Influence of an Exergaming Training Program on Reducing the Expression of IL-10 and TGF-β in Cancer Patients

Abstract

Objective: To evaluate the effect of exergaming in the plasma levels of adipokines (interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor-alpha [TNF-α]), Th1 (IL-2, IL-12, and interferon gamma [IFN-γ]), Th2 (IL-4 and IL-33), Th17 (IL-17 and IL-23), and regulatory T (Treg) (IL-10 and transforming growth factor-beta [TGF-β]) in cancer patients undergoing treatment.

Materials and Methods: We conducted a quasi-experimental control clinical trial using exergaming in all groups through the Xbox 360 Kinect™. The game used in this study was called Your Shape Fitness Evolved 2012. The volunteer participants played the game two to three times per week, for a total of 20 sessions. Forty-five volunteer participants were divided into 3 groups: cancer patients undergoing chemotherapy and/or radiotherapy treatment (chemotherapy and/or radiotherapy group CRG; n = 15); cancer patients who finished chemotherapy and/or radiotherapy treatment (cancer accompaniment group CAG; n = 15); and the control group (volunteers without a cancer diagnosis CG; n = 15). In the pre- and post-training period, all volunteers submitted to blood collection procedures using the enzyme-linked immunosorbent assay (ELISA). This test was used to obtain the levels of adipokines expression (IL-1β, IL-6, IL-8, and TNF-α) and the cytokine profiles Th1 (IL-2, IL-12, and IFN-γ), Th2 (IL-4 and IL-33), Th17 (IL-17 and IL-23), and Treg (IL-10 and TGF-β).

Results: After exergaming, the CRG showed significant reductions in proinflammatory cytokines (IL-6: P < 0.05; IL-10: P = 0.038; TGF-β: P = 0.049) and for CAG (IL-10: P = 0.034), as well as a reduction in the expression of cytokines related to the action of T lymphocytes.

Conclusion: Exergaming promoted changes in the expression of cytokine profiles IL-6, IL-10, and TGF-β, which correlated with the action profiles of CD4⁺ T lymphocytes.

Introduction

The National Comprehensive Cancer Network defines cancer-related fatigue (CRF) as a “distressing sensation, persistent, subjective fatigue or physical exhaustion, emotional and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and interferes with usual functioning.”¹ It is estimated that 96% of cancer patients should experience CRF levels during treatment, such as chemotherapy and/or radiotherapy.² CRF is not fully understood, but a possible cause can be attributed to a dysregulation of cytokine levels in cancer patients.³

Helper T lymphocytes (CD4⁺) are essential for carrying out an immunological function to protect the host against harmful agents. These cells are related to the production of cytokines, which can contribute to the differentiation of helper T lymphocytes. Currently, CD4⁺ T cells include a plurality of cell profiles, such as Th1, Th2, Th9, Th17, Th22, Th follicular (Tfh), and regulatory T (Treg).⁴ Regarding the synthesized cytokines, specific cells of the immune response are activated. For example, Th1 lymphocytes synthesize preferentially: interleukin (IL)-2, interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α)⁵; Th2 lymphocytes produce IL-3, IL-4, IL-5, and IL-13⁶; and Th17 lymphocytes produce IL-17 and IL-23.⁷ Several cytokines have shown CRF development, such as TNF-α, IL-6, IL-1β, IFN, and transforming growth factor-alpha (TGF-α).⁸ In addition, other factors can modify the immune response, such as the presence of disease, inflammation, and physical exercise.^8–10

The practice of physical exercise has direct effects on the cellular immune system because cytotoxic immune cells, such as natural killer (NK) and T cells, are mobilized for circulation.¹¹ Indirectly, exercise can affect the activity of immune cells that induce the release of regulatory cytokines from the immune system through muscle contractions. This process produces cytokines called myokines.¹² These myokines are usually released from muscles during exercise to regulate energy exchange and to promote metabolic adaptations in muscles and other organs.¹² Therefore, it is reasonable to state that exercise is one of the most common strategies used to improve health, which improves the inflammatory profile.¹³

