Prostate cancer (PCa) is the most common cancer, apart from skin cancer, diagnosed between men in the United States (1). It has been shown that chronic inflammation has a great effect on the onset and progression of PCa. Nuclear factor-kappa B (NF-kB), as a well-known mediator for chronic inflammation, is one of the effective marker for PCa in rats models (2). Molecular targeting by the NF-kB has been shown to suppress PCa progression (3). NF-kB, a transcription factor, also promotes the expression of cyclooxygenase-2 (COX-2). COX-2 is a rate-limiting enzyme in the biosynthesis of prostaglandins mediating the inflammatory processes. Inappropriate over-expression of COX-2 has been observed frequently in various premalignant and malignant tissues (4,5). On the other hand, p53 tumor suppressor protein is encoded in various organisms and protects the genome against mutation and consequently prevents cancer (6). The same function has been observed in rat models and other laboratory animals (7). p53 function or its signaling pathway has been reported to be defective against more than 50% of human cancers (6), and the rate of p53 mutation in human PCa is about 30% (8). In general, it has been shown that genetic, lifestyle, and diet play key roles in the incidence of PCa (9).
It has been suggested that exercise training has a potential role to reduce the risk of advanced PCa (10) and, consequently, the incidence of cancer-related death or mortality (11). Studies related to animal models of cancer often showed that exercise training inhibits tumourigenesis. In particular, moderate intensity exercise has been more effective by reducing the growth rate of cancer cells and the appearance of metastases (12), and this intensity was tolerated well by the animals (13). However, the fundamental mechanisms of the effect of exercise on the reduction or cessation of cancer growth have not been identified.
In addition, it is suggested that PCa is strongly influenced by various environmental factors such as diet. Green tea (Camellia sinensis) extract has bioactive ingredients with anticarcinogenic properties like catechins. In addition, the catechins have a potential role to reduce the risk of PCa (14–16). However, there is this ambiguity in knowledge, whether green tea can regulate chronic inflammation, which can help reduce or prevent PCa growth in the body. The researcher observed that green tea extract modulate NF-kB in vitro (17). However, the in vivo effects of green tea on NF-kB activation, and inflammation in the prostate needed further investigation. Based on our knowledge, no study has investigated the effect of exercise training and green tea supplementation in combination with each other on PCa. Therefore, the purpose of this study is to investigate, for the first time, the effect of aerobic exercise training and green tea extracts on the NF-kB, COX-2, and p53 protein levels of prostate tissue in the rats with PCa.
METHODS
Animals and experimental treatments
Sixty male Wistar rats of 2 months old (body weight of 250 ± 20 g) were purchased from Pasteur Institute of Iran (Karaj, Iran). Rats housed in a standard cage of polycarbonate (four rats per cage). The animal room was maintained at a temperature of 23°C ± 2°C with 12 to 12 h light–dark cycle. After 2 wk of acclimatization, the animals were assigned randomly into six equal groups (n = 10), including healthy control (HCt), cancer control (CCt), cancer training (CTr), cancer extract (CEx), cancer training+ cancer extract (CTr + CEx) and sham groups. It is worth mentioning that two rats died from each group during the intervention, and eight rats remained in per group. to ensure that the rats have cancer, a pilot study was conducted to induce PCa before conducting the research study, and after being ensured regarding the induction of cancer model, the original research was started. The animal experiments were conducted in accordance with National Institutes of Health’s Guide for the Use and Care of Laboratory Animals and were approved by the Ethics Committee Kurdistan University of Medical Sciences (Ir.muk.REC.1396.184).
In the pilot study, after implementing cancer inducing protocol in eight rats to assess the progression of cancer in three stages (2, 4, and 5 months after injection of N-Nitroso-N-methylurea [NMU]) (Figure 1), two rats in each stage were anesthetized with ketamine (50 mg·kg−1), and xylazine (4 mg·kg−1). After anesthesia, rats were sacrificed, and their prostate tissue was removed, and one portion was processed for histopathology studies and hence fixed in Bouin’s solution then embedded in paraffin. Sections (5-μm thick) were cut from the paraffin-embedded tissue, mounted on slides, and then stained with hematoxylin and eosin (H&E). Two rats died within a pilot study, and two rats were used in the control group. The results of this pilot study are mentioned in the results section (Figure 2).
