The data and samples of the patient sample used in our previous study were reclaimed The samples are collected between 01/2006 and 12/2012 (Departments of Neurosurgery and Neuropathology, University of Giessen, Germany) (19). At the time of diagnosis, the mean age of the 44 patients with GBM was 57.4±15.7 years (f:m=13:31), while the progression-free survival (PFS) time of the patients was 16 months. In the present study, all cases received a gross total resection. The overall survival (OS) of patients with MGMT promoter methylation was longer (OS, 23 months; range, 14.8–29.2) compared with those without MGMT promoter methylation (OS, 11 months; range, 5.5–16.5) (19). Inclusion criteria was diagnosed GBM, exclusion criteria were missing consent, missing files and Karnofsky-Index < 80. All patients provided written informed consent prior to enrolment in the study (Table I).

Nicotinehydrogentatrate (cat. no. N3260) and ACh were purchased from Sigma-Aldrich (Merck KGaA). GTS-21 (cat. no. 4557) was obtained from Tocris Bioscience, while α-BTX (cat. no. B1601) was purchased from Thermo Fisher Scientific, Inc. The A172, G28, U87 cell lines were purchased from ATCC. U87 cell line is a glioblastoma of unknown origin (HTB-14).

Tissue specimens from patients with histologically confirmed GBM were immediately collected after surgery. To isolate cells for the primary cell cultures, the tissues were finely chopped in DMEM (Dubelcco's modified Eagles medium) and trypsinized using an 0.05% EDTA/trypsin solution. The trypsin reaction was stopped by adding 10 ml DMEM and to obtain a single cell suspension, the minced tissues were passed through a 60-µm cell strainer (Sigma-Aldrich; Merck KGaA). Subsequently, cells were washed with PBS (Phosphate buffered saline). Following centrifugation, the cell pellet was resuspended in DMEM and the cells were then seeded into 25 cm² culture flasks (Greiner Bio-One GmbH) and cultured in an incubator at 37°C and 5% CO₂. Then, following the appropriate number of passages, the cells were expanded to 175 cm² flasks (Greiner Bio-One GmbH). For cell cryopreservation, the cells were first washed with PBS, centrifugated, resuspended in DMSO (Merck KGaA) and finally stored in liquid nitrogen. Frozen cells were thawed in a 37°C-water bath and were then cultured as adherent monolayers in 25-cm² flasks. The clinicopathological characteristics of patients are listed in Table I.

For total RNA isolation from frozen specimens, the RNeasy Lipid Tissue Mini Kit^® (Qiagen GmbH) was used. To ensure proper tissue dissociation, the specimens were treated with a lysis reagent, provided by the kit, for 2 min in a bead mill at 5,000 rpm. The RNA concentration was measured with NanoDrop^® 1000 spectrophotometer (Thermo Fisher Scientific Inc.). cDNA synthesis was carried out using 0.5 µg total RNA/sample as a template with the RT² First Strand Kit (Qiagen GmbH).

For the simultaneous analysis of 31 genes, including 28 target genes and three housekeeping genes, namely RPL13A, TBB and GAPDH, a Custom RT² Profiler PCR Array was used, containing lyophilized primers for each target gene (CAPH12576; cat. no. 330231; Qiagen GmbH). The analysis was carried out on the StepOnePlus quantitative PCR cycler (Thermo Fisher Scientific, Inc.) using the RT² SYBR^®-Green Mastermix (Qiagen GmbH) according to the manufacturer's recommendations. For the analysis, the qPCR results from all samples were merged and uploaded into the online analysis tool provided by the manufacturer. The online tool calculates the ΔΔCq-based fold change in the expression of each target gene normalized to that of the housekeeping genes.

To evaluate cell survival and proliferation, a MTT assay was performed following treatment of cells with GTS-21 (Tocris Bioscience), ACh or nicotine (both from Sigma-Aldrich; Merck KGaA). Briefly, cells were seeded into 96-well plates at a density of 10⁴ cells/well. All cell lines were treated with 6.25, 12.5, 25 or 50 µM GTS-21, ACh or nicotine. The negative control cells for the GTS-21 group were treated with 0.1% DMSO, while those of the ACh and nicotine groups with 0.1% H₂O. The MTT reagent (Sigma-Aldrich; Merck KGaA) was diluted in DMEM at a ratio of 1:10. Following treatment of cells with the corresponding compounds for 24, 48 and 72 h, the culture medium was removed and the cells were supplemented with 200 µl of MTT-DMEM solution and incubated at 37°C in an atmosphere of 95% air and 5% CO₂ for an additional 90 min. Subsequently, the cells were lysed in 150 µl isopropanol/hydrochloric acid solution (Sigma-Aldrich; Merck KGaA) for 15 min and the absorbance value in each well was measured using a microplate reader at a wavelength of 562 nm, with a reference wavelength of 630 nm.