Currently, other forms of physical exercise have been used for cancer patients, such as exergaming.¹⁴ It is a method of physical exercise through videogames, which include Xbox 360 Kinect™ games. It is a promising technology that uses interactive games that allow players to physically interact with avatars on the screen through physical movements, such as dancing, kicking, and jumping, allowing them to increase their physical activity and health levels.¹⁵ With this treatment modality, it was observed that CRF levels were reduced, the patients' quality of life improved, and the electromyographic pattern of the deltoid muscle of cancer patients also improved.¹⁴

The use of exergaming has been applied to healthy individuals, and changes in the profile of cytokines, such as IL-1β and TNF-α, resistin, and leptin, have been observed. This improved the health status and reduced the risk of cardiovascular disease in women aged 35–40 years.¹³ With this perspective, the present study was structured to compare the behavior of cytokines in the different stages of cancer treatment with healthy individuals after the practice of exergaming. Thus, the aim of this study was to evaluate the effect of exergaming through the Xbox 360 Kinect on plasma levels of adipokines (IL-1β, IL-6, IL-8, and TNF-α), Th1 (IL-2, IL-12, and IFN-γ), Th2 (IL-4 and IL-33), Th17 (IL-17 and IL-23), and Treg (IL-10 and TGF-β) from cancer patients undergoing chemotherapy and/or radiotherapy.

Materials and Methods

Study design

This was a quasi-experimental control trial that was conducted between June 2018 and November 2019. The study was approved by the Research Ethics Committee of the Federal University of Alfenas (Protocol number: 1.980.365) and registered with the Brazilian Clinical Trials Registry (RBR-9tmm4d). All volunteers were informed of the evaluations and training procedures involved in the study, and in agreement, all signed the free and informed consent form.

Participants

A total of 45 volunteers participated in the study. A sample of this study is part of a larger study entitled “Influence of Exergaming on the Perception of Cancer-Related Fatigue.”¹⁵ The volunteers were both male and female, and all resided in the city of Alfenas, Minas Gerais, Brazil.

The following inclusion criteria were adopted: cancer volunteers aged between 40 and 80 years who attended the oncology sector of Santa Casa de Alfenas. The participants were both male and female, with a diagnosis of cancer in stages 0, I, II, and III.

The groups were as follows: cancer patients who volunteered to participate in the study, who submitted to chemotherapy and/or radiotherapy (CRG: n = 15; age: 57.13 ± 16.74 years; body mass index [BMI]: 24.81 ± 4.63 kg/m²; diagnosis time: 23.92 ± 36.91 months; cancer diagnosis: gastrointestinal tract [%]: 7.14; breast: 42.86; abdominal and pelvic: 35.71; others: 14.29; cancer stage [%]: 0: 14.29; I: 21.43; II: 35.71; III: 28.57); volunteers who were under medical care with 6 months of treatment completion (CAG: n = 15; age: 63.29 ± 7.34 years; BMI: 24.72 ± 4.57 kg/m²; diagnosis time: 50.50 ± 53.92 months; cancer diagnosis [%] gastrointestinal tract: 6.66; breast: 20.00; abdominal and pelvic: 20.00; oropharyngeal: 26.67; others: 14.29; cancer stage [%]: 0: 8.33; I: 33.33; II: 50.00; III: 8.33); and volunteers without a cancer diagnosis (CG: n = 15; age: 56.73 ± 11.94 years; BMI: 26.24 ± 4.67 kg/m²). In all the groups, volunteers' age ranged from 40 to 80 years, were recruited via social media and pamphlets, and they were residents of the city of Alfenas, who had not practiced any type of physical activity in the previous 6 months and were included in the study.

Volunteers with a diagnosis of stage IV cancer were excluded from this study due to difficulties in understanding the practice of exercises through videogames. Volunteers with a history of viral infections, chronic injuries, rheumatological and hematological diseases, dysfunctions that restricted upper and lower limb movements, and those who refused to sign the informed consent form were also excluded.