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FIGURE 1: Schematic illustration of the study design. In the pilot study, we have investigated the incidence of early signs of cancer induced by carcinogens injection. Then, the main study was done (see the Methods section for more details).
FIGURE 2: A, The control group, which has a healthy and distinct cell wall. B, In this form, the cell is hyperplastic and the cell walls are broken and intracellular with the trachea. C, The amount of transfusion has been increased and the cell’s wall has become more ruptured, and neoplasm has been seen. D, It shows the stage of PCa in rats.
Initial weighing of the rats after the onset of cancer, final weighing of the rats before anesthetizing, and prostate gland weighing immediately after surgery was performed using a Sartorius ENTRIS 3202-1S with precision balance offering 3200 g weighing capacity with readability to 0.01 g.
PCa-inducing protocol
To induce the PCa in the rats, 40 rats (cancer groups) received an intake of cyproterone acetate (CA: 50 mg·kg−1·d−1 in sesame oil, Sigma-Aldrich, St Louis, MO) through injecting intraperitoneally for 18 sequential days as described in the previous studies (18). One day after the last injection of CA, that is, day 19, the rats received subcutaneous injections of testosterone propionate (TP) for 3 d (100 mg·kg−1·d−1; Sigma-Aldrich) for achieving the maximum stimulation in prostatic epithelial cells proliferation potential. On the day after the last TP injection, that is, day 22, the NMU (Sigma-Aldrich) was injected to the animals intraperitoneally at a dose of 50 mg·kg−1 in sesame oil. The NMU was wetted first with 3% acetic acid and then was diluted by normal saline to prepare a final concentration of 10 mg·mL−1 with pH 5.5 for injection (18). In the sham group, we injected solvents of carcinogenic substances, that is, sesame oil (50 mg·kg−1) instead of CA, normal saline (50 mg·kg−1) instead of TP, and sesame oil (50 mg·kg−1) instead of NMU.
Preparation and consumption of green tea extract
First, 250 g of green tea leaves were obtained from Guilan plantation (Guilan province, Islamic of Republic of Iran) in spring 2018, and was powdered by an electric mill (JL-280, Zhengzhou Yize, China). Then, 250 mL of 80% methanol (200 mL methanol +50 mL of water) was added. It was kept at the laboratory temperature (25°C–28°C) for 24 h. Then, the extract was filtered out by passing through (Whatman filter no. 1) and was dried at a temperature of 28°C to 30°C. The glass container was contained of the extract before and after the weighing extraction, and the amount of dry extract was expressed in grams. After complete dissolution, the treatment groups received %0.1 (10 mg per 100 mL of water) of green tea extract, equivalent to 1.34 mL of green tea extract for each rat, three times per week for 8 wk through oral gavage (19). Gas chromatographic analysis of green tea is shown in Figure 3. The identification of the main peaks from Figure 3 can be observed according to the retention times (min) for individual compounds.
FIGURE 3: Gas chromatographic analysis of this prepared solution confirmed the presence of the four major polyphenolic compounds (catechins) including epigallocatechin-3- gallate (retention time, 3.510), epigallocatechin (retention time, 13.218), epicatechin-3-gallate (retention time, 9.032–10.005), and epicatechin (retention time, 11.169), and other compounds including caffeine (retention time, 13.572), gallic acid (retention time, 4.732–6.075), α-tocoferol (retention time, 15.379), etc.
Gas chromatography–mass spectrometry
After preparing the green tea extract, the gas chromatography–mass spectrometry (GC/MS) method was used to determine the amount of catechins present in the extract. To this end, 0.3 g of dried extract was dissolved in 1 mL of water and was centrifuged (Centric 350 IVD) at 16,000g, 4°C, for 3 min. Then, 2 μL were injected in split mode. The GC/MS method was performed in the following way.