Statistical analysis was implemented using SPSS 20 (IBM Corp.). The comparison of different concentrations of GTS-21 and α-bungarotoxin were performed using Kruksal-Wallis-test (post hoc: Bonferroni). P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± SEM.

Using a custom-made qPCR array covering the majority of components of the cholinergic system, the expression levels of these genes were assessed in 44 tissue samples obtained from patients with primary GBM and compared with those in five tissue samples from healthy subjects (Fig. 1A). The results demonstrated that the expression of the majority of genes encoding the receptor subunits was significantly downregulated (fold changes, log₂ >-1; P<0.01). Additionally, the gene expression levels of ACHE and those of the dominant-negative form of CHRNA7 and the duplicate fusion gene product of CHRFAM7A were also significantly suppressed. In addition, the expression levels of three genes, namely CHRNB1, encoding the nicotinic β1 receptor subunit, CHAT, encoding choline acetyltransferase and CHRNA7, encoding the nicotinic α7 receptor subunit, remained unchanged.

The expression levels of the cholinergic system-related genes were also determined in the established GBM cell lines A172, U87 and G28 (Fig. 1B). CHRNA2 was not expressed in any of the three cell lines examined. By contrast, CHRNA1, CHRNA9 and BCHE were expressed in all cell lines. The most significant differences in the gene expression profile between primary GBM cells and cell lines were observed in CHAT expression, as CHAT was not expressed in A172, U87 and G28 cells compared with primary GBM cells. This finding could be attributed to the loss of ACh synthesis. Despite the similarities, the cell lines exhibited quantitative and qualitative heterogeneity in the expression of several genes, including CHRM2, which was expressed in G28, but not in A172 and U87 cells and the fusion gene product CHRFAM7A, which was not expressed in U87 cells.

Overall, the aforementioned findings revealed significant differences in the expression of cholinergic system-related genes in GBM. More specifically, the expression of CHRNA7 remained unchanged in all cell lines examined, indicating that these cell lines may serve as important tools for evaluating the role of CHRNA7 in GBM. Furthermore, the heterogeneity in the expression of CHRFAM7A could contribute to a better understanding of the participation of the dominant-negative regulator in the cholinergic system.

Subsequently, the present study aimed to investigate the effect of the activation of the α7-composed nAChR on cellular proliferation. Therefore, the three GBM cell lines A172, U87 and G28 were stimulated with increasing concentrations of GTS-21, a well-known partial agonist of α7 and α4β2 subunits, for 24 and 48 h. Since both α4 and β2 subunits were not expressed in the current cell model, GTS-21 served as a sole α7 agonist. Cell treatment with 50 µM GTS-21 significantly attenuated the proliferation of all cell lines at 48 h (Fig. 2). However, the extent of reduction differed among the different GBM cell lines (A172 cells, 63.3±7.5%; G28 cells, 39.4±5.3%; U87 cells, 47.2±4.8%). Additionally, GTS-21 exerted an antiproliferative effect on G28 cells even after treatment with 12.5 µM GTS-21 for 24 h (Fig. 2C). However, treatment of cells with equimolar concentrations of cholinergic agonists, namely nicotine and ACh, had no effect on cell viability (Fig. S1). In summary, pharmacological stimulation of the α7 receptor subunits with GTS-21 reduced GBM cell proliferation.

To investigate whether the antiproliferative effect of GTS-21 was associated with the α7 subunit, GBM cells were co-treated with 50 µM GTS-21 and increasing concentrations of α-BTX (1.2–100 µM), an antagonist of the homo-pentameric α7 receptor, for 48 h. The results showed that treatment of A172 and G28 cells with α-BTX had no effect on their proliferation capacity (Fig. 3A and C). However, the proliferation rate of U87 cells treated with 3.6 µM α-BTX was notably increased (174.6±21.71%; Fig. 3B). This finding indicated that the proliferative effects of GTS-21 could be abolished by α-BTX only in U87 cells.

A total of eight primary GBM cell cultures were treated with 6.25, 12.5, 25 or 50 µM GTS-21 for 48 h. The proliferation rate of primary tumour cells treated with different doses of GTS-21 was significantly reduced compared with A172, G28 and U87 cells (Fig. 4), suggesting that all primary cells responded to treatment. The median inhibition rate (loss of proliferation) in primary GBM cells was 51%, ranging between 36 and 84%.