Tumor growth reduction in Walker 256 tumor-bearing rats performing anaerobic exercise:participation of Bcl-2, Bax, apoptosis, andperoxidation

Carina de Lima, Luciana Alves, Fabíola Iagher, Andressa Franzoi Machado,Marcelo Kryczyk, Ricardo Key Yamazaki, Gleisson Alisson Pereira Brito,Everson Araújo Nunes, Katya Naliwaiko, and Luiz Cláudio Fernandes

Abstract: Physical activity has been used in cancer prevention and treatment. In this study, we investigated some of themechanisms by which anaerobic exercise reduces tumor growth. To do so, rats were trained for 8 weeks. Training consistedof jumping in a swimming pool for ten 30-s sets, with a load that was 50% of body weight attached to the back, 4 timesper week. At the sixth week, anaerobic exercise trained rats (EX group) were inoculated with a suspension of Walker 256tumor cells. Tumor weight, apoptotic tumor cells, tumor Bax and Bcl-2 protein expression, tumor lipid peroxidation, and tu-mor cell proliferation ex vivo were evaluated. Tumor weight was significantly lower in the EX group (∼30%) than in ratsthat did not undergo training (sedentary group) (p < 0.05). Apoptosis in the tumor cells of EX rats was 2-fold higher thanin the tumor cells of sedentary rats; in addition, Bax expression increased by 10% and Bcl-2 decreased by 13% in EX rats.Lipid peroxidation was 4-fold higher in the tumor cells of EX rats than in those of sedentary rats (p < 0.05). Tumor cellproliferation ex vivo was 29% lower in the EX group than in the sedentary group (p < 0.05). In conclusion, Walker 256tumor-bearing exercised rats presented more tumor cell apoptosis, a higher tumor content of lipid peroxides, pro-apoptoticprotein expression balance, and reduced tumor weight and cell proliferation ex vivo, compared with sedentary rats. Theseevents, together, account for the lower tumor growth we observed in the EX rats.

Key words: cancer, exercise, rats, apoptosis, Bax, Bcl-2, lipid peroxidation.

Résumé : L’activité physique est utilisée comme moyen de prévention et de traitement du cancer. Dans cet article, nous étu-dions quelques mécanismes par lesquels l’exercice physique à caractère anaérobie diminue la grosseur d’une tumeur. À cettefin, nous soumettons des rats à un programme d’entraînement de 8 semaines. Le programme consiste en 10 périodes de30 s de sauts, et ce, 4 fois par semaine, avec une charge fixée au dos équivalant à 50 % du poids du corps. À la 6e semainede l’entraînement anaérobie, on inocule les rats (EX) au moyen d’une suspension de cellules tumorales Walker-256. On éva-lue le poids de la tumeur, l’apoptose des cellules tumorales, l’expression des facteurs Bax et Bcl-2, la peroxydation lipidiquede la tumeur et la prolifération ex vivo des cellules tumorales. Comparativement aux sédentaires, le poids de la tumeur estsignificativement plus petit (~30 %) chez les animaux soumis à l’entraînement (p < 0,05 vs. S). L’apoptose des cellules tu-morales chez les rats du groupe EX augmente de 2 fois comparativement aux cellules des rats sédentaires (S); en outre, l’ex-pression du facteur Bax augmente de 10 % et celle du facteur Bcl-2 diminue de 13 %. La peroxydation lipidique de latumeur est 4 fois plus élevée chez les rats du groupe EX, comparativement à ceux du groupe S (p < 0,05). La proliférationex vivo des cellules tumorales est de 29 % plus faible chez les rats du groupe EX (p < 0,05 vs. S). En conclusion, la tumeurWalker-256 des rats soumis à l’entraînement présente une plus importante apoptose des cellules tumorales, un plus grandcontenu de peroxydes lipidiques, une meilleure balance de l’expression des protéines pro-apoptotiques, une tumeur plus lé-gère et une prolifération ex vivo inférieure des cellules tumorales, comparativement aux rats sédentaires. Ces phénomènesexpliquent la diminution du poids de la tumeur dans cette étude.

