Cancer Chemother Pharmacol DOI 10.1007/s00280-013-2348-x
Original Article
Mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy Maria Angélica Oliveira Mendonça · Fabrício O. Souto · Douglas C. Micheli · José Carlos Alves‑Filho · Fernando Q. Cunha · Eddie Fernando C. Murta · Beatriz M. Tavares‑Murta
Received: 25 April 2013 / Accepted: 1 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose To investigate the mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy. Methods Peripheral venous blood was collected at the time of diagnosis and immediately prior to the 4th cycle of an anthracycline-based chemotherapy regimen for patients diagnosed with different stages of breast cancer (n = 30), for experimental assays. Blood samples were also collected from a healthy control group (n = 17). Results IL-8 serum concentrations were higher in the patient group than in the control group (p = 0.02), and chemotherapy did not further affect this increase. Levels of TNF-α, IL-6, and IL-10 did not differ between controls and patients, or in relation to chemotherapy. Serum levels of nitric oxide (NO) metabolites were elevated following chemotherapy compared to levels detected prior to treatment (p = 0.01). When the supernatants of lipopolysaccharide-stimulated mononuclear cells and neutrophils obtained
from the patients were assayed for levels of nitrite, these levels were significantly higher and unchanged, respectively, compared with controls. Expression levels of the chemokine receptors, CXCR1 and CXCR2, were significantly reduced in patients compared to controls, and chemotherapy did not further affect these differences. Furthermore, filamentous actin content for IL-8-activated neutrophils was reduced with chemotherapy (median 8.85; range 3.38–13.43) compared to the content detected prior to treatment (median 9.23; range 2.86–22.16) (p = 0.001). Conclusion Elevated systemic levels of IL-8 and NO, desensitization to CXCR activation, and reduction in actin polymerization may affect neutrophil motility in patients before and after chemotherapy. Keywords Breast cancer · Chemotherapy · Cytokines · Nitric oxide · CXC receptors
Introduction M. A. O. Mendonça · D. C. Micheli · B. M. Tavares‑Murta Institute of Natural and Biological Sciences, Federal University of Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil Present Address: M. A. O. Mendonça (*) Faculty of Medicine, Federal University of Uberlândia, Avenida Pará, 1720, Bloco 2U, Uberlândia, MG 38400‑902, Brazil e-mail:
[email protected] F. O. Souto · J. C. Alves‑Filho · F. Q. Cunha Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil E. F. C. Murta Oncological Research Institute (IPON), Discipline of Gynecology and Obstetrics, Federal University of Triângulo Mineiro (UFTM), Uberaba, Brazil
Neutrophils are the main effector cells during the acute phase of an inflammatory response. Moreover, neutrophil migration is essential for maintaining other important functions such as phagocytosis and microbicidal activity [1]. Therefore, neutrophils are considered the most important leukocyte subpopulation in determining the risk and severity of infections [2]. Local production of TNF-α and IL-8 by cells present at the site of an infection has been shown to induce the activation and migration of circulating neutrophils to the inflammatory site [3]. However, high systemic levels of these cytokines can also inhibit neutrophil migration to the inflammatory site, by stimulation of nitric oxide (NO) production [4].
