Introduction

Astrocytoma is a form of primary tumor arising from astrocytes. Glioblastoma multiforme, the most aggressive astrocytoma, usually occurs more frequently in the adult brain than in the spinal cord. Because of this tumor’s potential to grow quickly, the median survival time is only 12–15 months following diagnosis, even if the patient receives optimal medical treatment (Ahmed et al. 2014), keeping in mind that truly effective drugs have yet to be developed to treat high-grade astrocytomas. This is partly because our present understanding of the pathology of astrocytomas has limited the development of therapeutic interventions.

Pituitary adenylate-cyclase activating polypeptide (PACAP) is a neuropeptide that exists in 38- and 27-amino acid residue forms (Miyata et al. 1989, 1990). PACAP belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family, and its closest paralog is VIP. PACAP and VIP share three types of receptors (PAC1-R, VPAC1-R, and VPAC2R), with both PACAP and its receptors widely expressed throughout the brain (Arimura and Shioda 1995; Shioda 2000). PACAP has a pleiotropic role in the central nervous system, functioning as a neurotransmitter, neuroprotectant, and promoting factors for neural stem cell differentiation (Shioda et al. 2006; Watanabe et al. 2007; Ohtaki et al. 2008). Moreover, PACAP regulates astrocytes as well as neurons (Masmoudi-Kouki et al. 2007; Nakamachi et al. 2011a).

PACAP also affects glioma cells. Based on elevated cAMP levels, Robberecht et al. first suggested a role played by PACAP receptor expression in human glioma cells (Robberecht et al. 1994). Later, the effect of PACAP on the proliferation of astrocytoma cells was studied by several groups, but conflicting results were obtained (Vertongen et al. 1996; Dufes et al. 2003; Sokolowska and Nowak 2008; D'Amico et al. 2013). We believe that these differences were caused by variations in the malignancy levels of the astrocytoma cells used in the respective studies. As such, the purpose of the present study is to clarify the relation between astrocytoma malignancy and the proliferative effects of PACAP in vitro using a number of different ascytoma cell lines.

Materials and Methods

Human Astrocytoma Cell Lines

Four human astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13) were obtained from the Health Science Research Resources Bank (Osaka, Japan). The KINGS-1 cell line was established from grade 3 astrocytoma tissue, while SF-126 and YH-13 were established from grade 4 astrocytoma tissue (or glioblastoma multiforme) according to the WHO classification of tumors of the central nervous system (Louis et al. 2007). The KNS-81 cell line was generated from astrocytoma tissue whose malignancy was not recorded.

Cell Culture

Stocks of the four cell lines were defrosted in a 37 °C water bath and then mixed with minimum essential medium (MEM) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 20 % fetal bovine serum (FBS). After centrifugation at 1,500 rpm and 4 °C, the supernatant was removed, the pellet was dissolved in MEM medium with 20 % FBS and antibiotic-antimycotic agents (Invitrogen) and cells were incubated in a 5 % CO2/air incubator at 37 °C. When the incubated cells reached subconfluence, they were detached by treatment with trypsin-EDTA (Invitrogen) for 5 min. The detached cells were collected and centrifuged at 1,500 rpm for 5 min at 4 °C. The pellet was suspended in the medium, and seeded on 100 mm culture dishes for passage, 60 mm dishes for immunocytochemistry, and 96-well plates for CyQUANT assay. Extra pellets were maintained at −80 °C for real-time PCR analysis.