Mots‐clés : cancer, exercice physique, rats, apoptose, Bax, Bcl-2, peroxydation lipidique.

[Traduit par la Rédaction]

Received 19 August 2010. Accepted 18 March 2011. Published at www.nrcresearchpress.com/apnm on 18 August 2011.

C. de Lima, L. Alves, A.F. Machado, M. Kryczyk, R.K. Yamazaki, G.A.P. Brito, K. Naliwaiko, and L.C. Fernandes. Department ofPhysiology, Biological Science Building, Federal University of Paraná, Curitiba-PR, Brazil.F. Iagher. Biological and Health Sciences Area, West of Santa Catarina University, Joaçaba-SC, Brazil.E.A. Nunes. Department of Physiology, Biological Sciences Center, Federal University of Santa Catarina, Florianópolis-SC, Brazil.

Corresponding author: Luiz Claudio Fernandes (e-mail: lcfer@ufpr.br).

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Appl. Physiol. Nutr. Metab. 36: 533–538 (2011) doi:10.1139/H11-047 Published by NRC Research Press

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Introduction

Cancer cachexia is a disease characterized by weight loss,atrophy and muscle loss, anemia, weakness, asthenia, and in-tense catabolism of carbohydrate, lipid, and protein stores(Argilés et al. 1997; Tisdale 2005). Loss of body weight andmuscle mass are 2 main features of cancer cachexia; the de-gree of muscle mass loss has been correlated with reducedsurvival rates in cancer patients (Burnham and Wilcox2002). These also occur with sedentary behavior and lack ofphysical activity, which lead to a low quality of life (Older-voll et al. 2004).There is quite a lot of evidence showing that exercise is an

important modifiable factor for cancer risk and treatment(Gago-Dominguez and Castelao 2006). It has been reportedthat exercise is able to modify tumor progression (Bacurauet al. 2007) and to improve physical fitness and quality oflife in cancer patients (Dimeo et al. 1997; Courneya 2003).It has previously been shown that exercise can preserve liverglycogen stores, prevent a decrease in body weight, and re-duce proteolysis-inducing factor expression (de Lima et al.2008), but the mechanisms underlying such findings werenot addressed in that study.Aerobic exercise is usually practiced by individuals want-

ing to achieve physical fitness, but resistance exercise, whichis predominantly anaerobic activity, has recently been in-cluded in the recommendations for physical fitness andhealth (Edwards et al. 2007). Both aerobic and anaerobic ex-ercise have been shown to reduce tumor growth and cancercachexia (Westerlind et al. 2003; Bacurau et al. 2007; deLima et al. 2008); however, how exercise affects tumorgrowth and cachexia is not fully known. The mechanism hasbeen partially explained by modulation of the immune system(Woods et al. 1999) and by neuroendocrine alterations (Peijieet al. 2003; Westerlind 2003). Furthermore, several studieshave shown that lipid peroxidation and free radicals increasein humans and animals after physical activity (Gago-Domi-nguez et al. 2005). It is known that cell membranes are proneto peroxidation and that lipid peroxides inhibit tumor cellgrowth (Gonzalez et al. 1993; Palozza et al. 2000), whichmight play a role in the anti-cancer effect of exercise on tu-mor growth.Walker 256 carcinoma is a mammary-gland-derived tumor

that is easily transplanted and is specific to the rat. It hasbeen used in several studies because of its ability to growrapidly and to induce many of the metabolic alterations pro-duced by human tumors (Black et al. 1994). Thus, to gain in-formation about the anti-cancer effects of anaerobic exerciseon tumor growth in vivo and ex vivo, we investigatedwhether anaerobic exercise affects lipid peroxidation, Baxand Bcl2 expression, tumor cell proliferation ex vivo, or thepercentage of apoptotic and necrotic cells in Walker 256tumor-bearing rats.

