BREAST CANCER SURGERY DECREASES SERUM BRAIN-DERIVED NEUROTROPHIC FACTOR CONCENTRATIONS IN MIDDLE AGED WOMEN: RELATIONSHIP TO THE SERUM C-REACTIVE PROTEIN CONCENTRATION

Brain-derived neurothrophic factor (BDNF) is the most abundant in the growth factor family (1, 2). For the first time this protein was purified from pig brain (3). Currently it is well established that BDNF which acts via protein tyrosine kinase receptor (TrkB) (4-6), is widely expressed in mammalian brain (7) and regulates neuronal development and function (1, 8, 9). It is considered that in the adult nervous system, BDNF plays predominantly functional role (10). BDNF is involved in memory formation including learning and behavior, synaptic plasticity, synaptic efficacy and neuronal connectivity and promotes the development of immature neurons and enhances the survival of adult neurons (1, 11). According to Monteggia et al. (9) the role of BDNF in the adult brain may be different from that in the developing brain. It was demonstrated that the loss of BDNF selectively in the brain of adult mice resulted in impaired hippocampal function, whereas the loss of BDNF at early stages of development contributed to hyperactivity as well as more severe impairment in hippocampal-dependent learning (9). On the other hand since the pioneering study by Neeper et al. (12), followed by Berchtold et al. (13) there is growing body of evidence that physical exercise inreases BDNF in various regions of rodent’s brain (14-16), which is considered as a benefitial response to physical training.

In the periphery BDNF is present in plasma, serum and platelets (10, 17) as well as that this protein is produced by vascular endothelial cells and by peripheral blood mononuclear cells (18). It is well established that serum BDNF concentration in humans is about 100-fold higher than in plasma (10, 19), due to the fact that the main source of BDNF in blood are platelets (20). It was demonstrated that BDNF can cross the blood-brain barrier (21, 22) in both directions via a high capacity saturable transporter system (21), although some authors question such a possibility (23, 24). Interestingly, a positive correlation between the brain and serum BDNF concentrations was reported (25). Moreover, a recent study reported a positive correlation between functional capacity of the central nervous system and plasma and/or serum BDNF concentration in humans (26, 27). In addition, it was shown that plasma and/or serum BDNF concentrations are significantly lower in patients suffering from major depression (28, 29).

Plasma and/or serum BDNF in human can be acutely increased by single bout of physical exercise (30-34). Moreover, it was recently reported that 5 weeks endurance training that has induced improvement of physical capacity in humans, involving increase in maximal oxygen uptake (VO2max), increase in power generating capability at the VO2max, decrease in lipid peroxidation and a slight decrease in insulin resistance, was accompanied by a significant increase in basal as well as the exercise-induced plasma BDNF concentrations in young healthy men (33). Interestingly, it has been recently reported that also in cases of Parkinson’s disease patients a moderate-intensity interval training lasting 8 weeks, resulted in an increase of serum BDNF level accompanied by a significant improvement of their health status (35, 36).

On the other hand it was reported that in some groups of subjects (37-39) elevated plasma and/or serum BDNF concentration may be considered as an early marker of pathological metabolic changes in the body, as originally proposed by Hristowa et al. (40). According to Hristowa (41) there is a link between the tissue and plasma levels of proinflammatory cytokines and the neurotrophins at a given stage of development of body metabolic disorders leading, finaly to carcinogenesis. This is why in the present study we aimed to establish the relationship between plasma and/or serum BDNF concentration with the body inflammatory status of the breast cancer patients - a group of patients with enhanced body inflammatory status (42), as juged based on the serum C-reactive protein (CRP) - a strong marker of inflammatory response (43-45). This seems to be especially interseting in view of the fact that a number of reports (although not all) shows that an elevated plasma or serum CRP levels at the time of diagnosis of breast cancer are associated with reduced overall and disease-free survival and with increased risk of death from breast cancer (46-48).

In the present study we aimed: (i) to establish the relationship between the pre-operative level of serum CRP with the plasma and serum BDNF concentrations in the breast cancer patients; (ii) to determine the impact of breast cancer surgery, known to increase the serum CRP level (49) on the post-operative plasma and serum BDNF concentrations determined at 24 h after the surgery.

We have hypothesized that plasma and serum BDNF concentrations in the breast cancer patients will be positively correlated with the serum concentrations of the C-reactive protein and that the surgery-induced increase in the serum CRP concentration will be accompanied by an increase in serum and/or plasma BDNF concentration in the cancer patients.

