The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells

Purpose There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. Methods Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. Results All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/β-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. Conclusions These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention (AU)

The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells.

PURPOSE: There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. METHODS: Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. RESULTS: All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/ß-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. CONCLUSIONS: These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.

Safety and feasibility of preoperative exercise training during neoadjuvant treatment before surgery for adenocarcinoma of the gastro-oesophageal junction.

Background: Neoadjuvant chemotherapy or chemoradiotherapy is used widely before tumour resection in cancer of the gastro-oesophageal junction (GOJ). Strategies to improve treatment tolerability are warranted. This study examined the safety and feasibility of preoperative exercise training during neoadjuvant treatment in these patients. Methods: Patients were allocated to a standard-care control group or an exercise group, who were prescribed standard care plus twice-weekly high-intensity aerobic exercise and resistance training sessions. The primary endpoint was the incidence of serious adverse events (SAEs) that prevented surgery, including death, disease progression or physical deterioration. Preoperative hospital admission, postoperative complications, changes in patient-reported quality of life and pathological treatment response were also recorded. In the exercise group, adherence to exercise and changes in aerobic fitness, muscle strength and body composition were measured. Results: The incidence of SAEs was not increased in the exercise group. The risk of failure to reach surgery was 5 versus 21 per cent in the control group (risk ratio (RR) 0·23, 95 per cent c.i. 0·04 to 1·29), the risk of preoperative hospital admission was 15 versus 38 per cent respectively (RR 0·39, 0·12 to 1·23) and the risk of postoperative complications was 58 versus 57 per cent (RR 1·06, 0·61 to 1·73). The exercise group attended a mean of 17·5 sessions, and improved fitness, muscle strength and Functional Assessment of Cancer Therapy - Esophageal (FACT-E) total score compared with the baseline level. Conclusion: Preoperative exercise training during neoadjuvant treatment in patients with GOJ cancer is safe and feasible, with improvements in fitness, strength and quality of life. Preoperative exercise training may be associated with a lower risk of critical SAEs that preclude surgery or result in hospitalization.

Leydig cell dysfunction, systemic inflammation and metabolic syndrome in long-term testicular cancer survivors.

BACKGROUND: Twenty to thirty percent of testicular cancer (TC) survivors have elevated serum levels of luteinising hormone (LH) with or without corresponding low testosterone levels (Leydig cell dysfunction) during clinical follow-up for TC. However, it remains to be clarified if this subgroup of TC survivors has an increased long-term risk of systemic inflammation and metabolic syndrome (MetS) when compared with TC survivors with normal Leydig cell function during follow-up. PATIENTS AND METHODS: TC survivors with Leydig cell dysfunction and a control group of TC survivors with normal Leydig cell function during follow-up were eligible for participation in the study. Markers of systemic inflammation and prevalence of MetS were compared between TC survivors with Leydig cell dysfunction and the control group. RESULTS: Of 158 included TC survivors, 28 (18%) had uncompensated Leydig cell dysfunction, 59 (37%) had compensated Leydig cell dysfunction and 71 (45%) had normal Leydig cell function during follow-up. MetS and markers of systemic inflammation were evaluated at a median follow-up of 9.7 years (interquartile range 4.1-17.1) after TC treatment. The prevalence of MetS was significantly lower among patients with compensated Leydig cell dysfunction during follow-up (12% versus 27%, p = 0.04), whereas there was no difference between TC survivors with uncompensated Leydig cell dysfunction and controls (33% versus 27%, p = 0.5). Apart from high-sensitivity C-reactive protein which was higher in TC survivors with uncompensated Leydig cell dysfunction during follow-up, there was no evidence of increased systemic inflammation in patients with Leydig cell dysfunction during clinical follow-up. Total testosterone at follow-up was significantly associated with MetS, whereas there was no association between LH and MetS. CONCLUSION: We did not find evidence that TC survivors with Leydig cell dysfunction during clinical follow-up had increased long-term risk of MetS. Total testosterone at follow-up was significantly associated with MetS. The study is registered at www.clinicaltrials.govNCT02240966.

Reproductive hormones and metabolic syndrome in 24 testicular cancer survivors and their biological brothers.