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Most chemoattractants bind specific G-protein-coupled receptors (GPCR) to induce complex cascades of events [5], including the activation of adhesion molecules expressed on the surface of neutrophils and endothelial cells. Upon cell adhesion, actin filaments are then assembled, thereby representing a fundamental step in neutrophil locomotion [6]. However, responses to IL-8 can be desensitized following prior exposure to IL-8, N-formyl-methionyl-leucyl-phenylalanine (fMLP), or C5a, and vice versa, a phenomenon referred to as “class desensitization” [6]. The desensitization of GPCR family proteins is an important determinant of the intensity and duration of agonist stimulation [7]. Patients with cancer can exhibit an altered immune response, either as a consequence of their own disease [8] or when induced by antineoplastic treatment [2]. Moreover, suppression of the immune system can render the host susceptible to infections and their associated complications, and also influence the overall outcome, for example, tumor growth [8]. Correspondingly, many types of tumors, including breast cancer, colon cancer, and melanoma, have been associated with a compromised functional maturation of natural killer cells during tumor growth [9]. In previous studies, patients with invasive cervical cancer were found to have a reduced capacity for neutrophil migration compared to healthy volunteers and patients with early stage cervical cancer [10]. Moreover, the surgical resection of tumors in patients with pre-invasive neoplasia was found to increase neutrophil and mononuclear cell migration, thereby suggesting that tumor cells produce soluble factors that inhibit leukocyte migration [10, 11]. In addition, patients with pre-invasive and invasive cervical neoplasia have been found to produce high levels of cytokines and NO in response to the tumor cells present. These results indicate that a tumor can act early to impair the innate immune response [12]. In patients with gynecological cancer, lower levels of superoxide production have been detected in the initial stages and evolved with the disease [13]. In stage I–III breast cancer patients, no significant differences in the neutrophil migration response were observed compared to a control group. However, reduced migration of neutrophils toward chemoattractant was observed following chemotherapy (CHT). Moreover, sera from breast cancer patients that had received CHT significantly inhibited the migration of healthy neutrophils, suggesting that circulating factors mediate this inhibitory effect [14]. In the present study, possible mechanisms involved in the impairment of neutrophil migration function in breast cancer patients before and after CHT were investigated. Accordingly, healthy volunteers and breast cancer patients with different stages of tumor were evaluated for the production of cytokines, NO, and levels of expression of chemokine receptors by neutrophils. Furthermore, during
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Cancer Chemother Pharmacol
the follow-up period, the same parameters, as well as filamentous actin content in neutrophils, were investigated in randomly selected patients after CHT.
Patients and methods Patients Women attending the Outpatient Mastology Service of the Federal University of Triângulo Mineiro (UFTM) as referred by the public health system were prospectively diagnosed with various stages of breast cancer. Exclusion criteria included previous treatment for disease or the use of immunosuppressive drugs. An anatomopathological diagnosis was determined according to guidelines established by the American Joint Committee on Cancer and the Committee of the International Union against Cancer, which includes an evaluation of tumor extension, presence of axillar nodes, and/or metastasis. Healthy female volunteers served as controls. This study protocol was approved by the UFTM Committee on the Use of Human Subjects, and written informed consent was obtained from all participating patients and volunteers. Chemotherapy (CHT) According to tumor stage, patients were initially treated with anthracycline-based CHT or surgery. CHT consisted of one of the following regimens: (1) (n = 18) adriamycin or epirubicin (50–60 mg/m2) administered with cyclophosphamide (500–600 mg/m2) or (2) (n = 9) administration of adriamycin or epirubicin (50–60 mg/m2), cyclophosphamide (500–600 mg/m2), and 5-fluorouracil (600 mg/m2). Four or six cycles of CHT were administered, with 21-day intervals between each cycle. In addition, total leukocyte count was monitored, and a level ≥2,000/mm3 was needed before the next cycle of CHT was started. Blood collection Peripheral venous blood samples were collected from controls upon enrollment in the study, and for patients, upon diagnosis (before CHT) and immediately prior to the 4th cycle of chemotherapy (after CHT). At each collection, two samples (5 ml) were collected, and one received 100 IU/ ml heparin. To obtain sera samples, blood was centrifuged (180×g) for 15 min; then, serum was stored at −70 °C. Supernatants obtained from cultured leukocytes Neutrophils and mononuclear cells were purified from heparinized blood samples using Hystopaque (density 1.119;
Cancer Chemother Pharmacol
Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. Cells (106/ml) were suspended in RPMI medium (containing 0.01 % bovine serum albumin) and stimulated with 1 μg/ml lipopolysaccharide (LPS) from E. coli serotype 026:B6 (Sigma, St Louis, Missouri, USA), then cultured in 96-well plates (Nunc, Roskilde, Denmark) at 37 °C and 5 % CO2. After 24 or 48 h, respectively, supernatants were centrifuged and stored at −70 °C until levels of nitrite were assayed. Cell viability was also assayed after cultures and reached 95 %, using trypan blue exclusion. Quantification of NO metabolites Concentrations of NO metabolites in serum samples were determined by enzymatically reducing the nitrate present with nitrate reductase, as previously described [15]. Briefly, aliquots (40 μl) of serum samples were incubated with the same volume of reductase buffer (0.1 M potassium phosphate (pH 7.5), 1 mM nicotinamide adenine dinucleotide, 4 U nitrate reductase/ml) for 20 h at 37 °C. A standard nitrate curve was obtained by incubating sodium nitrate (10–200 μM) with reductase buffer. Total nitrite content was determined using the Griess method [16]. Serum samples and samples obtained from leukocyte supernatants (40 μl each) were incubated with the same volume of freshly prepared Griess reagent (1 % sulfanilamide, 0.1 % naphthylethylenediamine dihydrochloride in 5 % phosphoric acid). Absorbance at 550 nm was recorded for each sample using a multiwell plate reader (Multiskan MCC340 MKII). Data are reported as the μM concentration of NO3− + NO2 present in serum samples and the μM concentration of NO2 in leukocyte supernatants. Quantification of cytokine concentrations in sera Concentrations of TNF-α, IL-6, IL-8, and IL-10 in serum samples were assayed using ELISAs as previously described [17]. Briefly, flat-bottomed 96-well microtiter plates were coated with antibodies (50 μl/well) specific for each cytokine that were diluted to 1–3 μg/ml in buffer binding solution and incubated overnight (4 °C). Plates were then washed (PBS/Tween 20) and nonspecific binding was blocked by incubating wells with PBS/1 % BSA (100 μl) for 120 min at 37 °C. Samples and standards were then loaded onto plates (50 μl/well) and incubated overnight (4 °C). Plates were washed with PBS/Tween 20; then, the appropriate biotinylated monoclonal anti-cytokine antibody was added. After 1 h, plates were washed again and avidin peroxidase (diluted 1:5,000) was added. After 30 min, the plates were washed and substrate [100 μl o-phenylenediamine dihydrochloride (OPD, Sigma, St. Louis, MO)] was added. The plates were subsequently incubated at room temperature for 15 min before H2SO4 (50 μl, 1 M)
was added to each well, and optical densities were measured at 490 nm using a multiwell plate reader (Multiskan MCC340 MKII, Flow Laboratories). Data are expressed as picograms of cytokine per milliliter serum, and the optical densities of the samples were compared with the standard curves generated. Expression of CXC receptors in neutrophils CXCR1 and CXCR2 expression was quantified using a FACSCalibur flow cytometer and CellQuest™ software (Becton–Dickinson, San Jose, CA). FITC-conjugated anti-CXCR1 antibody, PE/Cy5-conjugated anti-CXCR2 antibody (BD Pharmingen, San Diego, CA), and control antibodies (FITC- or PECy-5-conjugated IgG2b; BD Pharmingen, San Diego, CA) were used. Results are reported as the mean fluorescence intensity (MFI) and the percentage of neutrophils positive for expression of CXCR1 and CXCR2. Quantification of F‑actin Filamentous actin content in neutrophils obtained from breast cancer patients before and after CHT was examined by fluorescence microscopy. Briefly, after stimulation with or without IL-8 (10−7 M) for 10 min, neutrophil slides were prepared using a cytospin centrifuge. F-actin was then stained with TRITC-labeled phalloidin (Sigma, St Louis, MO). An epifluorescence microscope (Olympus BX40-F4; Tokyo, Japan) equipped with appropriate filters for FITC or TRITC, 100x/1.30 NA oil-immersion objectives, and a cooled, charge-coupled device CoolSNAP camera (Photometrics, Tucson, AR) was used to collect fluorescent images. All images were captured using identical camera settings with regard to: time of exposure, brightness, contrast and sharpness, and an appropriated white balance set according to the fluorescence filter. Images were acquired and analyzed using Image-Pro Plus 4.0 software (Media Cybernetics). Mean fluorescence density (MFD) was determined using a linear measurement of fluorescence exhibited by individual cells. All of the cells in at least five randomly chosen fields from each slide, performed in duplicate, were analyzed from at least ten individual experiments. Median and percentile values of MFD for each field were subtracted from the mean density of the area measured as background for each individual slide [18]. Statistical analysis Statistical analyses were performed using SigmaStat 2.03 software. Differences between two unpaired groups (e.g., controls and patients upon diagnosis) were tested using Student t test or the Mann–Whitney test, according to the
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distribution of the data. Paired groups (e.g., patients before and after CHT) were compared using a paired test or the Wilcoxon test, depending on the presence of normal or non-normal data distribution, respectively. Statistical significance was set at p