Materials and methods

Study designAll procedures involving animals were approved by the

Local Committee of Animal Welfare. Male Wistar rats (age,70 days) were maintained under controlled temperature (23 °C)and humidity, in a 12 h light:12 h dark cycle, and with food

and water ad libitum. They were separated into 2 tumor-bearinggroups: sedentary (n = 18) and anaerobic exercise trained(n = 18).Anaerobic exercise training was carried out in a swimming

pool apparatus, in which water temperature was maintainedbetween 29 °C and 32 °C. The apparatus is composed of 10individual swimming pools in which each rat swims individ-ually. The depth of the water column is maintained at 50 cm.The anaerobic exercise training protocol consisted of ten 30-ssets, each followed by 1 min of rest, with a load equivalent to50% of body weight attached to the back, 4 times a week for8 weeks.Rats were acclimated to the water environment for 2 days

before the exercise protocol was initiated. So that they couldfurther adapt to the water environment, rats underwent pro-gressive training, beginning with a load corresponding to30% of their body weight and reaching 50% at the eighth ex-ercise session. Because the load is too great for the rats tostay above the water surface by swimming, they must per-form serial jumps to keep breathing. In 30 s, the duration ofeach set, a rat performs 10 jumps, on average. This trainingprotocol is characterized by power and anaerobic exercise.Training sessions were performed on Mondays, Tuesdays,

Thursdays, and Fridays; all trained rats rested on the otherdays. After 6 weeks of training, sedentary and anaerobic ex-ercise trained rats were inoculated in the right flank with1 mL of a sterile suspension of 2 × 107 cells·mL–1 of Walker256 tumor cells, obtained from an ascitic tumor-bearing rat.In rats inoculated with Walker 256 tumor cells, the tumorgrows without causing apparent physiologic disturbances fora certain period of time (10 days), which is suddenly inter-rupted by the initiation of a period of rapid tumor growthand accentuated metabolic changes in the host, with eventualdeath at around 20 to 30 days. The anaerobic exercise trainedgroup continued the training after tumor cell inoculation until48 h prior to the end. On the fourteenth day after tumor inoc-ulation, the animals were killed by decapitation without anes-thesia. The tumor was removed and weighed, and differentsamples of the same tumor were used for cell culture, apop-tosis assay, lipid peroxidation, and Bax and Bcl-2 expression.All groups were decapitated 48 h after the last session of

exercise to eliminate the acute effect of the exercise sessionon cell metabolism.

Culture of Walker 256 tumor cells ex vivoThis procedure was performed according to the method of

Huang et al. (2000). Cells were obtained from rats bearing asolid tumor for 14 or 15 days. The whole tumor was removedand chopped with a scalpel. The cell suspension was filteredthrough a funnel with gauze. Red blood cells were dis-charged with a solution containing NH4Cl (15.5 mmol·L–1)and EDTA (5.5 mmol·L–1) in a 9:1 ratio. Tumor cells werecentrifuged at 1000g for 5 min and suspended in RPMI1640 medium. Then, tumor cells were cultured in RPMI1640 medium enriched with 10% fetal calf serum, containing100 U·mL–1 penicillin, 100 mg·mL–1 streptomycin, and0.05 mCi (2-14C)-thymidine. The cells were seeded in 96-well microplates at a concentration of 2 × 106 cells·mL–1.After 24 h of culture at 37 °C in 5% CO2, the cells were har-vested onto glass fiber disks and washed, using a Skatron cellhavester. Radioactive thymidine incorporation into DNA was

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determined by liquid scintillation counting in a Beckman LS6000IC scintillation counter. Each cell preparation was cul-tured in sixtuplicate, and the data were averaged.

Hydroperoxide content in the tumor tissueThe product of lipid peroxidation was measured using the

method described by Nourooz-Zadeh et al. (1994). Briefly,tumor tissue (0.2 g) was homogenized in 1 mL of methanoland centrifuged at 5000g for 5 min at 4 °C. The supernatant(0.09 mL) was added to 3 Eppendorf tubes containing(0.01 mL) methanol and to 3 Eppendorf tubes (0.01 mL)containing triphenylphosphine, and incubated for 30 min atroom temperature. To all the Eppendorf tubes, 0.9 mL of re-action solution (100 µmol·L–1 xylenol orange, 400 mmol·L–1

butylated hydroxytoluene, 25 mmol·L–1 sulfuric acid, and250 mmol·L–1 ferrous sulfate ammonium in 90% methanol)was added, and they were incubated for 30 min at room tem-perature prior to measurement at 560 nm. The concentrationof hydroperoxides was calculated by subtracting the methanolabsorbance samples from thetriphenylphospine samples. Thevalue obtained was then interpolated using a standard curveof hydrogen peroxide (100.0, 50.0, 25.0, 12.5, 6.2, 3.1, and1.6 µmol·L–1), which was submitted to the procedures de-scribed above for the determination of hydroperoxides. Dataare expressed as nmol of non-H2O2 hydroperoxides per mgprotein.