In the CRG, 21 volunteers with cancer were selected to participate in the exergaming exposure protocol, but there were some follow-up losses (deaths: n = 3; other diseases: n = 1; interruption of the intervention: n = 2), totaling 15 volunteers. In the CAG, there was no loss of follow-up, totaling 15 volunteers. In the CG, 19 volunteers without cancer were selected, but there were 4 losses (personal reasons: n = 2; interruption of the intervention: n = 2), totaling 15 volunteers.

Exergaming protocol

The practice of exergaming was performed using the Xbox 360 Kinect console (Microsoft, Albuquerque, NM), which was equipped with the game Your Shape Fitness Evolved 2012 (Ubisoft, Montreal, Canada). The exergaming took place at the Human Movement Analysis Laboratory at the Federal University of Alfenas (LAM-UNIFAL-MG), and the area was equipped with a 42-inch LED TV. The volunteers carried out 20 exergaming sessions, with a progressive increase in the duration of the games according to their tolerance, and reached a maximum duration of 50 minutes per session, included the realization of the games and the rest between activities. The exergaming protocol was performed two to three times a week, for a total of 8 to 10 weeks. Before starting the study, all volunteers received instructions on the handling of commands without using the videogame controls. Additionally, the participants received instructions on how to play the games chosen to practice exergaming.^14,15 The games were chosen based on the researchers' experience in previous pilot studies and specific interventions with patients at the physiotherapy clinic at UNIFAL-MG.

The games chosen for the intervention sessions were Wall Breaker, Stomp It, and Run the World, which belong to the game Your Shape Fitness Evolved 2012. The first 10 intervention sessions were carried out with 9 repetitions of the game Wall Breaker, which aims to improve coordination, agility, and speed. These skills are required to reach the cubes projected in front of the player, who performs alternating and crossed movements of the upper limbs. The game scores are obtained according to the execution time: the faster the execution, the higher the score. After the volunteers had a period of rest, nine repetitions of the game Stomp It were included. The objective of Stomp It is to improve the coordination and agility of the player to step on the lights that are directed around him, performing anterior and lateral movements of the lower limbs.

In the last 10 sessions, the game Run the World was added, which promotes the improvement of cardiovascular fitness and well-being through a walking simulation. The distance is determined by the game, and the player performs knee and hip flexion movements. This game was added to the study to minimize the possible lack or loss of interest among the volunteers.^14,15

The exergaming protocol followed the recommendations proposed by the American College of Sports Medicine. These recommendations include a prescription of physical exercise for cancer patients, who require activities between 75 and 150 minutes long, with a combination of moderate- and/or high-intensity exercises, every week. However, the activities were individualized for each volunteer, and the volunteer's capacity limit respected throughout the exergaming protocol.^16,17

Procedure for obtaining blood samples

Blood collection procedures were performed at the Central Laboratory of Clinical Analysis of the Federal University of Alfenas (LACEN-UNIFAL-MG). The blood samples of the volunteers were collected on an empty stomach. Approximately 20 mL of blood was obtained by venipuncture of the ulnar fossa vein before and after the exergaming intervention. In each case, the vein was chosen with the best location as a characteristic, which was determined by a trained professional using visual and tactile explorations.¹³ BD Vacutainer™ tubes were used to collect blood samples. These contain heparin, which allowed for the collection of plasma. Blood samples were centrifuged at 400 g for 10 minutes to allow the blood plasma to separate from the cell components.⁷ After the blood plasma was collected, all samples were stored in an Eppendorf and kept at −80°C to determine the amount of cytokines through the enzyme-linked immunosorbent assay (ELISA) test. Blood samples were analyzed no more than 72 hours after collection.

