Abstract Physical activity is associated with reduced risk of several cancers, including aggressive prostate cancer. The mechanisms mediating the effects are not yet understood; among the candidates are modifications of endogenous hormone levels. Long-term exercise is known to reduce serum levels of growth stimulating hormones. In contrast, the endocrine effects of acute endurance exercise include increased levels of mitogenic factors such as GH and IGF-1. It can be speculated that the elevation of serum growth factors may be detrimental to prostate cancer progression into malignancy. The incentive of the current study is to evaluate the effect of acute exercise serum on prostate cancer cell growth. We designed an exercise intervention where 10 male individuals performed 60 minutes of bicycle exercise at increasing intensity. Serum samples were obtained before (rest serum) and after completed exercise (exercise serum). The established prostate cancer cell line LNCaP was exposed to exercise or rest serum. Exercise serum from 9 out of 10 individuals had a growth inhibitory effect on LNCaP cells. Incubation with pooled exercise serum resulted in a 31% inhibition of LNCaP growth and pre-incubation before subcutaneous injection into SCID mice caused a delay in tumor formation. Serum analyses indicated two possible candidates for the effect; increased levels of IGFBP-1 and reduced levels of EGF. In conclusion, despite the fear of possible detrimental effects of acute exercise serum on tumor cell growth, we show that even the short-term effects seem to add to the overall beneficial influence of exercise on neoplasia.

Citation: Rundqvist H, Augsten M, Strömberg A, Rullman E, Mijwel S, Kharaziha P, et al. (2013) Effect of Acute Exercise on Prostate Cancer Cell Growth. PLoS ONE 8(7): e67579. https://doi.org/10.1371/journal.pone.0067579 Editor: Jean-Marc A. Lobaccaro, Clermont Université, France Received: August 10, 2012; Accepted: May 23, 2013; Published: July 5, 2013 Copyright: © 2013 Rundqvist et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The present study was supported by the funding of TG: the Swedish Research Council, the Swedish National Centre for Research in Sports, the Swedish Medical Association, the KI Foundation and the Wallenberg Foundation; AÖ: the Swedish Cancer Society and the Swedish Research Council including support for the Linné center and cancer research network ”Starget.” HR is supported by a Post Doc training grant from the Swedish Society of Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Prostate cancer is the second most frequent cancer diagnose in men in the world today. The highest incidence rates are found in the developed western countries and are 20 fold higher than the incidence rates found e.g. in South Central Asia and western Africa [1]. The discrepancy is partly due to the established use of PSA testing but lately a significant impact of life-style effects are being recognized [2]. Physical activity is an adjustable life-style factor associated with a reduced risk of several cancers, including prostate cancer [3]. A recent meta analysis comprising studies until 2012 suggests that being physically active is associated with a modest but significant reduction in risk of prostate cancer [4]. In addition, studies examining physical activity in relation to high-grade prostate cancer and prostate cancer mortality also reported a significant risk reduction [5]–[7]. The mechanisms mediating the effects of physical activity are not yet understood, although some candidates including weight control, improved immune cell function and modifications of endogenous hormone levels such as leptin, insulin and insulin like growth factor -1 (IGF-1) have been put forward [8]. Elevated serum levels of leptin, insulin and IGF-1 are all associated with high risk of prostate cancer incidence and progression [8]–[12] and long-term exercise is known to reduce serum levels of these and other endogenous hormones [13], [14]. Serum from endurance trained individuals on a low-fat, high-fiber diet has been shown to inhibit growth of an established prostate cancer cell line when compared to control serum [15]. More recent studies from the same group suggest that the mechanism behind the effect is mediated through the IGF-1 axis [16]. In contrast to long-term exercise, the endocrine effects of acute endurance exercise include increased levels of mitogenic factors such as growth hormone (GH) [17], various cytokines [18] including IL-6 [19], [20] and also increased bioavailability of IGF-1 [21], [22]. It can be speculated that the increase in serum growth factors induced by acute exercise may be detrimental to prostate cancer progression into malignancy. The incentive of the current study is to evaluate the effect of acute exercise serum on prostate cancer cell growth.

