J Mol Neurosci (2010) 42:450–458 DOI 10.1007/s12031-010-9374-5
Changes in the Expression of Pituitary Adenylate Cyclase-Activating Polypeptide in the Human Placenta during Pregnancy and Its Effects on the Survival of JAR Choriocarcinoma Cells R. Brubel & A. Boronkai & D. Reglodi & B. Racz & J. Nemeth & P. Kiss & A. Lubics & G. Toth & G. Horvath & T. Varga & D. Szogyi & E. Fonagy & J. Farkas & A. Barakonyi & Sz. Bellyei & L. Szereday & M. Koppan & A. Tamas
Received: 30 March 2010 / Accepted: 13 April 2010 / Published online: 7 May 2010 # Springer Science+Business Media, LLC 2010
Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino R. Brubel : D. Reglodi : P. Kiss : A. Lubics : G. Horvath : D. Szogyi : E. Fonagy : J. Farkas : A. Tamas (*) Department of Anatomy, University of Pecs, Szigeti u 12, 7624 Pecs, Hungary e-mail:
[email protected] A. Boronkai : B. Racz : S. Bellyei Department of Biochemistry and Medical Chemistry, University of Pecs, Pecs, Hungary A. Boronkai : S. Bellyei Department of Oncotherapy, University of Pecs, Pecs, Hungary J. Nemeth Department of Pharmacology and Pharmacotherapy, University of Debrecen, Debrecen, Hungary G. Toth Department of Medical Chemistry, University of Szeged, Szeged, Hungary
acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in firsttrimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents. Keywords JAR . Choriocarcinoma . RIA . PACAP38 . PACAP27 . Methotrexate
T. Varga : M. Koppan Department of Obstetrics and Gynecology, University of Pecs, Pecs, Hungary
Introduction
A. Barakonyi : L. Szereday Department of Medical Microbiology and Immunology, University of Pecs, Pecs, Hungary
Pituitary adenylate cyclase-activating polypeptide (PACAP) was first isolated in hypothalamic extracts, based on its potency to stimulate cAMP production (Miyata et al. 1989). PACAP occurs in two forms: 27 and 38 amino acid
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residues. Several subsequent studies have shown the role of PACAP not only in the regulation of pituitary hormone secretion but also in other reproductive functions (Counis et al. 2007; Isaac and Sherwood 2008; Koves et al. 2003; Sherwood et al. 2007; Szabo et al. 2002). PACAP is present in the uterus, where it supposedly plays a role in muscle contraction and blood supply (Steenstrup et al. 1996). Little is known about the effects of PACAP on the placenta. Both forms of the peptide, PACAP38 and PACAP27, along with PAC1 receptors, are present in the human pregnant uterus and placenta (Koh et al. 2005; Scaldaferri et al. 2000). PACAP causes relaxation of stem villous and intramyometrial arteries, suggesting a vasoregulatory role in the uteroplacental unit (Steenstrup et al. 1996). PACAP is suggested to play a role in decidualization, and the time-related localization of endometrial–uterine PACAP is implicated in facilitating endometrial blood flow and increasing the availability of metabolic substrates to the developing deciduoma or embryo (Spencer et al. 2001a, b). PACAP is an important trophic factor during embryonic development and in the mature nervous system (Waschek 2002). Based on currently available data, it seems that the placenta synthesizes PACAP in addition to the numerous growth factors and hormones that are important for the development and maintenance of pregnancy and for fetal development (Koh et al. 2003). The first aim of the present study was to determine the concentrations of PACAP38 and PACAP27 in human first-trimester and full-term placentas. One of the most well-known effects of PACAP is its survival-promoting actions, which are not restricted to neuronal cells (Racz et al. 2007, 2010; Somogyvari-Vigh and Reglodi 2004; Vaudry et al. 2009). Apoptosis and necrosis play crucial roles both in the normal development and differentiation of the placenta and in placental diseases (Hallmann et al. 2004). PACAP, dependent on the splice variant of its expressed receptor and on various other circumstances, may have differential effects on cell survival, differentiation, and proliferation (Lelievre et al. 2002; Nicot and DiCicco-Bloom 2001; Somogyvari-Vigh and Reglodi 2004; Vaudry et al. 2009). This is especially true for tumor cells (where different effects of PACAP have been described depending on the cell type and treatments used), from inhibiting apoptosis to inducing apoptosis or not affecting the growth of the cells at all (Aubert et al. 2008; Li et al. 2006; Zia et al. 1995). Previously, we have described that PACAP treatment sensitized JAR human choriocarcinoma cells to in vitro oxidative stress and hypoxia (Boronkai et al. 2009). This effect was not found in cells treated with ethanol or lipopolysaccharide, showing that the sensitizing effect of PACAP is dependent on the noxious stimulus. The second aim of our present study was
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to investigate whether PACAP influences the effects of methotrexate (MTX), the main chemotherapeutic agent used in choriocarcinoma, on the death of the JAR human choriocarcinoma cell line.
