCRPC (Fig. 1). Data were analysed using R software
(v4.0.2, The R Foundation), with the rstatix packages (v0.7.0,
Kassambra, 2021) for statistics, ggplot2 packages (v3.3.3,
Wickham, 2020) for visualisation and Desctools (v0.99.41,
Slgnorell, 2021) for the area under the curve (AUC) calculations.
Normality of the distribution for outcomes was tested using the
Shapiro–Wilk test and Q-Q plot. Analysis of covariance
(ANCOVA) was used to detect differences in post-intervention
outcomes after 6 months for baseline value [37]. All values are
presented as adjusted mean and 95% confidence interval. Tests
were two-tailed and significance was set at p< 0.05.
RESULTS
Patient characteristics
Patient characteristics are presented in Table 1. No significant
difference between groups at baseline was evident in body
weight, total LM, percent LM, LM index, FM, percent FM, BMI,
serum levels of three myokines (OSM, SPARC, and decorin), and
IGF-1, although serum levels of IGFBP-3 were significantly higher
in CON. The average percentage of exercise sessions completed in
EX was 82.5 ± 13.0% out of a total of 72 sessions.
Body composition
Adjusted body weight, total LM, percent LM, LM index, FM,
percent FM, and BMI adjusted for baseline did not show a
significant difference between the EX and CON groups after the
6-month intervention (Table 2).
Myokines and IGF-1/IGFBP-3
A total of seven different myokines (irisin, decorin, IL-6, IL-15,
SPARC, OSM, and myostatin), IGF-1, and IGFBP-3 were analysed
from serum acquired at baseline and after the exercise interven-
tion to examine the effect of exercise on serum levels of myokines
and the IGF-1 axis. However, due to a low recovery rate of irisin, IL-
6, IL-15, and myostatin in multiplexed magnetic bead-based
immunoassay, only OSM, SPARC, decorin, IGF-1, and IGFBP-3 were
able to be analysed (Table 2). After adjusting 6-month-
intervention serum levels for baseline levels, no significant
differences were observed in IGF-1, IGFBP-3 levels, or IGF-1/
IGFBP-3 ratio. However, there were significant differences
between groups in serum levels of OSM (P=0.050) and SPARC
(P=0.022) at post-intervention adjusted by baseline. Relative
SPARC levels (serum SPARC levels/body weight) increased
significantly in EX compared to CON (P=0.025), and there was
a trend for an increase in relative OSM (serum OSM levels/body
weight) (P=0.083).
Subgroup analysis for ARTA (CON-ARTA naïve, N=8; CON-
ARTA, N=4; EX-ARTA naïve, N=7; EX-ARTA, N=6) demonstrated
a significant increase in adjusted serum OSM and SPARC levels for
baseline in EX-ARTA naïve compared to the patients in CON-ARTA
(P< 0.05; Supplementary Table 1S). In addition, subgroup analysis
by chemotherapy (CON-NO Chemo, N=9, CON-Chemo, N=3; EX-
NO Chemo, N=10, EX-Chemo, N=3), revealed baseline adjusted
serum OSM and SPARC levels to be significantly higher in EX
Chemo group compared to CON Chemo group (P< 0.05;
Supplementary Table 2S). Furthermore, baseline adjusted serum
decorin levels were significantly higher in the EX-ARTA subgroup
compared to EX-ARTA naïve subgroup (P< 0.05; Supplementary
Table 1S), however, significantly lower baseline adjusted decorin
was observed in the EX-Chemo subgroup compared to CON-NO
Chemo subgroup (P< 0.05; Supplementary Table 2S).
Prostate cancer cell line growth analysis
Three-day cell growth kinetics are presented in Fig. 2A. Although
adjusted Cell Index at 72 h after administrating 6-month serum
from EX or CON groups did not show a difference, adjusted Cell
Index at 12 to 61 h revealed a significant decrease in EX compared
to CON at each time point. Therefore, the area under curve (AUC)
was calculated from the 72 h Cell Index plot (Table 3, Fig. 2B, C).