Testicular cancer survivors have impaired gonadal function and increased risk of metabolic syndrome when compared to healthy controls. However, because of the fetal etiology of testicular cancer, familial unrelated healthy men might not be an optimal control group. The objective of this study was to clarify if testicular cancer survivors have impaired gonadal function and increased risk of metabolic syndrome when compared to their biological brothers. A cross-sectional study of testicular cancer survivors (ClinicalTrials.gov number, NCT02240966) was conducted between 2014 and 2016. Of 158 testicular cancer survivors included, 24 had a biological brother who accepted to participate in the study. Serum levels of reproductive hormones and prevalence of metabolic syndrome according to International Diabetes Federation Criteria and National Cholesterol Education Program (Adult Treatment Panel III) criteria comprised the main outcome measures of the study. Median age was similar in testicular cancer survivors and their biological brothers [44 years (IQR 39-50) vs. 46 (40-53) years respectively (p = 0.1)]. In testicular cancer survivors, follow-up since treatment was 12 years (7-19). Serum levels of luteinizing hormone and follicle-stimulating hormone were elevated (p ≤ 0.001), while total testosterone, free testosterone, inhibin B and anti-Müllerian hormone were lower (p ≤ 0.001) in testicular cancer survivors than in their biological brothers. The prevalence of metabolic syndrome was similar and apart from HDL-cholesterol, which was lower in testicular cancer survivors (p = 0.01); there were no differences in the individual components of the metabolic syndrome between testicular cancer survivors and their brothers. In conclusion, gonadal function was impaired in testicular cancer survivors, while we did not detect any difference in the prevalence of metabolic syndrome between testicular cancer survivors and their biological brothers.

Running away from side effects: physical exercise as a complementary intervention for breast cancer patients

The number of breast cancer survivors increases every year, thanks to the development of new treatments and screening techniques. However, patients present with numerous side effects that may affect their quality of life. Exercise has been demonstrated to reduce some of these side effects, but in spite of this, few breast cancer patients know and follow the exercise recommendations needed to remain healthy. In this review, we describe the different breast cancer treatments and the related side effects and implications of exercise in relation to these. We propose that exercise could be an integrative complementary intervention to improve physiological, physical and psychological factors that affect survival and quality of life of these patients. For that reason, the main objective of this review is to provide a general overview of exercise benefits in breast cancer patients and recommendations of how to design exercise interventions in patients with different side effects (AU)

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Running away from side effects: physical exercise as a complementary intervention for breast cancer patients.

The number of breast cancer survivors increases every year, thanks to the development of new treatments and screening techniques. However, patients present with numerous side effects that may affect their quality of life. Exercise has been demonstrated to reduce some of these side effects, but in spite of this, few breast cancer patients know and follow the exercise recommendations needed to remain healthy. In this review, we describe the different breast cancer treatments and the related side effects and implications of exercise in relation to these. We propose that exercise could be an integrative complementary intervention to improve physiological, physical and psychological factors that affect survival and quality of life of these patients. For that reason, the main objective of this review is to provide a general overview of exercise benefits in breast cancer patients and recommendations of how to design exercise interventions in patients with different side effects.

Safety and efficacy of resistance training in germ cell cancer patients undergoing chemotherapy: a randomized controlled trial.

BACKGROUND: Bleomycin-etoposid-cisplatin (BEP) chemotherapy is curative in most patients with disseminated germ cell cancer (GCC) but also associated with toxic actions and dysfunction in non-targeted tissues. We investigated changes in muscle function during BEP and the safety and efficacy of resistance training to modulate these changes. METHODS: Thirty GCC patients were randomly assigned to resistance training (resistance training group (INT), n=15) or usual care (CON, n=15) during 9 weeks of BEP therapy. Resistance training consisted of thrice weekly sessions of four exercises, 3-4 sets/exercise of 10-15 repetitions at 12-15 repetition maximum load. The primary endpoint was muscle fibre size, assessed in muscle biopsies from musculus vastus lateralis. Secondary endpoints were fibre phenotype composition, body composition, strength, blood biochemistry and patient-reported endpoints. Healthy age-matched subjects (REF, n=19) performed the same RT-programme for comparison purposes. RESULTS: Muscle fibre size decreased by -322 µm(2) (95% confidence interval (CI): -899 to 255; P=0.473) in the CON-group and increased by +206 µm(2) (95% CI: -384 to 796; P=0.257) in the INT-group (adjusted mean difference (AMD), +625 µm(2), 95% CI: -253 to 1503, P=0.149). Mean differences in type II fibre size (AMD, +823 µm(2), P=0.09) and lean mass (AMD, +1.49 kg, P=0.07) in favour of the INT-group approached significance. The REF-group improved all muscular endpoints and had significantly superior changes compared with the INT-group (P<0.05). CONCLUSIONS: BEP was associated with significant reduction in lean mass and strength and trends toward unfavourable changes in muscle fibre size and phenotype composition. Resistance training was safe and attenuated dysfunction in selected endpoints, but BEP blunted several positive adaptations observed in healthy controls. Thus, our study does not support the general application of resistance training in this setting but larger-scaled trials are required to confirm this finding.