Immunocytochemistry

Cultured cells were fixed with 4 % paraformaldehyde in 0.05 M phosphate buffer, pH 7.2 for 30 min at 4 °C. After washing with phosphate buffered saline (PBS), the cells were incubated in 5 % normal horse serum with 0.25 % Triton X-100 in PBS for penetration and blocking. The cells were then incubated with primary antibody in blocking buffer for 18 h at 4 °C. The antibodies used were: rat anti-CD44 (1:1000; TONBO Biosciences, San Diego, CA, USA), mouse antiglia fibrillary acidic protein (GFAP, 1:500; Sigma, St. Louis, MO, USA), mouse anti-A2B5 IgM (1:200, EMD Millipore Corporation, Billerica, MA, USA), mouse antiglutamate synthase (GS, 1:500, EMD Millipore Corporation), mouse antineuronal nuclei (NeuN, 1:1,000; EMD Millipore Corporation), rat antimyelin basic protein (MBP, 1:500; EMD Millipore Corporation), rat anti-CD11b (1:500; Serotec, Oxford, UK), or PAC1-R (Suzuki et al. 2003). After incubation with Alexa488-labeled secondary antibodies (Invitrogen), cells were treated for 10 min with 4′,6-diamidino-2-phenylindole (DAPI, 1:10,000). After mounting with aqueous mounting medium on cover slips, the immunoreactivity of cells was measured with the aid of a fluorescence microscope (AXIO Imager Z1, Carl Zeiss, Germany). All of the above procedures were carried out on three cells from three independently prepared cultures.

Real-Time PCR Analysis

Total RNA was isolated by TRIzol Reagent (Invitrogen) from cell pellets prepared from each of the cell lines, and complementary DNA was generated by reverse-transcriptase (PrimeScript RT reagent kit, TaKaRa BIO INC., Kyoto, Japan). PCR primer sets for human PACAP, PAC1-R, VPAC1-R, VPAC2R, and GAPDH mRNA sequences are listed in Table 1. Real-time PCR was performed using SYBR Premix Ex Taq II reagent (TaKaRa BIO INC) and an ABI PRISM 7900 instrument (Applied Biosystems, Lincoln, CA, USA). Relative gene expression was calculated using the comparative delta Ct method, and normalized with respect to the GAPDH housekeeping gene. The specificity of PCR products was confirmed by dissociation curve with a single peak at the final PCR step.

Table 1 Primer sequences used for real-time PCR analyses
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CyQUANT Assay

Astrocytoma cells were seeded at 100 cells/cm2 on 96-well plates and treated with human PACAP38 (final concentration 10−13 to 10−7 M in PBS) or PBS as control. After 3 days of treatment, the medium was removed and plates were stored at −80 °C. The number of cells was measured by CyQUANT kit (Invitrogen) following the manufacturer’s instructions.

Statistical Analysis

Data were expressed as mean ± SEM. Multiple comparisons were made by ANOVA with Tukey-Kramer multiple-comparison tests. P values of less than 0.05 or less than 0.01 were considered to indicate statistical significance for all analyses.

Results

Characterization of Astrocytoma Cell Lines by Immunocytochemistry

We first checked the differentiation level of astrocytoma cells by immunostaining with astrocyte and glial progenitor markers given that malignant astrocytoma cell have been described to show an undifferentiated glial-like phenotype (Jellinger 1978; Piepmeier et al. 1993). Anti-GFAP (marker for mature astrocytes but not immature glial cells such as radial glia), anti-GS (marker for astrocytes and astrocytoma cells), and anti-A2B5 (glial progenitor cell marker) antibodies were used (Fig. 1). GFAP immunoreactivity was observed in the KNS-81 and KINGS-1 cell lines, but not in SF-126 or YH-13. On the other hand, GS immunoreactivity was detected in the KNS-81, KINGS-1, and SF-126 cell lines, but not in YH-13. In contrast, immunoreactivity to A2B5 was observed in SF-126 and YH-13, but not in KNS-81 and KINGS-1 cell lines. To confirm that these cells were derived from astrocytes, other cell markers, such as antibodies against NeuN (as a neuronal marker), MBP (as an oligodendrocyte marker), and CD11b (as a microglial marker) were also tested. Immunoreactivities against these antibodies were not observed in any of the cell lines (Fig. 2). CD44 is a receptor for hyaluronic acid and is used as a marker for cancer stem cells and as an indicator of high-invasive potential (Wiranowska et al. 2006; Anido et al. 2010). Only marginal CD44 immunoreactivity was observed in KNS-81 and KINGS-1 cells, but strong immunoreactivity was observed in SF-126 and YH-13 cells (Fig. 3). PAC1-R immunoreactivity was observed all cells, with the signal intensity increasing in the following order: KNS-81 < KINGS-1 < SF-126 < YH-13 (Fig. 3).