Materials and Methods Radioimmunoassay Human placentas were collected from aborted (9 weeks; n=7) and full-term (n=6) placentas. Samples were taken from the chorionic villi (fetal side) and the decidua (maternal side). In case of full-term births, samples from the umbilical cord were also collected (n=6). The procedure used was in accordance with protocols approved by the ethical committee (no. 2784,3117, University of Pecs; 8-28/92 009-10 I 8EKU, ETT TUKEB, Ministry of Health, Hungary). Tissue samples were weighed and homogenized in ice-cold distilled water. The homogenate was centrifuged (12,000 rpm, 4°C, 30 min), and the supernatant was further processed for radioimmunoassay (RIA) analysis of PACAP38 and PACAP27 contents, as previously described (Jakab et al. 2004; Hernadi et al. 2008; Nemeth et al. 2007). Briefly, the conditions were as follows: antisera: PACAP38 “88111-3” (working dilution, 1:10,000) and PACAP27 “88123” (dilution: 1:45,000); tracer: mono- 125 I-labelled ovine PACAP24–38 and mono-125I-labelled ovine PACAP27 prepared in our laboratory (5,000 cpm/tube); standard: ovine PACAP38 and PACAP27 ranging from 0 to 1,000 fmol/ml used as RIA standards; buffer: assay prepared in 1 ml of 0.05 mol/l (pH 7.4) phosphate buffer containing 0.1 mol/l sodium chloride, 0.25% (wt/vol) bovine serum albumin, and 0.05% (wt/vol) sodium azide; incubation time: 48–72 h of incubation at 4°C; separation solution: charcoal/dextran/milk powder (10:1:0.5 g in 100 ml of distilled water). Differences between PACAP contents were assessed by t test. Cell Culture JAR human choriocarcinoma cells were obtained from the American Type Culture Collection (Wesel, Germany). The cells were maintained as a monolayer-adherent culture in RPMI-1640 medium (Sigma, Hungary) containing 1% penicillin–streptomycin solution (Gibco, Hungary), 1% pyruvate solution (100 nM), and 10% fetal calf serum (Gibco) in a humified atmosphere of 95% air and 5% CO2 at 37°C. Cell Viability Test JAR cells were randomly assigned to one of 24 experimental groups. The control group of cells was incubated in RPMI
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medium (normal medium) without treatment. Other groups were incubated in RPMI medium containing PACAP1–38 in the presence or in the absence of the PACAP receptor antagonist and/or MTX. Briefly, cells were treated with 1 or 100 nM PACAP1–38 alone, and 100 nM or 1 µM PACAP6– 38 alone or in combination with 1 nM or 100 nM PACAP1– 38, respectively. Other groups were treated with the above concentrations of PACAP1–38 and/or PACAP6–38 together with 10 or 100 µM MTX. PACAP1–38 and PACAP6–38 treatments preceded the MTX treatments by 1 h. PACAP1–38 was synthesized as previously described (Gasz et al. 2006; Jozsa et al. 2005). Cells were exposed to the abovementioned concentrations of chemicals for 48 h. Evaluation of cell survival was performed immediately after termination of treatments. Experiments were repeated six times. The selected dose of MTX was based on our preliminary studies showing significant cell death in JAR cells with this concentration. The dose of PACAP was based on in vitro studies using PACAP as a protective (Somogyvari-Vigh and Reglodi 2004) or sensitizing agent against various toxic effects, where the range 1–100 nM was proven to be most effective (PACAP treatment sensitized JAR human choriocarcinoma cells to in vitro oxidative stress and hypoxia; Boronkai et al. 2009). The viability of JAR cells was determined by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) assay. The assay is based on the reduction of MTT into a blue formazan dye by the functional mitochondria of viable cells. The cells were seeded into 96-well plates at a density of 104 cell/well and cultured overnight before the experiment. After 48 h of treatment, the medium was removed, and fresh RPMI/ fetal calf serum containing 0.5% of the water-soluble yellow mitochondrial dye MTT was added. Cells were then incubated for 3 h at 37°C in an atmosphere of 5% CO2. After 3 h of incubation, the medium was removed, and the water-insoluble blue formazan dye formed stoichiometrically from MTT+ was solubilized by acidic isopropanol (Sigma). Optical densities were determined by an enzyme-linked immunosorbent assay reader (Anthos Labtech 2010; Anthos Labtech Instruments, Vienna, Austria) at a wavelength of 550 nm, with the values expressed in arbitrary units. All experiments were repeated six times. Results are expressed as percentages of control values.
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staining of the cells by conjugating a fluorescent group to annexin V apoptosis can be quantitatively detected using fluorescent microscopy or flow cytometry, and this assay has been used together with other criteria to detect apoptosis. The ratio of apoptosis was evaluated after double staining with fluorescein isothiocyanate (FITC)labeled annexin V (BD Biosciences Hungary) and PI (BD Biosciences Hungary) using flow cytometry. First, the medium was discarded, and the wells were washed twice with isotonic sodium chloride solution. JAR cells were removed from the plates using a mixture of 0.25% trypsin (Sigma), 0.2% ethylenediaminetetraacetate (Serva, Hungary), 0.296% sodium citrate, and 0.6% sodium chloride in distilled water. This medium was applied for 15 min at 37°C. Removed JAR cells were washed twice in cold phosphate-buffered saline and resuspended in binding buffer containing 10 mM Hepes NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Cell count was determined in Burker’s chamber by achieving a dilution in which 1 ml of solution contains 106 cells. One hundred microliters of buffer (105 cells) was transferred into 5-ml round-bottom polystyrene tubes. Cells were incubated for 15 min with FITC-conjugated annexin V molecules and PI. After this period of incubation, 400 µl of annexin-binding buffer (BD Biosciences Hungary) was added to the tubes, as described by the manufacturers. The samples were immediately measured by a BD FacsCalibur flow cytometer (BD Biosciences USA). Results were analyzed by Cellquest software (BD Biosciences USA). Quadrant dot plot was introduced to identify living cells, necrotic cells, and cells in early or late phase of apoptosis, according to previous descriptions. Necrotic cells were identified as single PI positive. Apoptotic cells were branded as annexin V–FITC-positive only, and cells in late apoptosis were recognized as double positive for annexin V–FITC and PI. Cells in each category were expressed as percentages of the total number of stained cells counted. Statistical Analysis All data are presented as mean ± standard error of the mean (SEM). Differences between groups were assessed with one-way analysis of variance, followed by Neuman–Keul’s post hoc analysis, and considered significant when P