Methods Ethics Statement Prior to the human exercise study, the experimental protocol was explained to all subjects and written, informed consent was obtained. The study was approved by the Ethics Committee of Karolinska Institutet (266/01) and conformed to the Declaration of Helsinki. The animal experiments were conducted in accordance with national guidelines and approved by the Stockholm North Ethical Committee on Animal Experiments (N19/08), all efforts were made to ameliorate suffering of the tumor bearing animals. Experimental Model for the Acute Exercise Study Ten healthy male subjects were included in the study. Their median (range) age, height and weight were 25 (18–37) yrs, 180 (170–190) cm and 77 (58–82) kg, respectively. Median (range) maximal oxygen uptake (VO2-max) determined prior to the experiments was 3.7 (3.1–4.3) L*min−1. The exercise was performed on an electro-dynamically loaded cycle ergo meter. The subjects performed about 65 min of exercise. For the first 20 min, subjects cycled at 60 rpm at a median (range) work rate of 125 (105–148) watts (W), chosen to correspond to 50% of VO2-max, after which the work rate was increased to 165 (138–195) W, corresponding to 65% of VO2-max for another 40 min. Teflon catheters were inserted into both femoral veins and the femoral artery, at the level of the inguinal ligament. Blood samples were drawn simultaneously from the femoral artery (fa) and the ipsilateral femoral vein (fv) as described in [23]. In short, resting samples and samples collected 120 min after the end of exercise were used for further analyses. Growth of Prostate Cancer Cells Growth rates of the cell lines NIH 3T3 and LNCaP cells, previously used in Augsten et al [24]; a total of either 2×103 fibroblasts or 5×104 LNCaP cells were seeded in 96-well plates. Fibroblasts were grown in DMEM supplemented with 5% FCS and either 5% rest or exercise serum, whereas LNCaP cells were cultured in RPMI 1640 medium supplemented with 5% FCS and either 5% rest or exercise serum for 24, 48 and 96 hours. 2×104 22rv1 and Du145 cells, previously used in [25] were cultured in RPMI 1640 medium supplemented with 5% FCS and either 5% rest or exercise serum for 96 hours. Cell numbers were determined by fixating the cells with 4% PFA for 20 minutes, followed by incubation with crystal violet (Chroma, Stuttgart, FRG) solution (0.1%, w/v, with ethanol 2%, v/v in 0.5 M Tris-C1, pH 7.8; 100 µl per well) for 20 min at room temperature. The stained cell layer was rinsed thoroughly with tap water, air dried and incubated with SDS solution (0.1%, w/v, with ethanol 50%, v/v, in 0.5 M Tris-C1, pH 7.8; 100 µL per well) for 30 min at room temperature. Meanwhile crystal violet was completely released from the cells into the supernatant. The supernatant was scanned in a DU Series 70 Beckman spectrophotometer (Beckman Coulter, Fullerton, CA, USA) and read at a fixed wavelength of 600 nm. Apoptosis and Proliferation Assays Redistribution of plasma membrane phosphatidylserine is a marker of apoptosis and was assessed with annexin-V-fluos staining kit (Roche Molecular Biochemicals, Mannheim. Germany). Briefly, 2×105 LNCaP cells were incubated with exercise or rest serum for 24 and 48 hours, collected, washed in PBS, pelleted and re-suspended in incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ) containing 1% Annexin V and PI. Samples were incubated for 10 min before analysis on a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using Cell Quest software (San Jose, CA, USA). For proliferation assessment, Click-iT EdU Microplate Assay kits were used (Invitrogen. Eugene, OR, USA). In short, LNCaP cells were seeded in a 96 well plate at 5×103 cells per well and allowed to settle overnight. EdU stock solution was prepared and diluted in exercise or rest media at a working concentration of 10 µM, 100 µL of supplemented media was added to the cells and allowed to incubate for 24 hours. Cell fixation and labeling was done according to manufacturers protocol. Quantitation and analysis was done on a VICTOR2 Multilabel Counter (PerkinElmer, Waltham, MA, USA). Xenograft Model of Tumor Growth Xenograft tumors were