Fig. 1
figure 1

Characterization of astrocytomas by immunostaining with astrocyte and glial progenitor markers. Cultured astrocytoma cells were immunostained with antibodies against GFAP, GS, and A2B5. Green shows immunoreactivity to antibodies. Nuclei counterstained with DAPI are shown in blue

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Fig. 2
figure 2

Characterization of astrocytomas by immunostaining with neuronal cell markers. Cultured astrocytoma cells were immunostained with antibodies against NeuN (neuronal marker), MBP (oligodendrocyte marker), and CD11b (microglia marker). Evidence of any green color would be indicative of immunoreactivity against these antibodies. Nuclei counterstained with DAPI are shown in blue

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Fig. 3
figure 3

Characterization of astrocytomas by immunostaining with anti-CD44 or anti-PAC1-R antibodies. Cultured astrocytoma cells were immunostained with antibodies against CD44 and PAC1-R. Green shows immunoreactivity to these antibodies. Nuclei counterstained with DAPI are shown in blue

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mRNA Levels of PACAP/PACAP Receptors in Astrocytoma

mRNA levels for PACAP and its receptor were measured in the four astrocytoma cell lines by real-time PCR analysis. While differences in ADCYAP1 (gene for PACAP) mRNA levels were not statistically significant between the cells lines (KINGS-1, 100 %; KNS-81, 82.1 %; SF-126, 111.3 %; and YH-13, 68.9 %) (Fig. 4a), large variations were seen for ADCYAP1R1 (gene for PAC1-R) mRNA levels, which in order of ranking were KINGS-1 < KNS-81 < SF-126 < YH-13, with normalized levels of 100, 130.3, 147.5, and 656.0 %, respectively. In this way, ADCYAP1R1 mRNA expression for the YH-13 cell line was significantly increased compared with the other astrocytoma cell lines (Fig. 4b). The VIPR1 mRNA level for YH-13 (4,720.3 %) was significantly higher than that for the other cell lines (KINGS-1, 100 %; KNS-81, 68.9 %; SF-126, 92.9 %) (Fig. 4c). VPAC2R mRNA was below the level of detection in all of the astrocytoma cell lines tested (data not shown).

Fig. 4
figure 4

mRNA levels of PACAP and its receptor in the four astrocytoma cell lines. mRNA level of ADCYAP1 (PACAP), ADCYAP1R1 (PAC1-R), and VIP1R in astrocytoma cell lines were measured by real-time PCR analysis with normalization to GAPDH mRNA level (n = 3 cultures for each result). **P < 0.01

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CyQUANT Proliferation Assay

The effect of PACAP on the proliferative activity of the four astrocytoma cell lines was measured by CyQUANT assay. When employed at concentrations ranging from 10−13 to 10−7 M, PACAP did not affect the number of KNS-81, KINGS-1, or SF-126 cells in culture compared with the PBS control groups. However, for YH-13 cells in culture, treatment with 10−11 M PACAP significantly increased the number of cells compared with PBS treatment (Fig. 5) indicating a proliferative effect of PACAP on YH-13 cells at this concentration.

Fig. 5
figure 5

Effect of PACAP on astrocytoma cell proliferation. Proliferation assays were performed with a CyQUANT kit after 3 days of PACAP treatment (10−13, 10−11, 10−9, and 10−7 M). Each value was normalized to values obtained for cells maintained in PBS alone. **P < 0.01 vs. PBS

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Discussion

This study was designed to compare the effects of PACAP and its receptor expression on the differentiation status of four human astrocytoma cell lines. GFAP is an intermediate filament protein that is expressed in mature astrocytes but not in immature glial cells such as radial glia (Pixley and de Vellis 1984). On the other hand, the A2B5 antibody binds to gangliosides and is used as glial progenitor marker (Fok-Seang and Miller 1992). Xia et al. (2003) suggested that astrocytoma cells share similar antigenicity with astrocytes, and that the A2B5-positive astrocytoma cells exhibited a higher recurrence rate than A2B5-negative astrocytoma. In our study, GFAP immunoreactivity was observed in KNS-81 and KINGS-1 cells, but not in SF126 or YH-13 cells. On the other hand, A2B5 immunoreactivity was observed in SF-126 and YH-13 cells, but not KNS-81 or KINGS-1 cells.