Apoptosis and necrosisThe percentage of tumor cells undergoing apoptosis was

evaluated using annexin-V FITC (produced by the Instituteof Biomedical Sciences, University of São Paulo, Brazil). Tu-mor cells undergoing necrosis were evaluated using 7-amino-actinomycin D (7-AAD) (BD Biosciences Pharmingen). Tu-mor cells (106), isolated from tumors after tumor extraction,were stained with annexin-V FITC for 15 min and with 7-AADfor 5 min, and were then analyzed using flow cytometry. Thepositive cells for annexin-V FITC were considered to be in anearly stage of apoptosis.

Western Blot analysis of Bax and Bcl-2Pieces of accurately weighed tumor tissue (0.1 g) were ho-

mogenized in 0.7 mL lysis buffer (100 mmol·L–1 Trizma base(pH 7.5), 10 mmol·L–1 EDTA, 10% sodium dodecyl sulfate,10 mmol·L–1 sodium pyrophosphate, 10 mmol·L–1 sodiumfluoride, 10 mmol·L–1 sodium orthovanadate, and 10 mg·mL–1

aprotinin). Then, the samples were boiled for 5 min and sub-jected (60 mg per lane) to SDS–PAGE (15% polyacrylamide),followed by electrophoretic transfer onto nitrocellulose mem-branes (Hybond-ECL; Amersham Biosciences, São Paulo,Brazil), using a semi-dry blotter apparatus (HOEFER-MiniVE; Amersham Biosciences). The membranes were incu-bated overnight with mouse monoclonal antibody to Bax andBcl-2 (ZYMED Laboratories) at a dilution of 1:500. Theywere then incubated with a secondary antibody-conjugatedmouse horseradish peroxidase (Santa Cruz Biotechnology,Santa Cruz, Calif.), diluted to 1:5000. Finally, the membraneswere subjected to enhanced chemiluminescence (SuperSignalSystems; Pierce). Blots were scanned, using Image J soft-ware, to detect relative band intensities.

Statistical analysisData are presented as mean ± SEM. Statistical analysis

was performed using Student’s t test; a value of p < 0.05was taken to indicate statistical significance.

Results

Tumor weight and Walker 256 tumor cell proliferation exvivoIn rats in the sedentary group, tumor weight at death was

24.30 ± 1.35 g (Fig. 1A). In rats in the anaerobic exercisetrained group, tumor weight at death was 17.03 ± 1.19 g.Therefore, exercise induced a 32% reduction in tumor growthin the anaerobic exercise trained group, compared with thesedentary group (p < 0.01). The incorporation of [2-14C]-thy-midine in Walker 256 tumor cells from rats in the sedentarygroup was 1458.00 ± 30.32 cpm and in the anaerobic exer-cise training group was 1128.00 ± 13.69 cpm (Fig. 1B).Anaerobic exercise training induced a reduction in cell prolif-eration ex vivo that was 29% lower than in the sedentary rats(p < 0.0001).

Hydroperoxides in Walker 256 tumor tissue, andpercentage of apoptotic and necrotic tumor cellsHydroperoxide content in the tumor tissue of sedentary

rats was 0.037 ± 0.007 nmol·mg–1 protein and in the tumortissue of anaerobic exercise trained rats was 0.164 ± 0.020nmol·mg–1 protein (Fig. 2A). Thus, Walker 256 tumor tissuelipid hydroperoxides increased 4-fold (p < 0.0001) in tumortissue from rats in the anaerobic exercise trained group, com-pared with that from the sedentary group. Apoptosis is ex-pressed as a percentage of apoptotic tumor cells detected asannexin-V-positive. The number of apoptotic cells in tumorsfrom the anaerobic exercise trained group was 2-fold higherthan in those from the sedentary group (p < 0.0001; Fig. 2B).Necrosis is expressed as a percentage of necrotic tumor cellsdetected as 7-AAD-positive. The number of necrotic cells intumor samples from the anaerobic exercise trained group was1.5-fold lower than in those from the sedentary group (p <0.0001; Fig. 2C).