generated by incubation of LNCaP cells in medium supplemented with rest serum or exercise serum for 48 hours and NIH-3T3 cells in medium supplemented with rest serum. Cells were trypsinized, re-suspended in medium, and counted with a cell counter (Chemometec, Allerod, Denmark). For co-injection analysis, 2×106 LNCaP cells and 0.5×106 NIH-3T3 cells were washed, combined, and re-suspended in 100 µL of cold PBS. Subsequently, the 100 µL cell suspensions were injected subcutaneously in the right lateral flank of 7–8 week-old, male SCID mice (strain CB17sc) from Taconic, DK, n = 10 per group. Animals were monitored daily, and tumor size measured every 3–4 days using external caliper. Experiments were terminated when individual tumors reached a volume of 1 cm3 or at day 49. Tumors were excised and split before either fixation in 4% paraformaldehyde overnight at 4°C followed by transfer to 70% ethanol, or snap frozen in liquid nitrogen. Analysis of Proliferation and Apoptosis in Tumor Samples Tissue sections from xenograft endpoint tumors were de-paraffinized and rehydrated in xylene (3×5 min), 99% ethanol (2×2 min), 96% ethanol (2×5 min), 70% ethanol (1×2 min), and then washed in distilled H2O. For apoptosis, the DeadEnd™ Colorimetric TUNEL System was used according to manufacturers instructions (Promega Corporation, Madison, WI, USA). For assessment of proliferation a PCNA histochemistry approach was used; antigen retrieval was done in 2 full racks of slides, boiled in a microwave oven 2×7 min at 650W in 10 mM citric acid buffer, pH 6.0. Sections were then left to cool for at least 30 min before being washed in PBS with 0.1% Tween-20 (PBT). Endogenous peroxidase activity was quenched by incubation in PBT containing 3% H 2 O 2 for 10 min, then washed in PBT (3×5 min). Slides were blocked in 20% goat serum diluted in PBT for 30 min and incubated overnight at 4°C with PCNA antibody (M0879 clone PC10, Dako Cytomation, (Dako Nordic a/s, Glostrup, Denmark) diluted 1∶100 in 20% goat serum diluted in PBT. After washing in PBT, slides were incubated with biotinylated goat anti-mouse antibody (E0432, 1∶500, Dako Cytomation) for 45 min at RT following incubation with the ABC kit (SK6100, Vectastain ABC-HRP, Vector Laboratories, Burlingame, CA, USA) for 45 min after washing in PBT. The signal was developed using diaminobenzidine hydrochloride, DAB (SK4100, Vector Laboratories), and sections were counter- stained with Mayer’s Heamatoxylin (Histolab Products AB, Gothenburg, Sweden), followed by washing in H 2 O, dehydration in ethanol (70%–95%–99%) and xylene. The slides were then mounted with Mountex mounting medium. Pictures from 5 different, randomly selected vision-fields per tumor were taken and the number of PCNA or tunnel positive structures was analyzed with ImageJ software. Serum Analysis Pools of exercise serum from 10 individuals and corresponding rest serum were analyzed with a human protein growth factor array kit (RayBiotech, Norcross, GA, USA). 200 µL of pooled exercise and rest serum was diluted with 1× blocking serum (provided in the kit) while the membranes were blocked for 1 hour in the same blocking buffer. 1 ml of diluted serum was added and incubated at room temperature for 2 hours. The membranes were then analyzed according to the manufacturer's instructions. Chemiluminiscens detection was done on the ImageQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK). Density of individual spots was quantified with Image J software. Individual serum concentrations of EGF and IGFBP1 were determined using a sandwich enzyme-linked immunoassay (ELISA; Abcam, Cambridge, UK) according to the manufacturer’s directions. The minimum detectable levels of these kits were 0.82 pg/mL for EGF and 2.74 pg/mL for IGFBP1. Statistics Student’s t-test was used to compare the effects of pre-exercise serum with effects of post exercise serum. A two way ANOVA was used to compare tumor growth between the two groups. Planned comparison was used (i.e., post hoc test) to identify significant interactions in the ANOVA model. Differences were considered significant at p<0.05. Unless otherwise stated, data is presented as mean ± SEM.