Determination of plasma levels of cytokines

To determine the plasma levels of adipokines (IL-1β, IL-6, IL-8, and TNF-α), Th1 (IL-2, IL-12, and IFN-γ), Th2 (IL-4 and IL-33), Th17 (IL-17 and IL-23), and Treg (IL-10 and TGF-β), the ELISA test was used according to the manufacturer's instructions (DuoSet Kit, Quantikine; R&D Systems, Minneapolis, MN). For each set of samples, 100 μL of the sample and standard was used. A standard curve was constructed, and the cytokines were tested, generating a correlation between 0.98 and 0.99. When necessary, the samples were diluted within the linear range of values required for the methods used in this study. A reading of the ELISA plate was performed using the SpectraMax 384 Plus, Molecular Devices^®, an automatic reader, with the results being obtained by the difference between the 450 and 570 nm absorbances.

Statistical analysis

The Statistical Package for the Social Sciences (v. 20.0; SPSS, IBM Corp., Chicago, IL) was used for the statistical analysis of the data. The descriptive variables are presented as mean ± standard error of the cytokine average of the 45 volunteers. The linear concentrations of cytokines were transformed on a logarithmic scale. All sample data were tested for normality using the Shapiro–Wilk test.

The effect of the exergaming intervention on the cytokines in all groups was compared through an analysis of variance (ANOVA) using a three-way repeated-measures model. The ANOVA was applied to Mauchly's sphericity test. In cases of sphericity violation, the Huynh–Feldt correction was adopted. The post hoc Bonferroni test was used to compare inter- and intra-group analysis. A significance level of 5% was present in all variables.

Results

The results obtained from the clinical and anthropometric data of the volunteers in this study are part of a larger study that sought to analyze the influence of the exergame on the perceived fatigue and on the electromyography of the leg muscles of cancer patients and volunteers without cancer.¹⁵

Among the results presented in this study are the dosages related to the cytokines and chemokines involved with the inflammatory process (Fig. 1), namely IL-1β, IL-6, IL-8, and TNF-α. In the CRG, it was observed that there is a significant statistical reduction of the synthesis of IL-6 in peripheral blood when comparing the amount from before training and after training. In addition, there was a tendency to decrease the other cytokines in this same group when assessing the amount from before and after physical activity. However, in the other groups, there were no relevant changes in the expression of these cytokines.

FIG. 1.

Representation of the values expressed as mean and SEM of inflammatory cytokines in peripheral blood, IL-1β (A); IL-6 (B); IL-8 (C), and TNF-α (D). *P < 0.05 versus CRG pre-training. CAG, cancer accompaniment group; CG, control group; CRG, chemotherapy and/or radiotherapy group; IL, interleukin; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha.

By evaluating the levels of cytokines related to the T lymphocyte activity profile (Table 1), for example, Th1 (IL-2, IL-12, and IFN-γ), Th2 (IL-4 and IL-33), Th17 (IL-17 and IL-23), and Treg (IL-10 and TGF-β), volunteers from the CRG and CAG groups showed a reduction in IL-10 expressions (P = 0.038 vs. post-CRG; P = 0.034 vs. post-CAG) and TGF-β (^#P = 0.049 vs. post-CRG) when compared before and after the training protocol (Fig. 2).

Table 1.

Representation of Concentrations of Cytokines Measured in Peripheral Blood and May be Related to the Response Profiles of CD4 T Lymphocytes

Cytokines	Pre-training			Post-training
	CG	CRG	CAG	CG	CRG	CAG
IL-2	0.85 (0.33)	0.82 (0.20)	0.67 (0.21)	0.82 (0.30)	0.38 (0.32)	0.48 (0.19)
IL-12	2.33 (0.12)	2.31 (0.17)	2.18 (0.11)	2.10 (0.23)	2.11 (0.27)	1.93 (0.23)
IFN-γ	1.39 (0.29)	0.76 (0.47)	0.78 (0.40)	1.84 (0.25)	0.06 (0.40)	0.26 (0.37)
IL-4	0.61 (0.31)	0.15 (0.32)	0.09 (0.25)	0.37 (0.27)	0.09 (0.25)	0.18 (0.26)
IL-33	2.14 (0.35)	2.11 (0.39)	2.36 (0.35)	1.59 (0.43)	2.13 (0.42)	1.99 (0.39)
IL-17	1.08 (0.26)	0.77 (0.26)	0.94 (0.23)	0.78 (0.28)	0.71 (0.25)	0.84 (0.22)
IL-23	3.39 (0.24)	3.48 (0.38)	3.44 (0.35)	2.87 (0.42)	2.71 (0.56)	2.98 (0.46)
IL-10	1.56 (0.21)	1.38 (0.19)*	1.22 (0.14)*	1.23 (0.20)	0.90 (0.16)	0.84 (0.15)
TGF-β	1.95 (0.37)	2.36 (0.43)#	2.32 (0.42)	2.69 (0.42)	1.32 (0.41)	1.61 (0.38)

