Physical Activity and Natural Anti-VIP Antibodies: Potential Role in Breast and Prostate Cancer Therapy

Background There is convincing evidence from numerous clinical and epidemiological studies that physical activity can reduce the risk for breast and prostate cancer. The biological mechanisms underlying this phenomenon remain elusive. Herein we suggest a role for naturally produced antibodies reactive with the vasoactive intestinal peptide (VIP) in the suppression of breast and prostate cancer, which we believe could offer a possible molecular mechanism underlying control of these cancers by physical exercise. Methodology and Results We found that sera from individuals having breast and prostate cancers have decreased titers of VIP natural antibodies as demonstrated by a lower reactivity against peptide NTM1, having similar informational and structural properties as VIP. In contrast, sera collected from elite athletes, exhibited titers of natural NTM1-reactive antibodies that are significantly increased, suggesting that physical activity boosts production of these antibodies. Significance Presented results suggest that physical exercise stimulates production of natural anti-VIP antibodies and likely results in suppression of VIP. This, in turn, may play a protective role against breast and prostate cancers. Physical exercise should be further investigated as a potential tool in the treatment of these diseases.

Taken together, these observations strongly support the notion that VIP plays an important role in breast and prostate cancer pathogenesis and suggests that elevated concentrations of VIP in the circulation may represent a risk factor for these cancer types.
The present study was carried out in order to compare reactivity with peptide NTM1 of sera collected from individuals with breast and prostate cancer and sera from elite athletes. Obtained results showed a significant difference in NTM1 reactivity of sera from elite athletes, healthy controls and cancer patients.

Results
Reactivity of sera collected from subjects with breast and prostate cancer with peptide (NTM1s) 4 -SOC 4 was determined by the ELISA immunoassay ( Figure 1 and Table 1). Statistical analysis revealed that this reactivity in sera from cancer patients was significantly lower in comparison with the reactivity of control sera P(0.02) and P(3.7e-05) in breast and prostate cancer, respectively). In Table 2 is given the total IgG content determined for breast and prostate cancer patients.

Discussion
There is now strong and consistent evidence from several studies conducted worldwide that regular physical activity reduces breast cancer risk by 20% to 30% and that a dose-response effect exists [49]. Long-term athletic training during the college and precollege years lowers the risk of breast cancer throughout the life span [50], [51], [52]. For tertiary cancer prevention, observational studies suggest that breast cancer survivors performing exercise (e.g., 2-3 h of brisk walking/week) have a 40-67% reduction in breast cancer recurrence and all-cause mortality compared with inactive survivors [53], [54], [55]. Similar data were reported for prostate cancer where an average risk reduction ranged from 10%-30% [56], [57]. Recently, the evaluation of physical activity in relationship to prostate cancer mortality among 2,750 men diagnosed with prostate cancer showed that a modest amount of vigorous activity, such as biking, tennis, jogging, or swimming for 3 hours a week, may substantially improve prostate cancer-specific survival [58], [59].
However, the potential biologic mechanisms through which physical activity may decrease the risk of breast and prostate cancer are still elusive. Several putative etiologic pathways, including those involving steroid hormones, chronic inflammation, growth factors, lymphokines and insulin resistance were suggested (reviewed in Ref. [49]). On the other hand, there are mounting evidences that VIP pathway plays an important role in pathogenesis of breast and prostate cancer, indicating that elevated concentration of VIP, a facilitator of breast and prostate cancer, in the circulation may contribute to these diseases and that suppression of this peptide could have positive effect [24], [25], [26], [27], [28], [29].
The level of circulating VIP is controlled by natural anti-VIP antibodies [39], [40], [41], [42], [43] and we hypothesized that these suppressive antibodies could contribute to a better control of breast and prostate cancer and that lack of these antibodies could contribute to the progression of these diseases. In order to test this assumption we compared reactivity with peptide NTM1 of sera collected from cancer patients and healthy control. Results given in Table 1 and Figure 1 show that this reactivity is significantly lower in cancer patients in comparison with healthy control subjects.
Paul and Said showed that natural anti-VIP antibodies were present in plasma from 29.6% of healthy human subjects who habitually performed aerobic muscular exercise (running, cycling, swimming, aerobic dancing, and/or weight training, 3 or more workouts per week for a year or more prior to study entry), compared to 2.3% of healthy subjects who did not [37]. Herein, Figure 1. Results of ELISA. The absorbance values (OD) obtained for sera of cancer patients, athletes and healthy control subjects with peptide NTM1. Antibodies recognizing peptide NTM1 are significantly more prevalent in serum samples from athletes compared to control subjects (swimming P(2.9e-06), water polo P(0.0001), volleyball P(8.1e-07), rowing P(1.8e-06), wrestling P(0.0009), and karate P(3.2e-07); Mann-Whitney test). The absorbance values for sera from cancer patients are significantly lower in comparison with the values obtained for control sera (P(0.02) and P(3.7e-05) in breast and prostate cancer, respectively; Mann-Whitney test). doi:10.1371/journal.pone.0028304.g001 presented results show that a significantly higher percentage of trained athletes had increased titers of NTM1 reactive antibodies, 23 out of 28 (82.1%) females and 23 out of 26 (88.5%) males compared to non-athletes. This finding expanded our recent report on male water polo elite athletes [38] and strongly supported the suggestion that physical exercise represents a stimulus of the immune system which boosts production of natural VIP/NTM1-reactive antibodies.
The total IgG content in all cancer patients (1162.7 mg/ml) and in elite athletes (11.262.9 mg/ml) was in the normal range (7-16 mg/ml) and comparable with normal values suggesting that that difference in the different age distribution of the athletic subjects (18)(19)(20)(21)(22)(23)(24)(25)(26) and cancer patients (5663.9) did not significantly affect global function of the immune system, allowing comparison of immunological data obtained for these two studied groups. Thus, the reduced levels of natural antibodies recognizing peptide NTM1 in breast and prostate patients was specific and may contribute to disease progression.
The antigenic stimulus for the formation of natural VIP/NTM1reactive antibodies could not be identified from our studies; however, several studies have demonstrated that acute exercise is associated with increased plasma levels of VIP [30], [31], [32], [33], [34], [35], [36]. It is thus plausible to theorize that these antibodies may have been produced in response to an increase in VIP levels during exercise, providing increased antigenic stimulus. In line with this hypothesis is that physical activity provides a potential source of the natural anti-VIP/NTM antibodies that could contribute to the control of breast and prostate cancer.
In order to keep the conclusions derived from this study under its real extent, it is necessary take into account that elite athletes are different from sedentary and even from physically active subjects, not just because of the exercise routine, but mainly for genetic predisposition allowing remarkable physiological and biological indexes. For this reason, we can't exclude the possibility that these specific genetic makeup of elite athletes could also partially contribute to their increased production of natural VIP/ NTM1-reactive antibodies.
Herein we suggest a possible role for naturally produced antibodies reacting with peptides VIP and NTM1 in the control of breast and prostate cancer, which we believe could offer a possible molecular mechanism underlying positive effects of physical exercise in these cancers. Presented results strongly suggest further research of physical exercise as an important natural approach against breast and prostate cancer. Because little is known about the optimal level of exercise in combating of these cancers, variations in the mode, intensity, duration, and frequency of exercise prescriptions also requires future research.