GC/MS was performed with an Agilent Technologies 7890A GC system coupled to an Agilent Technologies 7000C triple quadrupole mass spectrometer. The extract was analyzed on an HP5MS (30 m × I.D. 0.25 mm × film thickness 0.25 μm). The analytical situation is mentioned below: The oven temperature was maintained at 40°C for 0 min, and increased to 190°C for 2 min at 3°C·min−1, then continued up to 250°C at 10°C·min−1, for 3 min; splitless status was performed; helium (99.999% purity) was applied as the carrier gas at a rate of 1 mL·min−1. The mass spectrometer was conducted in an electron impact method. The scan range was 50 to 450 m/z. The scan rate was 0.2 s per scan. The temperatures at ionization region and junction were 230°C and 280°C, respectively.
Aerobic training protocol
During this study, the animals in training groups performed low to moderate intensity aerobic exercise training on the treadmill, 5 d·wk−1 for 45 min·d−1 (15 min work in three sets, interspersed 2-min rest periods between sets). It should be mentioned that based on the protocol as training intensity was low, therefore there was no need to warm-up and cooldown. Exercise intensity increased weekly by 1 from 3 to 10 m·min−1. The intensity of the given exercise was equal to 60% of maximal aerobic capacity (20) (exercise intensity domain representing low-to-moderate) for animals with PCa (21). Figure 1 shows how the overall research protocol is performed.
Biochemical assays for NF-kB, COX-2, and p53
Forty-eight hours after the last exercise training session and green tea extract intake, all animals were anesthetized by the combination of ketamine (50 mg·kg−1) and xylazine (4 mg·kg−1) and were killed, and then their prostate tissue was excised by performing surgery and weighed. After weighing the prostate tissue, it was immersed in liquid nitrogen immediately and was stored at −80°C until being used. For performing biochemical assays, 100 mg of tissue was cut and then was transferred into microtubes containing 500 μM of solution containing phosphate-buffered saline (pH 7.4) and antiproteases PI (Protease Inhibitor) (CAS number 535140; Calbiochem) and PMSF (phenyl-methyl-sulfonyl fluoride) (CAS number 329-98-6;Merk, Germany), then it was homogenized for 2 min by a homogenizer (Heidolph DAIX 900, Germany). Tissue homogenate liquid was prepared by centrifuging for 12 min at 10,000g and at 4°C. After preparing the tissue homogenate liquid, they were placed in separate microtubes and were placed into −80°C freezer. The protein contents of NF-kB (cat number. CSB-E13148r, sensitive. 0.39 ng·mL−1, intra-assay precision. Coefficient of variation % < 8%), COX-2 (cat number. CSB-E13399r, sensitive. 0.39 ng·mL−1, intra-assay precision. Coefficient of variation % < 8%) and p53 (cat number. CSB-E08336r, sensitive. 3.12 pg·mL−1, intra-assay precision. Coefficient of variation % < 8%) in the homogenate of prostate were measured by the specific ELISA kits developed for rats according to their manuals (Cusabio Biotech, China).
Statistical analysis
Statistical analysis was performed using the SPSS software (version 20). All values are presented as mean ± SD. One-way ANOVA with post hoc Tukey’s test was performed to compare the means between groups. P values ≤0.05 were considered statistically significant.
RESULTS
The pilot study results are mentioned as follows (Fig. 2). Histologic images of prostate tissue in the pilot study indicated that this method induced PCa after 5 months (Fig. 2D).
Changes in body weight of rats at the beginning and end of the study are presented in Table 1. The results revealed that there was a significant decrease in body weight in CCt, CTr, CEx, and CTr + CEx groups compared to HCt group at the beginning of the intervention (P = 0.0001, 0.001, 0.02, and 0.0001; respectively). Also, body weight was increased significantly in HCt, CTr, CEx, and sham groups at the end of the intervention compared to the beginning (P = 0.001, 0.01, 0.002, and 0.001; respectively) (Table 1).
TABLE 1: Changes of body weight during the experimental period.
Changes in prostate weight of rats at the end of the study are presented in Table 2. The results revealed that there was a significant increase in prostate weight in CCt group compared with CEx, CTr, CEx + CTr, HCt, and sham groups (P = 0.001). Also, prostate weight significantly decreased in CEx group compared with CTr and CEx + CTr groups (P = 0.001 and 0.004, respectively) (Table 2).
TABLE 2: Changes of prostate weight at the end of the experimental period.