Muscle dysfunction in cancer patients.

BACKGROUND: Muscle dysfunction is a prevalent phenomenon in the oncology setting where patients across a wide range of diagnoses are subject to impaired muscle function regardless of tumor stage and nutritional state. Here, we review the current evidence describing the degree, causes and clinical implications of muscle dysfunction in cancer patients. The efficacy of exercise training to prevent and/or mitigate cancer-related muscle dysfunction is also discussed. DESIGN: We identified 194 studies examining muscular outcomes in cancer patients by searching PubMed and EMBASE databases. RESULTS: Muscle dysfunction is evident across all stages of the cancer trajectory. The causes of cancer-related muscle dysfunction are complex, but may involve a wide range of tumor-, therapy- and/or lifestyle-related factors, depending on the clinical setting of the individual patient. The main importance of muscle dysfunction in cancer patients lies in the correlation to vital clinical end points such as cancer-specific and all-cause mortality, therapy complications and quality of life (QoL). Such associations strongly emphasize the need for effective therapeutic countermeasures to be developed and implemented in oncology practice. Significant progress has been made over the last decade in the field of exercise oncology, indicating that exercise training constitutes a potent modulator of skeletal muscle function in patients with cancer. CONCLUSION: There are clear associations between muscle dysfunction and critical clinical end points. Yet there is a discrepancy between timing of exercise intervention trials, which can improve muscle function, and study populations in whom muscle function are proven prognostic important for clinical end points. Thus, future exercise trials should in early-stage patients, be powered to evaluate clinical outcomes associated with improvements in muscle function, or be promoted in advanced stage settings, aiming to reverse cancer-related muscle dysfunction, and thus potentially improve time-to-progression, treatment toxicity and survival.

IL-6 regulates exercise and training-induced adaptations in subcutaneous adipose tissue in mice.

AIM: The aim of this study was to test the hypothesis that IL-6 regulates exercise-induced gene responses in subcutaneous adipose tissue in mice. METHODS: Four-month-old male IL-6 whole body knockout (KO) mice and C57B wild-type (WT) mice performed 1 h of treadmill exercise, where subcutaneous adipose tissue (AT) was removed either immediately after, 4 h or 10 h after exercise as well as from mice not running acutely. Moreover, AT was sampled at resting conditions after 5 weeks of exercise training. RESULTS: AT leptin mRNA decreased immediately after a single running exercise bout in both genotypes and returned to baseline within 10 h of recovery in IL-6 KO mice, but not WT mice. Leptin mRNA content decreased in WT and increased in IL-6 KO mice with training, but without significant alterations in leptin protein. Acute exercise induced a decrease in the AT TNFα mRNA content in WT, but not in IL-6-KO mice, while training lowered resting levels of TNFα mRNA in both genotypes. In addition, an exercise-induced decline in AT PPARγ mRNA content was absent in IL-6 KO mice and in line training increased PPARγ mRNA only in IL-6 KO mice. CONCLUSION: The present findings indicate a role of IL-6 in regulating exercise- and training-induced leptin and PPARγ expression in adipose tissue. In addition, while IL-6 is required for TNF-α mRNA reduction in response to acute exercise, IL-6 does not appear to be mandatory for anti-inflammatory effects of exercise training in adipose tissue.

Duration and level of transgene expression after gene electrotransfer to skin in mice.

In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations. Level and duration of transgene expression after gene electrotransfer to skin is essential and here we present data from two independent quantitative studies. Using in vivo bioimaging of a far-red fluorescent molecule, Katushka, allowing for continuous monitoring of local gene expression, compared with measurements of a systemic transgene, that is, serum erythropoietin (EPO) after gene electrotransfer with EPO to skin, we found a significant increase in transgene expression (P< 0.01) with a peak 9 days (Katushka) and 14 days (EPO) after transfection. Duration of expression could be 3-4 weeks, which is a suitable time frame for vaccinations and is applicable, for example, in gene therapy for wound healing or treatment of cancer.

Therapeutic levels of erythropoietin (EPO) achieved after gene electrotransfer to skin in mice.