PATIENTS AND METHODS

Patients

Eighteen patients with recently diagnosed breast cancer aged between 30 and 65 years (mean ± SE) age 49.1 ± 1.6 years, weight 69.8 ± 2.2 kg, BMI 25.8 ± 0.8 kg m–2) were studied. The studied patients before the surgery had normal hemoglobin concentration amounting to 13.4 ± 0.4 g%, as well as normal erythrocyte and leukocyte counts, amounting to 4.6 ± 0.1 M µl–1 and 5.9 ± 0.3 k µl–1, respectively. All patients were scheduled for breast cancer surgery and were of ASA grades I and II. Patients with history of neuropsychiatric disorders, diabetes, COPD, asthma, renal and hepatic failure, previously treated with chemotherapy, receiving hypnotic or analgesic drugs were excluded from the study. Written informed consent was obtained from each patient and the study was approved by the local Ethics Committee 101/KBL/OIL/2007.

Assessment of depression and anxiety levels

The patients were screened for signs of anxiety and depression with the Hospital Anxiety and Depression Scale (HADS) questionnaire (50). They were asked to complete it during the preoperative anesthetic visit.

Determining the menopausal status

Basing on the WHO recommendations (51), menopause was diagnosed in 4 patients, 14 were premenopausal.

Drawing of the blood samples

In the morning on the day of the operation and the day after the operation blood samples were drawn from the patients. The samples (10 ml) of blood were taken from the antecubital vein for the plasma and serum BDNF and serum CRP and complete blood count.

Anesthesia

Patients were premedicated with midazolam 7.5 mg given by mouth 30 minutes before the operation. Before the induction patients were given fentanyl 0.1 mg and precurarisation (atracurium 5 mg). Anesthesia was then induced with thiopental 2 – 5 mg kg–1, and after the administration of suxamethonium 1 mg kg–1 the trachea was intubated. Anesthesia was maintained with 66% nitrous oxide in oxygen supplemented with sevoflurane 0.5 – 1.0% and repeated doses of fentanyl and atracurium. At the end of the operation ketoprofen 100 mg and ondansetron 4 mg were given intravenously. No opiate antagonists or acetylcholinesterase inhibitors were employed.

Surgery extension and duration

Twelve patients underwent modified radical mastectomy (Madden), 3 breast conservative surgery (lumpectomy and axillary dissection), 2 axillary dissection, and 1 lumpectomy and removal of the sentinel node. The mean duration of operation from the skin incision to the last stitch was 58 minutes and ranged from 25 to 95 minutes.

Postoperative course

After the operation the patients remained for one day in the postoperative ward. Postoperative analgesia was achieved by intramuscular morphine 10 mg (on average 3 ± 1 times) and intravenous metamizole 2 g every 8 hours. No important postoperative complications were noted in any of the patients. Two patients developed fever (> 38.0°C) during the first day after the operation.

Brain-derived neurotrophic factor measurements

The brain derived neurotrophic factor concentration was measured in serum [BDNF]s and in plasma [BDNF]p using enzyme-linked immunosorbent assay (ELISA) kit BDNF Emax – immuno Assay System Promega (Madison, USA) with a range of standard from 7.8 – 500 pg ml–1 (sensitivity 16.6 pg ml–1) and with < 3% cross-reactivity with others neurotrophines, catalog no. RK-028-28.

C-reactive protein assay

Serum C-reactive protein concentration [CRP]s was assayed using Human ELISA Kit, Phoenix Pharmaceutical, Inc. USA, with a range of standard from 0.375 – 50 ng ml–1, catalog no. EK-072-62.

Statistics analysis

In this study the data are presented as mean ± SE. All statistical analyses were performed using nonparametric tests. The significance of differences between pre- and post-surgery concentrations was tested using a Wilcoxon test for paired samples. Correlation between two variables was tested using Spearman’s correlation analysis. Significance was set at P < 0.05. All analyses were performed using statistical packet STATISTICA 13.1; StatSoft, Tulsa, USA).

RESULTS

Serum brain-derived neurotrophic factor concentrations measured before and 24 hours after the surgery

Serum BDNF concentration before and after the surgery is presented in Fig. 1 panel A. Significant decrease (P = 0.004) in [BDNF]s concentration was found 24 hours after the surgery in relation to the pre-surgery status (Fig. 1A).

Fig. 1. (A): Plasma brain-derived neurotrophic factor concentration [BDNF]p before (PRE) and 24 h after the surgery (POST) (n = 18). (B): Serum brain-derived neurotrophic factor concentration [BDNF]s before (PRE) and 24 h after the surgery (POST) (n = 18).