The findings showed that there was a significant difference in NF-kB between groups (P = 0.003). The NF-kB level in CCt group significantly increased compared with the HCt group (P = 0.02). In addition, there was a significant decrease in CTr compared to CCt group (P = 0.001) and also there was a significant decrease in CTr compared to CEx group after 8 wk of intervention (P = 0.05) (Fig. 4A). There was a significant decrease in levels of p53 in CTr, CEx, CTr + CEx compared to CCt groups (P = 0.001, 0.02 and 0.004; respectively) (Fig. 4B). Based on the results, there were no significant differences in the level of COX-2 after 8 wk of aerobic exercise training and oral administration of the green tea extract (P = 0.1) (Fig. 4C).
FIGURE 4: The levels of nuclear transcription factor kappa B (NF-kB) (A); p53 tumor suppressor protein (p53) (B) and cyclooxygenase-2 (COX-2) (C) after 8 wk of aerobic exercise training (Tr) and green tea extract (Ex). HCt, Healthy control; Data are presented as mean ± SD. *Significant (P ≤ 0.05).
DISCUSSION
The purpose of the current study was to determine the effect of exercise training and green tea extract on the level of NF-kB, COX-2 and p53 in prostate tissue of rat model with PCa. The results showed the NF-kB factor in the cancer control group was higher than the healthy control group. Also, exercise training led to reducing the NF-kB level compared to the cancer control group. In addition, exercise training and green tea extract supplementation and their simultaneous combination simultaneously reduced p53 protein level compared with the cancer control group. There was no significant difference between the studied groups in the level of COX-2 protein.
Based on previous studies the blocking inflammation is a key factor to prevent PCa progression (8,9). The NF-kB is the main factor in both inflammation and cancer, which needs to be adjusted (22). Although there is no direct evidence, Yu et al. (23) stated that NF-kB expression may be related to carcinogenesis. The NF-kB pathway influences many cellular responses that are attributed to carcinogenesis, such as regulation of cell cycle, apoptosis, and inflammation, and it closely interacts with hormonal homeostasis. Targeting NF-kB may have important prevention or therapeutic values against PCa and inflammation. We showed that exercise training decreased prostate inflammation through reducing or suppressing NF-kB in rats with PCa. However, exercise training can decrease the levels of low-grade systemic inflammation, following the hypothesis that these effects are related. In this regard, various experimental researches have tried to decrease low-grade inflammation in cancer, or in people at high risk of cancer, through exercise training (3,24). The results from these studies state that a decrease in systemic levels of CRP, TNF-α, IL-6, NF-kB, and other proinflammatory factors may be obtained by administrating exercise interventions, but these interventions are needed to be done for long duration (24,25). Totally, the previous studies show different results regarding the beneficial effects of exercise training on the cancers probably due to different experimental designs used in their studies. However, various studies showed that exercise training has the potential to prevent the proliferation of cells or to stimulate the apoptosis in a human PCa cell line (26). Also, there is a relationship between the average of running distance during exercise and the progression of tumors to high-grade prostatic intraepithelial neoplasia and local invasion (27). Exercise training may exert beneficial effects to reduce tumor volume through several mechanisms, including the modulation of hormones (circulating levels of leptin, androgen, IGF-1, insulin, etc.), reduction of the systemic inflammation, and improvement of antioxidative defense and immune surveillance (28).
NF-kB can lead to the expression of COX-2 in inflammation. Increased abnormal expression of COX-2 has been observed in different tissues with cancer (29). Studies have reported that physiological stress and diseases, such as cancer can increase COX-2 expression, as well as NF-kB activation. The results of the present study indicated that there was no difference between intervention groups in the COX-2 level. Creation of this adjustment is considered as one of the probable mechanisms, so that exercise training leads to a decrease in NF-kB and can be effective in weight control, improving the immune system and antioxidant, inducing apoptosis, thus, NF-kB has a key role in the signaling pathway for expression of COX-2. This pathway can lead to a decrease in the expression of COX-2. Also, when exercise training intensity was increased, both COX-2 expression and NF-kB activation were increased significantly (30). In the present study, the training intensity was low-moderate on rats’ aerobic capacity. Therefore, the training intensity may have an important role for no change in the level of COX-2 in rats with PCa. Esser et al. (27) demonstrated that 6 wk of endurance training decreased the COX-2 level in mice with cancer.