Ethics statement
Informed consent and local ethics committee approval was obtained for these human studies. All patients provided written informed consent. The Ethical Committee for Clinical Trials of the Faculty of Pharmacy in Belgrade is responsible for the ethical conduct of this study.

Human subjects
Cancer patients. Sera samples were collected from 15 subjects with breast and 17 subjects with prostate cancer (disease stage, values of tumor markers and the age are given in Table 2). Sera were collected immediately after confirmed diagnosis and before the start of any therapeutic intervention.
Athletic subjects. Sera samples were collected from 54 Serbian elite international athletes (26 males and 28 females) engaged in the following types of sports activities: wrestling, water polo, rowing, karate, kick boxing, swimming and volleyball. Female and male elite athletes were engaged in the sport training in average 9 and 11 years, respectively. All athletes were tested at the end of basic preparatory mezocycle (a phase of training with duration of between 4-6 weeks) in a period of recuperative microcycle (shorter training period of about 7-10 days for active recuperation of the athletes) [60]. We expected that in this latter period, production of natural antibodies which clear VIP from circulation should have reached higher levels.

Peptide conjugates synthesis
The synthesis of the (NTM1s) 4 -SOC 4 conjugate was carried out manually by stepwise solid phase peptide synthesis using the Boc-Gly-OCH 2 -Pam resin (1 g, 0.25 mmol/g capacity). Sequential oligopeptide carrier (SOC 4 ), formed by the repetitive Lys-Aib-Gly, is applied to display analyzed peptides. The synthetic procedure starts with the step-by-step couplings of the protected residues (Boc/Bzl) corresponding to the SOC 4 carrier to the resin. Lysine was introduced as Boc-Lys(Fmoc)-OH. After removal of the Fmoc protective groups from the Lys-N e H 2 groups by 20% piperidine in dimethylformamide, synthesis of the epitope FTDNAKTI was carried out (Boc/Bzl) by the simultaneous attachment of each residue in four copies.

ELISA
ELISA was performed with peptide (NTM1s) 4 -SOC 4 by the following procedure: polystyrene microtest plates (Sarstedt, Germany) were incubated overnight at 4uC with 100 ml of peptides (0.5 mg/well) diluted in carbonate buffer, pH 9.6. Plates were washed with phosphate-buffered saline (PBS)-0.05% Tween and non-specific sites were blocked with 200 ml PBS containing 5% bovine serum albumin (BSA) for 2 h at room temperature. After six washings, serum specimens were added to the wells (100 ml/well). Sera were diluted 1:100 in 5% BSA in PBS. Plates were incubated for 3 h at room temperature. After six washings with PBS-0.05% Tween, 100 ml of goat anti-human IgG alkaline phoshatase-conjugated antibodies (Sigma), diluted 1:2500 were added and the plates were incubated for 30 minutes at room temperature. After six washings, pNPP (p-Nitrophenyl Phosphate) substrate was added and the absorbance (OD) measured at 492-620 nm after 15 minutes. Each sample was tested independently twice and the mean values are reported.

Statistical analysis
The significance of the differences in O.D. values for individuals with breast and prostate cancer, elite athletes and control subjects was calculated by the non-parametrical Kruskal-Wallis. The two groups are unpaired with uneven variance and therefore they were considered as part of a two-tailed, heteroscedastic matrix. For each comparison, the level of significance p for a directional test is given.