Tumor Bax and Bcl-2 expressionBax protein expression in the tumor tissue from the anae-

robic exercise trained group was 10% higher than in tumorsfrom the sedentary group (p < 0.05; Fig. 3A). The expressionof Bcl-2 in tumors from the anaerobic exercise trained groupwas 13% lower than in tumors from the sedentary group (p <0.001; Fig. 3B).

DiscussionPhysical activity, during or after cancer treatment, is now

considered an effective way of restoring physical and psycho-logical function (Oldervoll et al. 2004; Courneya et al. 2007;Newton and Galvão 2008). Despite the high prevalence ofphysical and emotional impairment in cancer patients, physi-cal exercise is still not a common component of cancer ther-apy (Oldervoll et al. 2004).It has previously been shown that anaerobic exercise is

able to decrease tumor growth and cancer cachexia in rats(de Lima et al. 2008); this was accompanied by an increase

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in the activity of immune system, but at that time othermechanisms involved in high-intensity anaerobic exerciseand tumor development were addressed. Regular physical ac-tivity promotes important changes that might help to decreasetumor growth, including an improvement in innate and adap-tive immune cell functionality, as mentioned in reviews ofthese subjects (Westerlind 2003). Also, it is known thatchronic exercise can decrease insulin, estradiol, free testoster-one, and insulin-like growth factor-1 (Leung et al. 2004), andcan increase insulin-like growth factor binding protein-1plasma concentrations (Ngo et al. 2003). High insulin-likegrowth factor-1 plasma levels have been associated with highrates of tumor cell proliferation and a reduction in apoptoticcells (Yu and Rohan 2000).Bacurau et al. (2007) showed that the metabolic profile of

Walker 256 tumor cells, harvested from rats that performed in-tense treadmill exercise before and during Walker 256 cancer,is not the same as that from sedentary rats. However, the nov-elty of our work is that it has shown that the inner tumor envi-ronment can promote tumor cell death. Several mechanismshave been linked to the reduction in tumor growth related tophysical activity; however, few data have been published re-garding this observation in Walker 256 tumor-bearing rats.Based on our data ex vivo, we hypothesize that the anaerobicexercise training somehow induced alterations in Walker 256tumor cells. These alterations might be responsible for the

reduction in cell proliferation. However, investigation is re-quired to determine the mechanisms behind this event.Tumor growth occurs because the accumulation of cells re-

sulting from proliferation exceeds the loss of cells resultingfrom apoptosis. In our study, 8 weeks of anaerobic exercisetraining led to a significant reduction in tumor weight. Otherstudies have demonstrated a reduction in tumor growth andan improvement in the state of the individual with moderate-and high-intensity aerobic exercise protocols (Westerlind etal. 2003; Bacurau et al. 2007). Thus, the downregulation ofapoptosis can lead to cancer development (Hanahan andWeinberg 2000). One of the earliest events in apoptosis is

Fig. 1. Walker 256 tumor weight and cell proliferation. (A) Walker256 tumor weight (g) 14 days after inoculation in sedentary (S) andanaerobic exercise trained (EX) rats. (B) Proliferation of Walker 256tumor cells ex vivo (cpm) harvested from S and EX Walker 256tumor-bearing rats. Cells were isolated from tumors and cultivatedfor 24 h in the presence of (2-14C)-thymidine. Data are presented asthe mean ± SE of 18 rats per group. *, p < 0.0001 vs. S.