Discussion Prostate cancer develops over a long period of time with early PIN lesions that may or may not progress into prostate cancer. It is believed that this transition from benign hyperplasia to malignant carcinoma is affected by lifestyle factors. Previous studies suggest that serum from individuals that have been active over long periods of time may exert an inhibitory effect on prostate tumor cell growth in vitro [27]. The present study is the first to evaluate the acute serum effects of endurance exercise on prostate cancer cell growth. In the current study we present novel data showing that serum obtained directly after a bout of strenuous exercise, despite its mitogenic potential, reduced proliferation of prostate cancer tumor cells. The effect on prostate cancer cell growth was significant both in comparisons of pools of serum from 10 individuals and in comparisons of the individual serum pairs. Acute exercise serum had no apoptotic effect on the tumor cells. This is in contrast with the effect of chronic exercise serum described by Barnard et al. where apoptosis was induced through p53 activation [16]. Rather than inducing apoptosis we found that acute exercise serum altered the rate of active DNA synthesis measured by incorporation of EdU, indicating that the inhibitory effect is primarily on tumor cell proliferation. Interestingly, the effect on tumor cells in vitro is robust enough to be extrapolated to the in vivo situation. Exercise serum-treated LNCaP cells initially displayed impaired tumor growth compared to LNCaP cells pre-incubated in rest serum. As would be expected, the inhibitory effect seemed to be reversible and was lost once the xenografts were established. In line with this, we found no difference in the number of proliferating or apoptotic cells when tumors were harvested. From the growth factor array, two factors correlated to the growth data and emerged as possible candidates to convey the exercise effect; reduced levels of EGF and increased levels of IGFBP-1. EGF is known to stimulate the growth of LNCaP cells [28]. Increased EGF receptor expression is found in prostate cancer and is associated with poor prognosis [29]. Compounds inhibiting EGF receptor activity in prostate cancer are currently undergoing clinical trials [30]. EGF is traditionally considered to be released in a paracrine fashion from the local microenvironment and not provided through circulation. Whether circulating levels of EGF is of clinical relevance to prostate cancer progression has, to our knowledge, not been investigated. The IGF binding proteins modulates the bioavailability of IGF-1 and 2. IGF-1 is known to regulate e.g. cell proliferation and apoptosis [31] and high levels of circulating IGF-1 have been associated with increased prostate cancer risk [11], [32]. It has previously been shown that serum from long-term endurance trained individuals exerts an inhibitory effect on LNCaP growth, attributed to low levels of IGF-1 and high levels of IGFBP-1. [16], [33], [34]. In the studies mentioned, addition of IGFBP-1 to the control serum reduced tumor cell growth to the same extent as exercise serum. In studies of acute exercise in healthy individuals, and also in prostate cancer patients undergoing androgen deprivation [35], serum levels of IL-6, GH and IGF-1 increase [21], [22]. In the current study there is no detectable increase in serum IGF-1. This may be due to a transient mode of action that is already back to normal at two hours after exercise, when the current samples are taken. Solid tumors consist not only of malignant epithelial cells. Growth and progression of tumors are dependent on a number of associated cell types such as fibroblasts, vascular and immune cells. The current study did not identify effects of exercise serum effect on the growth of fibroblasts. However, more studies are needed to evaluate the effect of exercise on the supportive role of the cells of the tumor microenvironment. The current data show a robust effect of acute exercise on growth of an established prostate cancer cell line. In the future, follow up studies on the molecular basis for the differential effects of rest and exercise serum on prostate cancer cell growth are highly motivated, preliminary data suggest that compensating for the lower levels of EGF in the post exercise serum by adding rhEGF partly reverse the growth inhibitory effect of exercise serum (data not shown), thereby supporting the idea that multiple factors altered by exercise are involved. Furthermore, analyses of the effect of exercise serum on transformation of pre-malignant prostate epithelial cells would provide important additional information on the role of exercise in prostate cancer risk and progression. In conclusion, despite the fear of possible detrimental effects of acute exercise serum on tumor cell growth, we show that even short-term effects seem to add to the overall beneficial influence of exercise on neoplasia.

Supporting Information Figure S1. Growth inhibition of prostate cancer cells by exercise serum is not mediated through induction of apoptosis. A) Flow cytometry of LNCaP cells stained with AnnexinIV and PI, squares giving distinct subsets of viable (low left), necrotic (top left), early (low right) and late (top right) apoptotic cells. B) Quantification of total (early apoptotic+late apoptotic) number of apoptotic cells after 24 hours incubation with rest and exercise serum, based on the number of hits in the different subsets defined in A). C) Quantification of total (early apoptotic+late apoptotic) number of apoptotic cells after 48 hours incubation with restand exercise serum based on the number of hits in the different subsets defined in A). https://doi.org/10.1371/journal.pone.0067579.s001 (PDF) Figure S2. Growth inhibition of prostate cancer cells by exercise serum is not mediated through increased serum levels of cortisol.♮ denotes a significant (p<0.05) increase in s-cortisol directly after exercise. This increase has returned to normal levels in the exercise serum samples used in the analysis of prostate cancer cell growth (serum obtained 2 hours post exercise). https://doi.org/10.1371/journal.pone.0067579.s002 (PDF)

Author Contributions Conceived and designed the experiments: HR ER TG AÖ. Performed the experiments: HR MA AS ER SM PK. Analyzed the data: HR MA ER SM AS TP. Contributed reagents/materials/analysis tools: MA AS PK TP TG. Wrote the paper: HR MA TG AÖ.