Total cell growth AUC (0–72 h) was significantly reduced with the
presence of serum obtained after 6 months of an exercise
intervention in EX compared to CON after adjusting for baseline
AUC (P=0.029). In addition, the adjusted 6-month timepoint
intervention AUC at 0 to < 24 h, 24 to < 48 h and 48 to 72 h
periods exhibited a significant decrease in EX compared to CON
(P=0.016, P=0.006, and P=0.039, respectively) (Table 3, Fig. 2C).
DISCUSSION
In this sub-study comprised from the INTERVAL-GAP4 rando-
mised controlled trial [6], we examined chronic adaptations in
potential muscle-induced candidates for tumour suppression in
men with mCRPC undertaking 6 months of exercise training. We
confirmed that the first 6 months of an exercise intervention vs
control produced an increase in myokine expression, specifi-
cally OSM, SPARC, and body weight relative SPARC, as well as a
Table 1. Baseline characteristics of exercise and usual care
control group.
CON (n=12)
(mean ± SD)
EX (n=13) P-value
(mean ± SD)
Age 76.9 ± 7.1 72.6 ± 7.0 0.140
Height (m) 1.7 ± 0.1 1.7 ± 0.1 0.608
Body weight (kg) 82.1 ± 13.4 93.7 ± 20.8 0.164
Total lean mass (kg) 49.1 ± 8.2 53.1 ± 10.4 0.364
Percent lean
mass (%)
59.8 ± 4.0 57.0 ± 3.9 0.099
Lean mass index
(kg/m
2
)
16.7 ± 2.1 17.6 ± 1.9 0.292
Total fat mass (kg) 26.9 ± 6.7 33.4 ± 10.5 0.118
Percent fat mass (%) 34.4 ± 4.7 37.1 ± 4.4 0.143
Presence of nodal
metastasis
98–
Presence of bone
metastasis
810–
Presence of nodal
and bone metastasis
55–
ARTA (e.g.,
abiraterone and
enzalutamide) naïve
patients
87–
Patients on ARTA 4 6 –
BMI (kg/m
2
) 28.0 ± 4.0 31.1 ± 4.6 0.149
IGF-1 (ng/ml) 841.1 ± 636.6 806.5 ± 547.0 0.905
IGFBP-3 (ng/ml) 13273.6 ± 5871.5 6987.9 ± 2054.7 <0.001
Oncostatin M (ng/ml) 6.6 ± 4.9 4.5 ± 3.0 0.503*
SPARC (pg/ml) 495.7 ± 158.4 408.1 ± 82.1 0.605*
Decorin (ng/ml) 64.7 ± 7.3 63.0 ± 10.6 0.695
Three patients from the CON and EX group each commenced chemother-
apy (Docetaxel or Cabazitaxel) during the exercise period. ARTA Androgen
receptor-targeted agents. *Indicates Wilcoxon-rank test used for statistical
analysis otherwise independent t-test.
J.-S. Kim et al.
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borderline increase in relative OSM. We also examined the
effect of serum collected at baseline and 6 months on prostate
cancer cell line DU145 growth. Although we cannot definitely
conclude a direct relationship between exercise training
elevated myokines and prostate cancer cell line growth, the
6-month serum drawn from the EX reduced DU145 cancer cell
growth compared to CON.
While there are numerous hypotheses, the mechanistic details by
which exercise influences tumour biology are unknown. In our prior
review [5], we provide a rationale for the influence of muscle-
induced myokines as anti-cancer agents acting through several
pathways to drive apoptosis and suppress proliferation and
metastasis. Furthermore, our team recently completed a single-
group trial involving prostate cancer patients receiving ADT, and we
Table 2. Adjusted physical outcomes and serum OSM, SPARC, decorin, IGF-1, and IGFBP-3 levels. All outcomes are for the initial 6-month phase
adjusted for baseline.