Fig. 2. Lipid peroxidation, apoptosis, and necrosis. (A) Lipid perox-idation determined in the tumor tissue of S and EX Walker 256tumor-bearing rats. (B) Percentage of apoptotic cells in tumors fromS and EX Walker 256 tumor-bearing rats. (C) Percentage of necroticcells in tumors from S and EX Walker 256 tumor-bearing rats. Dataare presented as the mean ± SE of 18 tumors for each group. *, p <0.0001 vs. S.

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the redistribution of phosphatidylserine from the inner to theouter leaflet of the cell membrane. In the early phases ofapoptosis, the cells still have full membrane integrity, andannexin-FITC binds to the externalized phosphatidylserine.Tumor cells from anaerobic exercise trained Walker 256tumor-bearing rats have a higher proportion of apoptotic cellsand a lower proportion of necrotic cells than tumor cellsfrom sedentary rats. These data show that besides the de-crease in tumor cell proliferation, the exercise protocol ap-plied here stimulated, somehow, the apoptotic process inWalker 256 tumor cells. Apoptosis is a silent death, andplays an essential role in controlling cell numbers in manydevelopmental and physiological settings. In contrast, cellularnecrosis leads to cell membrane rupture, extracellular fluidrelease, and stimulation of the inflammatory process (Curtinet al. 2002). Elevation in the necrosis process in tumor tissueincreases pro-inflammatory mediators, such as prostaglandinE2, a known promoter of angiogenesis and tumor growth (Liet al. 2002). Apoptosis has been a potential target for cancertherapy during the past 30 years. Some authors have pointedout that the failure of tumor cells to undergo apoptosis trans-lates into malignant potential and chemotherapeutic resist-ance (Huerta et al. 2006). Apoptosis occurs after a cascade

of cell signaling and caspase-mediated events that regulatepro-apoptotic and anti-apoptotic proteins, which can be trig-gered by 2 pathways: the extrinsic pathway that is inducedby death-receptor activation, and the intrinsic pathway that ismediated by mitochondria cytochrome c release. The Bcl-2protein family plays a central role in controlling the intrinsicpathway, and includes proteins that suppress apoptosis (Bcl-2)and promote apoptosis (Bax). The switching, on or off, ofapoptosis can be determined by the ratio of pro-apoptoticand anti-apoptotic proteins (Hu and Kavanagh 2003). Here,we found that anaerobic exercise decreased Bcl-2 expression,an anti-apoptotic protein, and increased Bax expression, apro-apoptotic protein, promoting an intracellular pro-apop-totic condition. This result was corroborated by a flow cy-tometry experiment, which showed a significant increase inannexin-V-positive apoptotic cells in the anaerobic exercisetrained group. Based on the alterations of Bax and Bcl-2 pro-tein concentrations, the tumor cell apoptosis induced by anae-robic exercise seems to be associated with an increase inintrinsic pathway activity.The increment in tumor cell apoptosis correlates with the

degree of peroxidation within the tumor (Valko et al. 2004).The initiation of lipid peroxidation is caused by an attack ofany species that has sufficient reactivity to abstract a hydro-gen atom from the polyunsaturated fatty acid moiety of mem-brane phospholipids (Mund et al. 2007). Acute aerobic andanaerobic exercises are potent inducers of lipid peroxidation,because during high oxygen consumption, approximately4%–5% of the oxygen consumed during respiration is notcompletely reduced to water, forming free radicals instead(Bloomer and Goldfarb 2004). Therefore, as oxygen con-sumption increases during exercise, a parallel increase occursin free radical production and lipid peroxidation (Clarksonand Thompson 2000). Here, tumors from anaerobic exercisetrained rats showed higher concentrations of lipid peroxida-tion products than those from sedentary ratsIn conclusion, this study demonstrates that Walker 256

tumor-bearing rats that underwent anaerobic exercise pre-sented more tumor cell apoptosis, a higher hydroperoxide tu-mor content, a pro-apoptotic protein expression balance, andreduced tumor cell proliferation ex vivo, compared with sed-entary rats. These events together, along with a previousstudy showing improvement in the immune system (de Limaet al. 2008), account for the lower tumor growth we observed.

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538 Appl. Physiol. Nutr. Metab. Vol. 36, 2011

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