Plasma brain-derived neurotrophic factor concentration measured before and 24 h after the surgery

Plasma BDNF concentration before and after the surgery is presented in Fig. 1B. No significant effect of the surgery on the [BDNF]p concentration determined 24 h after the surgery was found (Fig. 1B).

Serum C-reactive protein concentration [CRP]s measured before and 24 h after the surgery

Serum C-reactive protein concentrations measured before and 24 h after the surgery is presented in Fig. 2. Note a significant increase in [CRP]s (P = 0.002) at 24 h after the surgery.

Fig. 2. Serum C-reactive protein concentration [CRP]s before (PRE) and 24 h after the surgery (POST) (n = 13).

Correlations
Serum brain-derived neurotrophic factor versus platelets count

A significant positive correlation was found between the serum brain-derived neurotrophic factor concentration [BDNF]s and the platelets count (PLT) measured before (PRE) r = 0.65; P = 0.003 (Fig. 3A) and 24 h after the surgery (POST) r = 0.52; P = 0.027 (Fig. 3B).

Fig. 3. Correlations between the serum brain-derived neurotrophic factor concentration [BDNF]s and the platelets count (PLT) measured before (PRE) (Fig. 3A) and 24 h after the surgery (POST) (Fig. 3B); (n = 18).

Serum brain-derived neurotrophic factors versus serum C-reactive protein

A significant positive correlation was found between the serum brain-derived neurotrophic factor concentration [BDNF]s and the serum C-reactive protein [CRP]s concentration measured before (PRE) r = 0.56; P = 0.046 (Fig. 4A) and 24 h after the surgery (POST) r = 0.57; P = 0.044 (Fig. 4B).

Fig. 4. Correlations between the serum brain-derived neurotrophic factor concentration [BDNF]s and the serum C-reactive protein concentration [CRP]s measured before (PRE) (Fig. 4A) and 24 h after the surgery (POST) (Fig. 4B); (n = 13).

Depression and anxiety levels

Anxiety and depression levels were assessed by the HADS scale (50). The mean total HADS score was 16.00 ± 2.0. The average anxiety and depression levels as assessed by the HADS subscales were respectively 9.94 ± 1.16 for anxiety (i.e. a borderline result) and 6.83 ± 0.80 for depression (value in the normal range). No correlation between the plasma or serum brain-derived neurotrophic factor concentrations determined before the surgery and the total as well as the subscales i.e. A and D of the HADS scores was found in the present study.

DISCUSSION

In the present study we have evaluated the effect of breast cancer surgery in middle aged women on the serum and plasma brain-derived neurotrophic factor concentration in relation to the serum C-reactive protein [CRP]s concentration at 24 hours after the surgery.

Surprisingly, little is known in the literature on the effect of surgery on the serum and/or plasma BDNF concentrations ([BDNF]p and [BDNF]s, respectively) in humans. It was reported by Vutskits et al. (52) that a minor surgery (lumbar or cervical discectomy) resulted in a significant decrease in the BDNFp concentration at 24 hours after the surgery.

In the present study, contrary to the findings by Vutskits et al. (52), we have found that the breast cancer surgery had no significant effect on the [BDNF]p measured 24 hours after the surgery, when compared to the pre-operative level (Fig. 1B). It should be noticed however that the mean plasma BDNF concentration in the present study was by about 30 percent lower then before the surgery. Therefore the direction of changes in [BDNF]p measured in the present study at 24 hours after the surgery was similar to that reported by Vutskits et al. (52), however due to large inter-individual variation in the [BDNF]p this difference was statistically not significant. This suggests that breast cancer surgery can decrease plasma BDNF concentration as well as reported by Vutskits et al. (52) in patients operated for lumbar or cervical discectomy. In the present study we have found however, that the breast cancer surgery resulted in a significant decrease (P = 0.004) of serum BDNF concentration [BDNF]s measured on the morning before and 24 hours after the surgery (Fig. 1A). The decrease in the [BDNF]s in our study was accompanied by a significant decrease in the platelet count (PLT) (254.7 ± 12.2 versus 228.8 ± 9.7 k µl–1, respectively before and after the surgery). This observation is in accordance with the findings by Fujimura et al. (20), showing that platelets are the main source of BDNF in blood. Interestingly, we have also found a close correlation between PLT count and the [BDNF]s both before as well as after the surgery (Fig. 3) (10).