^*P < 0.05 versus post-training; ^#P < 0.05 post-CRG.

CAG, cancer accompaniment group; CG, control group; CRG, chemotherapy and/or radiotherapy group; IL, interleukin; TGF-β, transforming growth factor-beta.

FIG. 2.

Representation of the values of mean and SEM of Treg profile cytokine concentrations before and after the training protocol with exergames. (A) IL-10 concentrations in the different experimental groups. (B) TGF-β concentrations. *P < 0.05 versus CRG post-training; **P < 0.01 versus CAG post-training. TGF-β, transforming growth factor-beta; Treg, regulatory T.

When assessing the behavior of cytokine associations in profiles (Fig. 3), it is possible to observe in Figure 3A, which refers to the control group, a maintenance of the cytokine synthesis of the Th1 profile. The Th2 and Th17 profiles reduced the expressions, and there was an increase in the cytokines from the Treg profile. With respect to the CRG (Fig. 3B) and CFA (Fig. 3C) groups, these groups maintained synthesis in the Th2 and Th17 cytokines, but the concentrations of Th1 and Treg cytokines were substantially reduced, which can be seen more clearly in the representation of the heat map in Figure 3 (right panel).

FIG. 3.

Left: Representation of concentration mean values on a logarithmic scale of the behavior of cytokine concentrations in the response profiles of CD4 T lymphocytes, comparing the moments before and after training protocol with exergaming. (A) CG; (B) CRG; (C) CAG. Right: Heat map representation of the behavior of cytokines related to the performance profiles of CD4 T lymphocytes. Evidence of alteration in the intensity pattern of the concentration averages after the training protocol with exergaming, highlighting a balance between the Th1 and Treg profiles.

Discussion

According to Dimeo et al.,¹⁸ most cancer patients experience energy loss and restricted physical performance. It is estimated that this problem affects up to 70% of those diagnosed with cancer who are undergoing treatment with chemotherapy and radiation, or after surgery. To further aggravate the situation, fatigue can lead to other diseases such as depression and immune-mediated diseases.¹⁹

For patients who experience fatigue, rest and reduced daily activities are often recommended. However, prolonged rest may, instead of improving the clinical picture, perpetuate fatigue even further, as physical inactivity induces intense muscle catabolism.^18,20

It is evident that the action of physical activity corroborates with the different aspects of cancer, whether in the identification of genes that can participate in cancer prevention (as the first gene related to the prevention of colon cancer after the practice of physical activity has already verified,²¹ as well as supporting the vascularization of tumors and improving the results of chemotherapy and radiotherapy treatments.^22,23

This study aimed to understand the changes in cytokine expression at the systemic level and also to understand the immunological mechanisms that cause cancer. In addition, it aimed to study the extent to which exergaming is able to modify the immune response and participate in tumor biology.

It is known that an effective antitumor immune response is necessary and that the components of this response work synergistically, that is, the components of the innate and acquired immune response must work together. The most prominent cells in the innate immune response are macrophages, dendritic cells, and NK cells²⁴ and in the acquired immune response the T and B lymphocytes are the most prominent.²⁵ This study aimed to evaluate the expressions of various cytokines profile responses related to the T helper lymphocytes and macrophages, via systemic dosing.