CON (n=12) EX (n=13) P-value
Adjusted mean 95% confidence interval Adjusted mean 95% confidence interval
Physical Outcomes
Body weight (kg) 89.5 [87.6, 91.5] 86.7 [84.9, 88.6] 0.052
Total lean mass (kg) 50.7 [49.4, 51.9] 50.6 [49.4, 51.8] 0.948
Percent lean mass (%) 57.7 [56.4, 59.0] 58.4 [57.1, 59.6] 0.454
Lean mass index (kg/m
2
) 17.0 [16.6, 17.4] 17.2 [16.8, 17.6] 0.625
Total Fat Mass (kg) 32.1 [30.0, 34.1] 29.8 [27.9, 31.8] 0.126
Percent Fat Mass (%) 36.7 [35.1, 38.2] 35.9 [34.4, 37.5] 0.513
BMI (kg/m
2
) 30.0 [29.4, 30.7] 29.2 [28.6, 29.8] 0.068
Serum IGF-1, IGFBP-3, and myokine levels
IGF-1 (ng/ml) 924.85 [687.47, 1162.24] 878.29 [650.23, 1106.36] 0.772
IGFBP-3 (ng/ml) 10186.76 [8554.44, 11819.08] 7884.04 [6329.68, 9438.40] 0.068
IGF-1:IGFBP-3 ratio 0.10 [0.05, 0.15] 0.14 [0.09, 0.19] 0.307
OSM (ng/ml) 4.88 [2.17, 7.59] 8.71 [6.11, 11.30] 0.050
SPARC (pg/ml) 410.58 [362.18, 458.97] 492.66 [446.28, 539.04] 0.022
Decorin (ng/ml) 67.08 [62.92, 71.23] 63.75 [59.76, 67.74] 0.246
Relative IGF-1 (ng/m/kg) 11.25 [8.31, 14.19] 10.17 [7.34, 12.98] 0.586
Relative IGFBP-3 (ng/ml/kg) 120.40 [101.35, 139.45] 97.71 [79.57, 115.84] 0.120
Relative OSM (ng/ml/kg) 0.06 [0.03, 0.09] 0.10 [0.07, 0.13] 0.083
Relative SPARC (pg/ml/kg) 4.85 [4.32, 5.37] 5.73 [5.22, 6.23] 0.025
Relative Decorin (ng/ml/kg) 0.78 [0.74, 0.83] 0.77 [0.73, 0.82] 0.770
All 6-month outcomes are adjusted for the baseline value.
Fig. 2 Cell proliferation data. A Time-course changes of DU145 Cell growth in Cell Index adjusted for baseline values at each time point
(adjusted mean±adjusted SE). The error bar indicates an adjusted SE. BAdjusted Area Under Curve (AUC) over 72 h incubation. CAdjusted
Area Under Curve at different time points (0 to <24 h, 24 to < 48 h, 48 to 72 h). Due to contamination of a serum sample from one patient in
the EX group, serum samples from only 12 patients in the CON group and 12 patients in the EX group were analysed. The grey bar indicates a
95% confidence interval of adjusted values. *P< 0.05, **P< 0.01.
J.-S. Kim et al.
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demonstrated positive alterations in myokine concentrations with
subsequent growth suppression of prostate cancer cell lines [14].
While this finding is intriguing, the question remained whether such
anti-cancer effects of exercise could be induced in patients with
more advanced prostate cancer. Patients with mCRPC have a very
high disease load combined with accumulated treatment toxicities,
resulting in considerable deconditioning, reduced muscle and bone
mass and elevated fat mass. Should an anti-cancer systemic
mechanism produced through exercise therapy be evident, then
this could be a particularly attractive strategy for patients with
mCRPC to slow disease progression.
The current study is the first to examine myokine expression
before and at the 6-month exercise intervention in patients with
mCRPC incorporating resistance and aerobic training. We focused
on the endocrine function of skeletal muscle [15], given that
preclinical studies demonstrated a positive role of myokines in
cancer cell suppression [19,20,24–27,30] and a retrospective
study in prostate cancer patients showed a positive association
between skeletal muscle mass and progression-free survival [38].
The results of serum myokine analysis in the current study showed
significant elevation of serum SPARC and OSM levels in the EX
group compared to the CON group, which is consistent with our
previous report [14] in predominantly localised prostate cancer
patients.
In addition, although this is speculative due to the limited
sample number, our subgroup analysis for different treatments
provided interesting observations regarding myokine expression.
As androgen is a critical factor in muscle growth, we expected
ARTA (androgen receptor-targeted agent), commonly prescribed
as the first-line treatment for castrate-resistant prostate cancer,
may have an impact on exercise-induced serum myokine levels.