Based on the results of the present study, the p53 protein level in exercise training, green tea extract, and combined exercise training with green tea extract groups was lower than the cancer control group that this decrease was more significant in combined exercise training with green tea extract group. The decrease in p53 level can be attributed to the increase in apoptosis (31). Similar to our study, Ghorbanzadeh et al. (26) indicated a significant increase in p53 expression in diabetic rats. The p53 increases to prevent malicious effects when cell faces such factors as stress oxidative, reactive oxygen species, etc. Therefore, considering the reduction of p53 in exercise training and green tea extract groups, the improvement of cellular conditions is indicated, and the combination of these two intervention leads to a more favorable condition of the cell.
Anticarcinogenic effects of EGCG has been shown at several stages of the carcinogenesis process. EGCG is a powerful antioxidant and effective inhibitor for carcinogen activation, and its ability to limit tumor promotion has been well established (32). EGCG exerts proapoptotic effects through inhibiting NF-kB in vitro by NF-kB–inducing kinase (NIK) and subsequent activation of IkB-kinase (IKK)/NIK signaling complex, as it has been shown in human lung cancer cells (33). Jacob et al. (34) indicated that green tea may have chemopreventive effect on rats with PCa. The protective effect of green tea extract can be due to the inhibition effect on releasing free radicals during carcinogen, improving the amount of antioxidant defense (35,36).
Also, we demonstrated that green tea decreased prostate inflammation through attenuation of NF-kB. It can be stated that the main mechanism of green tea includes increasing proapoptotic signals, such as Bax effectively, decreasing proinflammatory cytokines and inflammatory infiltration, which are responsible for the incidence of the prostate hyperplasia (37). The anti-inflammatory effects of green tea were mediated through inhibition of NF-kB activity and protein expressions. In agreement with the previous studies, the results of our study show a beneficial effect of hydroalcoholic extract of green tea alone and combined with exercise training (11,36). Combination of exercise training with green tea extract has not yet been studied on cancer, but a similar study has been conducted on the effect of exercise training combined with supplements on cancer, which it has been mentioned before. Similar to our study, Ghorbanzadeh et al. (26) indicated that the administration of crocin and exercise training reduced the p53 level of pancreas tissue after 8 wk of intervention.
Our study had several limitations, including a continuous infusion of carcinogens for 22 d, which could lead to death in rats and or causing stress. Also, the response of rats to these chemicals in the incidence of cancer and the lack of target organs in response to the injected substance could be another limitation of this study. The intensity of exercise training may also be the other study limit; we only examined low to moderate intensity. Future studies are needed to determine how exercise training intensity may influence the level of these proteins. In addition, it would be better to investigate other genes and protein activity involved in the signaling pathway. Future investigations must be used larger sample size to study whether low-moderate exercise training is suitable as well, and also to determine whether the protective effect that provided by exercise training in rats with cancer can be generalized to other strains. Also, there is a need to perform the study of effects of exercise training on immune function and signaling pathways related to prostate carcinogenesis to develop our understanding in terms of the mechanisms involved to reduce the risk of cancer, which could be generalized to different organs as well. In summary, a combination of exercise training with green tea extract led to a significant decrease in the p53 level and a significant decrease in NF-kB protein level as an inflammatory agent inflammation. Inflammation plays a key role in cancer. Therefore, the administration of these two interventions can be used in human clinical trials.
CONCLUSIONS
In conclusion, the results of this study showed that the low-to-moderate aerobic exercise training and/or green tea extract led to reduced inflammation during PCa in rat models, because exercise training, green tea extract, and the combination of these two interventions caused a decrease in the NF-kB level in these rats. Inflammation plays an important role in the progression of cancer, and combination exercise training with green tea extract caused a significant decrease in the level of inflammatory protein. Both of these interventions can provide an effective potential for the suppression of PCa in rats. Finally, our results pieces of evidence regarding the advantages of administrating green tea extract as well as aerobic exercise training.
Iran National Science Foundation (INSF) has financially supported this work with code approval number 96001504. The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation, and do not constitute endorsement by ACSM. There are no conflicts of interests.
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