GS is a an enzyme that plays a key role in the metabolism of nitrogen. It is a commonly used marker for astrocytes and astrocytoma cells, where an inverse correlation between histological malignancy and degree of GS expression has been observed (Akimoto 1993; Anlauf and Derouiche 2013). Here, GS immunoreacitivty was observed in KNS-81, KINGS-1, and SF126 cells, but not in YH-13 cells (Fig. 1). Moreover, immunoreactivity for CD44, which is known as an astrocytoma stem cell marker, was weak in KINGS-1 cells, and stronger in SF-126 and YH-13 cells (Fig. 3). Taken together, these immunohistochemistry observations suggest that the malignancy of the astrocytoma cell lines used here (in the order of strength) is YH-13, followed by SF-126, KINGS-1, and KNS-81. These findings also imply that the malignant character of the original astrocytoma tissue used to prepare these cell lines was maintained. Immunoreactivity for other neuronal cell markers (NeuN, MBP, and CD11b) was not observed in the four cell lines (Fig. 2), indicating that they are cells from astrocytomas and not other types of brain tumor cells.

PACAP mRNA levels were not significantly different among the astrocytoma cell lines, but PAC1-R and VPAC1-R mRNA levels in YH-13 were significantly greater than the others (Fig. 4). Similary, strong PAC1-R immunoreactivity was also observed in YH-13 cells (Fig. 3). In consideration of the character of the astrocytoma cell lines, PAC1-R and VPAC1-R mRNA levels of cells with high-malignant potential were greater than those of low-malignant potential.

The results of the CyQUANT assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells (Jones et al. 2001), showed that PACAP, employed at a concentration of 10−11 M, stimulated an increase in the number of YH-13 cells compared with control PBS-treated cells, but this effect was not seen in the other cell lines (Fig. 5). These differences in sensitivity to PACAP seem to correlate with PAC1-R and VPAC1-R expression levels. As such, conflicting results from previous reports on the effects of PACAP on astrocytoma proliferation and malignancy (see “Introduction” section) may be explained in part by differences in the expression levels of PACAP receptors.

We previously reported that PAC1-R immunoreactivity was increased in reactive astrocytes induced by brain ischemia or spinal cord injury (Tsuchikawa et al. 2012; Nakamachi et al. 2013). PACAP treatment of cultured reactive astrocytes induced by scratch injury significantly increased the number of Ki67-positive proliferating cells in vitro (Nakamachi et al. 2011b). These data imply that malignant astrocytes and reactive astrocytes display similar characteristics, making it possible therefore to choose between low-malignant type astrocytoma cell lines (e.g., KNS-81) and high-malignant type cell lines (e.g., YH-13) as models of resting and reactive astrocytes, respectively.

It has also been reported that PACAP protects astrocytes against oxidative stress-induced apoptosis (Masmoudi-Kouki et al. 2011). Although the results in this study provide some information showing that PACAP increased the number of highly malignant astrocytoma cells, it remains unclear whether PACAP stimulates proliferation or survival. In addition, it is known that PACAP induces the differentiation of neural stem cells into astrocytes (Watanabe et al. 2007; Nakamachi et al. 2011a), implying that PACAP may influence differentiation status or, in other words, the malignancy level of astrocytoma cells. Further studies focusing on the function of PACAP on astrocytoma cells is therefore warranted.

In conclusion, we have shown here that PACAP receptor expression is correlated with malignant potential, and that this effect may underlie differences in PACAP sensitivity exhibited by astrocytoma cell lines. Further study on the effects of PACAP on astrocytoma cells could help to elucidate characteristics of malignant astrocytoma cells, on their high proliferative and invasive potential, and on their resistance to chemotherapy.