Regarding the levels of inflammatory cytokines, namely IL-1β, IL-6, IL-8, and TNF-α, the CRG showed a significant reduction for IL-6, despite they reduced the other cytokines at an insignificant level from the statistical point of view. Studies indicate that reducing inflammatory cytokines could be related to a positive response against the tumor because the pro-tumor environment is inflammatory^7,26–29 as well as mitigating CRF. This is because after the cancer patients finished exercising, they showed reductions in IL-6, TNF-α, and protein chain reaction (PCR), and also improved the perception of fatigue.⁹ However, after physical activity sessions, IL-6 levels may be elevated due to the muscular production of this cytokine.^12,30,31 What could justify the nonincrease of IL-6 in our study would be the fact that the intensity of activity provided by exergaming is not effective in activating the muscles to the point of producing such a cytokine as IL-6.

A study by Amorim et al.¹³ showed that women who underwent training with exergaming using a dance game twice a week for 1 hour per day, for a period of 1 month, provided results that were sometimes similar to those that were not in agreement, because the levels of TNF-α and PCR were reduced. However, the levels of IL-1β and IL-6 increased after the training period. In addition to these adipokines, it was shown that the training promoted a reduction in leptin, an important negative modulator of TNF-α production, and consequently a reduction in inflammation.³²

Analyzing the cytokine expressions of the Th1, Th2, Th17, and Treg profiles in peripheral blood, it can be seen that the practice of exergaming provided a substantial reduction in the Th17 cytokines (IL-17 and IL-23) and a statistically significant reduction in cytokines of the Treg profile (TGF-β and IL-10).

In view of our results, there are reports in the literature that reinforce our findings. In the post-exercise responses, Cox et al.³³ found that the concentrations of IL-2, IL-4, and IL-12 were small and not substantially different between the groups that practiced physical activity and the groups that were sedentary. However, the concentrations of IL-8 and IL-10 were lower in individuals undergoing training. In preclinical studies, it was seen that physical activity is an adjuvant measure for the treatment of cancer because the development of experimental tumors has been significantly attenuated, and it has also polarized the immune response to an antitumor pattern. That is, the activation of Th1 lymphocytes, with the synthesis of IL-12, IFN-γ, and IL-2, and M1 macrophages, with a primordial synthesis of IFN-γ and IL-12, and reducing the activation of Treg lymphocytes (TGF-β and IL-10), Th17 (IL-17), and M2 macrophages (IL-4).^34,35 The results of this study confirm these findings, given the reduction of cytokines Treg (TGF-β and IL-10) and Th17 (IL-17 and IL-23) at the systemic level, which are also cytokines that stimulate M2 macrophages. Alternatively, a study by Tofighee et al.³⁶ found no changes in the synthesis of IL-17 in practitioners of a high-intensity physical activity. However, it was shown that the elderly subjects practicing physical activities for 16 weeks reduced the levels of IL-17, IFN-γ, and IL-4.³⁷ The data are similar to those found in our study groups.

It is evident in this study that the regular practice of physical activity with exergaming can be an alternative and viable strategy of cancer patients. It can be used as an adjunct to conventional therapies, chemotherapy, radiotherapy, and surgery, as well as new therapies such as immunotherapies. However, there is a need for further studies in which better immunological marker regimens are standardized, such as surface markers and transcription factors to characterize the T helper lymphocyte profiles.

This study has some limitations. As we were not in a referral center for the cancer treatment, it was difficult to standardize the number of chemotherapy treatments and the cancer stages of these volunteers. In addition, regarding the cardiovascular and metabolic responses during the intervention protocol, they were not measured, as the session was carried out in a self-controlled way by the volunteer, in case of discomfort. The levels of plasma cytokines measured by the ELISA test do not have reference values; therefore, the variability between volunteers with or without cancer is present and makes it difficult to measure the responses profiles.

Conclusion

After the exergaming protocol adopted in this study, there were changes in the expression of cytokines IL-6, IL-10, and TGF-β in the volunteers with cancer who were receiving treatment. In addition, there was a reduction in the cytokine concentrations of the TCD4⁺ lymphocyte profiles. The authors encourage more research on immunological changes promoted by the practice of physical activity with exergaming in special groups and patients with chronic diseases.