Gene electrotransfer refers to gene transfection by electroporation and is an effective non-viral method for delivering naked DNA into cells and tissues. This study presents data from gene electrotransfer with erythropoietin (EPO) to mouse skin. Nine-week-old female NMRI mice received one, two or three intradermal injections of 50 microg EPO plasmid and were subsequently electroporated. With plate electrodes and 100 microg of EPO, a significant increase in hemoglobin (P<0.01) was observed compared with controls. The level of hemoglobin peaked after 5 weeks but stayed significantly elevated for more than 3 months. Serum EPO was significantly increased (P<0.001) 24 h after the transfection and remained significantly different compared with controls until the maximum level of serum EPO was reached after 2 weeks. Eight weeks after the transfection serum EPO returned to baseline. In this study, we have established that gene electrotransfer to skin of even small amounts of DNA can lead to systemically therapeutic levels of protein. This means that in addition to DNA vaccinations, there is a potential utility for electroporation in alleviating systemic diseases such as cancer and protein deficiency disorders.

Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser(79)) were analysed, as was fatty acid oxidation (FAO). Finally, we electroporated a Bdnf vector into the tibialis cranialis muscle of mice. RESULTS: BDNF mRNA and protein expression were increased in human skeletal muscle after exercise, but muscle-derived BDNF appeared not to be released into the circulation. Bdnf mRNA and protein expression was increased in muscle cells that were electrically stimulated. BDNF increased phosphorylation of AMPK and ACCbeta and enhanced FAO both in vitro and ex vivo. The effect of BDNF on FAO was AMPK-dependent, since the increase in FAO was abrogated in cells infected with an AMPK dominant negative adenovirus or treated with Compound C, an inhibitor of AMPK. Electroporation of a Bdnf expression vector into the tibialis cranialis muscle resulted in increased BDNF protein production and tropomyosin-related kinase B (TrkB(Tyr706/707)) and extracellular signal-regulated protein kinase (p44/42 Thr(202)/Tyr(204)) phosphorylation in these muscles. In addition, phosphorylation of ACCbeta was markedly elevated in the Bdnf electroporated muscles. CONCLUSIONS/INTERPRETATION: These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing lipid oxidation in skeletal muscle via activation of AMPK.

Control by pulse parameters of DNA electrotransfer into solid tumors in mice.

Electrotransfer (electroporation) is recognized as one of the most promising alternatives to viral vectors for transfection of different tissues in vivo for therapeutic purposes. We evaluated the transfection efficiency of reporter genes (green fluorescent protein and luciferase) in murine subcutaneous tumors using different combinations of high-field (HV) (600-1400 V cm(-1), 100 mus, 8 pulses) and low-field (LV) (80-160 V cm(-1), 50-400 ms, 1-8 pulses) pulses and compared it to protocol using eight identical pulses of 600 V cm(-1) and 5 ms duration (electro-gene therapy, EGT). Expression of GFP was determined using a fluorescent microscope and flow cytometry and expression of luciferase by measuring its activity using a luminometer. The EGT protocol yielded the highest expression of both reporter genes. However, a careful optimization of combinations of HV and LV pulses may result in similar transfection as EGT pulses. With the combination protocol, relatively high fields of LV pulses were necessary to obtain comparable transfection to the EGT protocol. Expression of reporter genes was higher in B16 melanoma than in SA-1 fibrosarcoma. Our data support the hypothesis that both electropermeabilization and electrophoresis are involved in electrotransfer of plasmid DNA, but demonstrate that these components have to happen at the same time to obtain significant expression of the target gene in tumors.

Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation.

Electroporation-based gene transfer (electro gene transfer (EGT)) is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. Induction of expression using the Tet-On system was previously established; however, attempts to reach a predefined target dose - a prescription, have not been reported. We set three target haemoglobin levels (10, 12 and 14 mmol/l, base level was 8.2 mmol/l) and aimed at them by transferring the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle, and varying (1) DNA amount, (2) muscle mass transfected and (3) induction with the Tet-On system. Results showed that (a) using GFP, luciferase and EPO low DNA amounts were needed. In fact, 0.5 microg of DNA to one TC muscle led to significant Hgb elevation - this amount extrapolates to 1.4 mg of DNA in humans, (b) three prescribers hit the targets with average Hgb of 10.5, 12.0 and 13.7 mmol/l, (c) different approaches could be used, (d) undershooting could be corrected by retransferring, and (e) overshooting could be alleviated by reducing dose of inducer (doxycycline (dox)). In conclusion, this study shows that using EGT to muscle, a preset level of protein expression can be reached. This is of great interest for future clinical use.