The serum concentrations of the BDNF in the present study amounting before surgery to 25 523 ± 1416 pg ml–1 and 21 551 ± 998 pg ml–1 at 24 h after surgery (Fig. 1A), were in the range of values measured in healthy individuals (10, 17, 25, 47). The plasma concentrations of the BDNF amounting to 590 ± 191 pg ml–1 before and 412 ± 149 pg ml–1 after the surgery (Fig. 1B) were also close to the values reported in patients before and after minor surgery (46) and are in the range of results measured by Lommatzsch et al. (10) in a relatively larger group of healthy individuals. Although some researches have reported a substantially lower values of [BDNF]p and/or [BDNF]s in healthy individuals. Those differences most likely arise from methodological procedure in assaying plasma and/or serum BDNF concentrations (53). Therefore, one should be rather cautious when comparing the absolute values of plasma and/or serum BDNF concentrations determined in various study using varied measurements methods.

It was recently postulated that elevated [BDNF]p and/or [BDNF]s in humans (over-secretion) might be an early symptom of pathological metabolic changes in the body (39). Suwa et al. (38) have reported an elevated [BDNF]s in newly diagnosed female patients with type 2 diabetes mellitus. Moreover, Levinger et al. (37) recently found that elevated plasma BDNF concentration positively correlated with risk factors for metabolic syndrome and type 2 diabetes mellitus in middle age group of subjects. In accordance with this observations in the present study we have found a positive correlation between the serum C-reactive protein concentration - a marker of inflammatory and infective status (43-45) - and the [BDNF]s (Fig. 4A). A significant correlation between [CRP]s and [BDNF]s was present both before as well at 24 hours after the surgery (Fig. 4A and 4B). It is a new and original observation in humans. It is of interest that despite of a significant decrease in the [BDNF]s as well as a significant increase in the [CRP]s noticed 24 hours after the surgery the correlation between the [CRP]s and the [BDNF]s was still present (Fig. 4B). This illustrates that in our patients the serum BDNF concentration was closely related to the magnitude of the inflammatory reactions in the body. It should be noticed that the strength of the correlation between [CRP]s and the [BDNF]s after the surgery, although still significant, is poorer than before the surgery (Fig. 4B). Furthermore it should be added that the surgery-induced decreases in serum BDNF did not correlated with the surgery-induced increase in [CRP]s (r = 0.06, P = 0.8). This indicates that in the post operative stage some factors, most likely related to the surgery, are affecting the relationship between [CRP]s and the [BDNF]s. Nevertheless, the significant correlation between [CRP]s and the [BDNF]s found in our study in the breast cancer patients, especially in the pre-surgery state (Fig. 4A) suggest that high [BDNF]s level in these patients should be considered with causion, especially when it is accompanied by a high serum CRP levels.

Systemic inflammatory response, of which CRP is a non-specific marker, seems to be an epiphenomenon of cancer growth and progression. The results of many studies have shown convincingly, that elevation of inflammatory markers, like CRP, is inherently linked to poorer prognosis and greater tumour aggressiveness (54). This relationship has been confirmed also in breast cancer (46-48). In fact CRP level is an element of some clinical scoring systems used to predict outcomes in oncology, e.g. the Glasgow Prognostic Score (GPS) and the modified Glasgow Prognostic Score (mGPS) (55). The patophysiologial link between systemic inflammation and cancer progression is not entirely understood, while it can be assumed that more aggressive tumours may induce more pronounced immunological response and inflammation, it has also been shown, that an exaggerated inflammatory response may itself promote metastasis, and worsen the outcomes by depleting the body’s resources (54).

Elevated plasma or serum CRP levels at the time of diagnosis of breast cancer are associated with reduced overall and disease-free survival and with increased risk of death from breast cancer (46-48). According with this scenario, our results suggest that a high [BDNF]s present in breast cancer patients in the pre-operative state might belong to the early body defence response in these patients.

The positive correlation between [CRP]s and [BDNF]s found in the breast cancer patients participating in our study (Fig. 4), is also surprising in view of the previous findings of our group showing a significant increase in basal as well as exercise-induced increase in [BDNF]p concentrations, accompanied by several positive responses to endurance training including an increase in maximal oxygen uptake (VO2max), increase in power generating capability at the VO2max, decrease in lipid peroxidation and a slight decrease in insulin resistance after 5-weeks of endurance training. Therefore, the present study provides further evidence, that the increase in plasma and/or serum BDNF level might have opposite meaning in patients with metabolic disorders (37-41) than in young healthy individuals (33). It was reported that plasma and/or serum BDNF concentrations are significantly lower in patients suffering from depression (28, 29, 56). In the present study we have also evaluated the level of anxiety and depression using the Hospital Anxiety and Depression Scale (HADS) questionnaire (50). The average anxiety and depression levels as assessed by the HADS subscales in the present study were respectively 9.94 ± 1.16 for anxiety and 6.83 ± 0.80 for depression. The mean total HADS score was 16.44 ± 1.87. The HADS questionnaire is a screening tool for psychiatric disorders validated in many populations, among others in breast cancer patients (57). Results equal or greater than 11 on either subscale are considered to be clinically significant, while results between 8 and 10 are classified as borderline (50). Among the patients enrolled in this study 10 scored 11 or more on the HADS anxiety subscale and 3 scored 11 or more on the depression subscale. The results are similar to obtained in other studies performed in patients with breast cancer (58). In the present study we have found no correlation between the plasma or serum BDNF concentrations determined before the surgery and the total as well as the subscales i.e. A and D of the HADS scores (50).

