GABA_B receptor regulates proliferation in the high-grade chondrosarcoma cell line OUMS-27 via apoptotic pathways

The human high-grade chondrosarcoma cell line OUMS-27 [21], characterized by short tandem repeat analysis (access code; CVCL_3090), was obtained from Okayama University.

Cells were grown in supplemented Dulbecco’s modified Eagle medium with 10% (v/v) heat inactivated fetal bovine serum and 1% antibiotic-antimycotic (100×, Thermo Fisher Scientific, Waltham, MA, USA) under an atmosphere of 95% air and 5% CO₂ at 37 °C. Cells were confirmed to be free of mycoplasma infection using an e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Gyeonggi-do, Korea).

OUMS-27 cells were harvested by low speed centrifugation at 800 rpm and washed twice in phosphate-buffered saline (PBS) without trypsin. The harvested cells were fixed with 50 mL 4% (w/v) paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Following brief rinsing with PBS, the cells were blocked with 3% bovine serum albumin/PBS. Immunohistochemistry was performed for the GABA_A receptor and its subunits, GAD, and GABA using a goat polyclonal antibody directed against the GABA_A receptor subunits α2, α3, β1, and γ3 and GABA_B receptor R1 subunit (diluted 250×; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal antibodies directed against GAD65 (diluted 1000×; Chemicon International, Temecula, CA, USA) and GABA (diluted 250×; Chemicon International), mouse monoclonal antibody directed against GAD67 (diluted 1000×; Chemicon International), and guinea pig polyclonal antibody directed against the GABA_B receptor R2 subunit (diluted 1500×; Chemicon International). The specificity of these antibodies has been reported previously [22‐24]. Briefly, sections pre-washed with PBS were incubated with normal donkey or goat serum (diluted 50×) for 30 min at room temperature (RT), followed by overnight incubation with each primary antibody at 4 °C. The sections were rinsed with PBS and incubated with Alexa Fluor™ 488 donkey anti-goat IgG secondary antibody (diluted 300×; Molecular Probes, Eugene, OR, USA) for the GABA_A receptor subunits and GABA_B receptor R1 subunit, Alexa Fluor™ 488 goat anti-rabbit IgG (diluted 300×; Molecular Probes) for GABA and GAD65, Alexa Fluor™ 488 goat anti-mouse IgG (diluted 300×; Molecular Probes) for GAD67, and Alexa Fluor^R 546 goat anti-guinea pig IgG (diluted 300×; Molecular Probes) for the GABA_B receptor R2 subunit for 60 min at RT in the dark. Subsequently, the sections were rinsed with PBS. The sections, except those used for double-staining, were treated with 100 μg/mL RNase A in PBS for 1 h at 37°C and counterstained with 10 μg/mL of propidium iodide (PI, Molecular Probes) diluted in phosphoric and citric acid buffer for 3 min at RT. After several rinses with PBS, immunoreactivity was examined using a confocal laser microscope (LSM510, Zeiss Co., Ltd., Oberkochen, Germany) equipped with a 488-nm argon laser. Sections incubated with non-immune sera from the same species as the primary antibody served as negative controls.

OUMS-27 cells were harvested by low speed centrifugation at 800 rpm and washed three times in PBS without enzyme. Total RNA was extracted from the harvested cells using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized using Omniscript reverse transcriptase (Qiagen GmbH) according to the manufacturer’s instructions. The reverse transcription reaction mixture contained 1 μM oligo-d (T)12–18 primer, 10 U RNase inhibitor, 0.5 mM of each dNTP, and 4 U Omniscript reverse transcriptase. PCR was performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster, CA, USA). The PCR reaction mixture (25 μL) contained 1× GoTaq Green Master Mix reaction buffer (pH 8.5), 400 μM dNTPs, 3 mM magnesium chloride_, (Promega, Madison, WI, USA), 2 μL cDNA solution, and 0.2 μM of each primer. The primer sequences used for GAD65, GAD67, the GABA_A α1–6, β1–3, γ1–3, δ, π, θ, and ε subunits, and GABA_B R1a, R1a/b, and R2 are shown in Table 1. The PCR products were separated on 1.5% agarose gels, followed by staining with 0.1% ethidium bromide solution for 10 min and illuminated using an ultraviolet transilluminator.

OUMS-27 cells were plated at a concentration of 1 × 10⁴ cells/well on 96-well plates one day before each drug treatment. They were subsequently incubated for 48 h in culture medium containing one of the following GABA receptor ligands: 100 μM GABA and GABA_A receptor agonist, 50 μM Muscimol(MUS) GABA_B receptor agonists, 100 μM R-(+)-Baclofen(BFN)or 10 μM SKF 97541 [3-aminopropyl (methyl) phosphine acid] (SKF), 100 μM GABA+ GABA_A receptor antagonist, 100 μM (−)-bicuculline methochloride(BMC)or GABA_B receptor antagonist, 1 μM CGP54626(CGP). After seeding the cells, 5-bromo-2′- deoxyuridine (BrdU) was added to a final concentration of 1 μM in each sample and incubated for 2 h. Thereafter, DNA synthesis was assayed by cell proliferation enzyme-linked immunosorbent assay (ELISA) and BrdU (Roche Molecular Biochemicals, Basel, Switzerland) was estimated by colorimetric detection according to the manufacturer’s instructions. Colorimetric analysis was performed using an ELISA plate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

First, 1 × 10⁶ OUMS-27 cells, both adherent and suspended, harvested after 24 h treatment with various concentrations of CGP or dimethyl sulfoxide (DMSO) (10, 100, 250, 500 μM CGP), were fixed in 1% (w/v) paraformaldehyde in PBS (pH 7.4). The APO-DIRECT™ apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s protocol. The APO-DIRECT™ assay is a single-step method for labeling DNA breaks with FITC-dUTP. Cells isolated from each step were incubated at − 20 °C in 70% (v/v) ethanol for 30 min. Apoptotic cells were analyzed by FACScan flow cytometry and BD Diva Software Version 4.1 (BD Biosciences).