Interestingly, our subgroup analysis for ARTA and ARTA naïve
showed a significant increase of serum OSM and SPARC in the EX-
ARTA naïve subgroup compared to the CON-ARTA subgroup,
suggesting that ARTA may impact exercise-induced OSM and
SPARC levels possibly due to ARTA impacts on muscle size and
physiology. Furthermore, our ARTA subgroup analysis demon-
strated significantly increased serum decorin levels in the EX-ARTA
group compared to EX-ARTA naïve; however, a decreased trend of
decorin levels was observed in EX-Chemo compared to EX-NO
Chemo, suggesting exercise-induced alteration of serum decorin
may partially be impacted by chemotherapy. However, although
these observations from our subgroup analysis are noteworthy,
further research with larger patient numbers is required to fully
elucidate these interactions.
Given that body composition, in terms of fat and muscle mass,
influences cytokine levels in the blood [39,40], it was somewhat
surprising that we did not observe improvements in body
composition. Although the intensity and volume prescribed to
these patients were considerably high, a lack of differential
response may be due to the disease and treatment load these
patients are experiencing, compromising their ability to adapt
with morphological changes. Similarly, a lack of change in body
composition was also evident in our previous study involving
prostate cancer patients with bone metastases following a
3-month exercise intervention [41]. The interference of aerobic
exercise on adaptations to resistance training may also have
occurred in these patients with mCRPC, as we have reported
previously for men with nonmetastatic prostate cancer being
treated with ADT [42]. Whether or not this impacted the
magnitude of increase in myokines and subsequent growth
suppression in the cell line experiments cannot be determined.
We also observed a reduced prostate cancer cell line (DU145)
growth after directly applying resting serum acquired following
6 months of exercise. For clinical relevance, we recruited patients
under very strict criteria [6] and used the androgen-insensitive,
metastatic prostate cancer cell line DU145 as this cell line
originated from a 69-year-old Caucasian male with metastatic
prostate cancer, which shares similar characteristics with mCRPC.
Furthermore, it should be noted that resting serum was collected
after at least 48 h of complete rest, and the acute physiological
arousal from the last exercise session did not affect the results. Our
previous report also observed reduced prostate cancer cell line
growth by directly applying resting exercise-conditioned serum
acquired from predominantly localised prostate cancer patients
[14], suggesting exercise adaptation-induced systemic milieu
alteration might positively influence tumour biology. Furthermore,
previous studies by Barnard et al. [7], Leung et al. [11], and
Ngo et al. [12] also reported reduced prostate cancer cell line
(LNCaP) growth with the presence of resting serum obtained from
active, healthy individuals and healthy persons following a short-
term period of exercise and dietary intervention. Although these
reports demonstrated the potential role of exercise in inducing
changes in the IGF-1 axis [7,11,12], serum IGF-1 and IGFBP-3
levels did not change in our cohort, suggesting that the serum
myokine level changes due to exercise training are more likely
candidate drivers suppressing DU-145 growth rather than the IGF-
1 axis. Nevertheless, consistent reduction of prostate cancer cell
growth in previous studies and the current study provides
important insight in the field of exercise oncology that not only
should exercise be considered as a strategy to improve health-
related outcomes for prostate cancer patients, but also as a
potential daily-dosage strategy to create a tumour-suppressive
environment.
The current study has a number of strengths and limitations
worthy of comment. First, we used a randomised control trial
design to investigate the expression of multiple myokines
resulting from exercise. Second, DXA was used for body
composition assessment providing accurate measures of fat and
lean tissue. Third, by using RTCA, we were able to detect cell
growth differences in multiple time points. This is important as
previous studies that observed cell growth after applying human
serum used end-point analysis and were unable to monitor cell
growth kinetics. Fourth, the study provided clinically relevant
Table 3. Area under curve from adjusted Cell Index adjusted for baseline.
Time frame (hour) CON (n=12) EX (n=12) P-value
Adjusted mean (Cell
Index*Time(h))
95% confidence
interval
Adjusted mean (Cell
Index*Time(h))
95% confidence
interval
0–72 195.00 [172.71, 217.28] 155.34 [133.05, 177.62] 0.029
0–24 25.72 [23.40, 28.03] 20.87 [18.55, 23.18] 0.016
24–48 62.06 [54.19, 69.94] 43.05 [35.17, 50.92] 0.006
48–72 108.83 [96.65, 121.01] 90.06 [77.85, 102.24] 0.039
The 6-month AUC outcomes are adjusted for baseline AUC. Due to contamination of a serum sample from one patient in the EX group, serum samples from
only 12 patients in the CON group and 12 patients in the EX group were analysed.