Regarding the observed decrease in the [BDNF]s at 24 h after surgery we have also considered the plasma volume expansion 24 h after the surgery as a possible cause of the observed decrease in the [BDNF]s. However, even after correcting the plasma and serum BDNF concentration for the plasma expansion (calculated according to Dill and Costill, see ref. 59) the decrease in the [BDNF]s at 24 h after the surgery was still significant (P < 0.03). Another possible mechanism of the post-operative decrease in the [BDNF]s could be the effect of anesthetics on its release as shown by Vutskits et al. (52). In the present study the anesthetic regime (thiopental + N2O/O2 + sevoflurane + fentanyl) was similar to the thiopental + isoflurane + sufentanyl group of the Vutskits’ study (52). As mentioned above, Vutskits et al. (52) observed a decrease of [BDNF]p at 24 h following surgery, a phenomenon that we have not observed in the present study.

The observed in the present study significant decrease in the [BDNF]s at 24 h after surgery by about 15% (Fig. 2) could cause some functional disturbances in central and peripheral nervous system (56). Interestingly, it has been recently reported that deeper decreases in plasma BDNF levels during major spine surgery was associated with the incidence of postoperative delirium in older adults (60). These authors postulated that changes in plasma BDNF levels during surgery may provide a biomarker for postoperative delirium (60). Furthermore the surgery-induced decrease in serum BDNF migh affect the functioning of patient’s central and peripheral nervous system in the subsequent weeks (months) of the post-operative period. Although so far little is known regarding the effect of temporal decrease in the plasma or serum BDNF level on the functioning of brain in the breast cancer patients, nevertheless in view of some basic study concerning the effect of BDNF on the function of the central nervous system (1, 2, 8, 9, 28, 29), we cannot exclude such possibility. This is in accordance with the very recent reports showing high prevalence of depression (61, 62) and suicides (63) among breast cancer patients.

Despite of potential harmful effect of the post-operative decrease of serum BDNF level on the functioning of the central and peripheral nervous system in the breast cancer patients, one should also consider, beneficial effects of the decreased serum BDNF level to the system of these patients. It has been reported that brain-derived neurotrophic factor could stimulate breast cancer cell growth and metastasis via tyrosine kinase receptors TrkA, TrkB, and the p75NTR death receptor (64). Concerning the impact of BDNF on the invasion, migration, and proliferative activities of human lung SCC cells (65). Therefore, a decrease of the BDNF bioavailability in breast cancer patients after the surgery might contribute to the improvement of clinical outcome and enhance survival of the patients.

Further studies involving larger group of patients are needed to establish the ultimate role of the BDNF, including the Val66Met BDNF polymorphism (66), in the health status of the breast cancer patients.

In summary: we have concluded that serum BDNF concentration in breast cancer patients positively correlates with serum CRP both before and at 24 h after the surgery. Morover, breast cancer surgery decreases serum BDNF concentration at 24 h after operation and increases serum CRP. The surgery-induced decrease in serum BDNF level in the breast cancer patients on the one hand might lead to some disturbances in functioning of their central and peripheral nervous system including an increase of depression - frequently reported in those patients in subsequent phases of the post-operative period, but on the other hand a decrease in BDNF bioavailability in the breast cancer patient might lead to an attenuation of breast cancer cell growth and metastasis and enhancement of survival of breast cancer patients.

Abbreviations: BDNF, brain-derived neurotrophic factor; BMI, body mass index; CRP, C-reactive protein; HADS, Hospital Anxiety and Depression Scale; PLT, platelet count; (p), plasma; (s), serum; PRE, morning before surgery; POST, morning 24 h after surgery; TrkB, protein tyrosine kinase receptor.

Acknowledgments: This study was supported by a grant nr 245/KFiB/2009 from the University School of Physical Education, Cracow, Poland. Technical support by Malgorzata Wilczek M.D., and Justyna Zapart-Bukowska Ph.D., is kindly acknowledged.

Conflict of interests: None declared.

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