J.-S. Kim et al.
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evidence for tumour suppression using serum acquired from
prostate cancer patients with mCPRC as prior to this data for
myokine expression research has been limited to either healthy
cohorts or those with metabolic disease, or patients with less
advanced disease. However, as we used a multimodal exercise
program, we cannot determine which exercise mode or if both
contributed to the adaptation in myokine expression. Moreover,
we analysed the data and samples available in our ongoing trial,
INTERVAL-GAP4, which limits the volume of serum available for
individual experiments and so this study was confined to
investigate serum levels of myokines and prostate cancer cell
growth. As there is limited research investigating myokine
expression in patients with advanced prostate cancer, we made
our initial sample size and power calculation based on available
data from our previous trial in prostate cancer patients with
localised disease [14]. Unfortunately, for the current study in
patients with advanced disease our post-hoc analysis revealed
that we only achieved statistical power of 65% in the adjusted
AUC of Cell index, which likely reflects differences between the
two patient groups in exercise responses. In addition, although we
reported an increase in serum myokine levels and a significant
reduction of DU145 cell growth after applying exercise-
conditioned human serum, the current study is limited with
regard to in-depth intercellular mechanistic measures to address
the tumour-suppressive role of exercise-induced myokines or
potential interaction between the treatments and myokines.
In conclusion, this study provides preliminary evidence for
enhanced myokine expression, and a tumour-suppressive effect
of serum collected from mCRPC patients after 6 months of
vigorous, multimodal exercise. Future trials are needed to
further elucidate the influenceofexerciseonmyokineexpres-
sion, particularly specifics of exercise prescription such as
threshold exercise intensity, volume, and mode. Furthermore,
more in-depth intercellular mechanistic research involving the
applicationofbothacuteandchronically exercise-conditioned
human serum is required to enhance our understanding of the
direct tumour-suppressive role of myokines in patients with
prostate cancer.
DATA AVAILABILITY
The data are available for bona fide researchers who request it from the authors.
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ACKNOWLEDGEMENTS
The authors thank Harry Perkins Institute, Nedlands, WA, Australia, for providing
prostate cancer cell line DU145.
AUTHOR CONTRIBUTIONS
J-S Kim and RU Newton had full access to all the data in the study and take
responsibility for the integrity of the data and accuracy of the data analysis. J-S Kim,
RU Newton, DA Galvão, and DR Taaffe conceptualised the study. J-S Kim and RU
Newton designed the study. RU Newton, NH Hart, and DA Galvão collected the
clinical information, body composition and fasting blood samples. NH Hart and SA
Kenfield supervised and directed the overall study’s implementation, data collection,
and data monitoring. J-S Kim analysed the data. J-S Kim and RU Newton were
responsible for the drafting of the manuscript. RU Newton, NH Hart, DA Galvaõ,
E Gray, SA Kenfield, and DR Taaffe provided administrative, technical, or material
support. J-S Kim, RU Newton, NH Hart, DA Galvão, E Gray, CJ Ryan, SA Kenfield,
F Saad, and DR Taaffe reviewed and edited the manuscript prior to submission.
FUNDING
This work was funded by the Movember Foundation. National Health and Medical
Research Council Centre of Research Excellence (NHMRC-CRE; APP1116334) funded
the additional materials involving serum analysis and cell work through Centre for
Research Excellence in Prostate Cancer Survivorship. J-S.K is supported by the NHMRC
Centre for Research Excellence in Prostate Cancer Survivorship Scholarship. Open
Access funding enabled and organized by CAUL and its Member Institutions.
COMPETING INTERESTS
The authors declare no competing interests.
ETHICS APPROVAL
The study was conducted according to the guidelines of the Declaration of Helsinki
and approved by the Edith Cowan University Human Research Ethics Committee (ID:
13236 NEWTON).
CONSENT TO PARTICIPATE
Informed consent was obtained from all subjects involved in the study prior to
inclusion.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41391-022-00504-x.
Correspondence and requests for materials should be addressed to Robert U.
Newton.
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