OUMS-27 cells were plated at concentration of 1 × 10⁴ cells/well on a 96-well plate one day before CGP treatment. Cell viability was measured after treatment with 10 μM CGP or DMSO for 24 h using the Cell Titer-Blue cell viability assay (Promega). The activities of caspase-3, caspase-8, and caspase-9 were measured using the Caspase-Glo™ Assay luminescence kit (Promega). The fluorescent intensities for the cell viability assay and luminescent intensities for caspase activity were measured using the GloMax-Multi detection system (Promega). The activity of each caspase was adjusted to account for the corresponding cell viability data as described previously [25].

OUMS-27 cells were grown in each well with various concentrations of CGP or DMSO for 24 h. Harvested cells were fixed in cold 70% (v/v) ethanol. CycleTEST™ PLUS DNA reagent kit (BD Biosciences) was used according to the manufacturer’s protocol. The cell cycle distribution in all sample cells was measured by flow cytometry (EPICS Elite ESP; Beckman Coulter Co., Brea, CA, USA) and the percentage of cells in each phase of the cell cycle was analyzed using a Multi Cycle for Windows Version 3.0 (PHOENIX, San Diego, CA, USA).

After treatment with 10 μM CGP or control for 24 h, OUMS-27 cells were harvested and proteins were extracted. The cells were homogenized in 1 mM Tris-HCl (pH 7.5) containing 150 mM sodium chloride, 5 mM EDTA, 1% (w/v) sodium dodecyl sulfate (SDS), 1 mM phenylmethane sulfonyl fluoride, 1% (w/v) sodium deoxycholate, and 0.5% (w/v) protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). After centrifugation, the protein concentration in the cell supernatants was determined using the Qubit^a protein assay kit (Molecular Probe) and a Qubit^a.2.0 fluorometer. Aliquots containing 2.5 or 5 μg protein were then boiled in loading buffer containing 50 mM Tris (pH 6.8), 6% 2-mercaptoethanol, 2% SDS, 10% glycerol, and 0.004% bromophenol blue. Each aliquot was loaded onto an 8% polyacrylamide gel. After electrophoresis, the gels were transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated with 1% bovine serum albumin in TBS containing 0.1% Tween-20 overnight to block non-specific binding, followed by incubation with GAD65, GAD67, total Akt, phospho-Akt-Ser473, phospho-Akt-Thr308, ERK 1/2, phospho-ERK1/2, JNK, phospho-JNK, p38 (diluted 1000×, Cell Signaling Technology, Danvers, MA, USA), phospho-p38 (diluted 2500×, BD Biosciences) or anti-GAPDH antibodies (diluted 3200×, MAb 6C5, HyTest, Turku, Finland). After rinsing the membranes, horseradish peroxidase (HRP)-linked anti-rabbit IgG and HRP-linked anti-mouse IgG (Cell Signaling) secondary antibodies were applied and the chemiluminescent reaction was performed using ECL Plus western blotting detection reagents (GE Healthcare, Little Chalfont, UK). Protein expression was detected using the LAS-3000 Lumio image analyzer (Fuji Photo Film, Tokyo, Japan). Signal intensities were further analyzed using Multi Gauge software (version 3.0; Fuji Photo Film).

OUMS-27 cells were treated with 10 μM CGP or DMSO for 24 h. Total RNA was extracted from each cell using the RNeasy mini kit (QIAGEN, Hilden, Germany), and cDNA was synthesized using QuantiTect reverse transcription kit (QIAGEN). Primers in the Human PrimerArray Cell Cycle series (Takara Bio, Inc., Shiga, Japan) were used to amplify loci involved in cell cycle regulation. Real-time PCR was performed with the Thermal Cycler Dice Real Time system (Takara Bio, Inc.) according to the manufacturer’s instructions. Data were normalized to the expression of glyceraldehyde-3- phosphate dehydrogenase and expressed as the mean ± standard deviation (SD).

Whole-cell patch-clamp recordings for Ca²⁺ currents in OUMS-27 cells treated with 100 μM GABA and 1 μM CGP were conducted as previously described [26‐28]. Patch pipettes were pulled from borosilicate glass capillary tubes (GC120F15, Clark Electromedical Instruments, Pangbourne, UK) using a vertical electrode puller (PP-83, Narishige Scientific Instrument Laboratories, Tokyo, Japan), which exhibited resistance between 4 and 6 MΩ. The filling solution of the patch-pipettes contained 80 mM cesium chloride, 65 mM cesium-methane sulfate, 2 mM EGTA, 10 mM HEPES, 4 mM ATP-Mg, and 0.2 mM GTP-Tris (pH 7.3). The bath solution for recording consisted of 135 mM tetraethylammonium chloride, 10 mM barium chloride, 10 mM HEPES, 10 mM D-glucose, and 20 mM sucrose (pH 7.3 adjusted with 1 N hydrochloric acid). Ca²⁺ channel currents were routinely evoked with a 100 ms voltage step from − 60 to 0 mV in increments of 10 mV. GABA-induced Ca²⁺ channel currents were measured using a patch-clamp amplifier (EPC-9, HEKA Elektronik, Lambrecht, Germany) which was controlled by a Macintosh computer (Power Macintosh G3, Apple Computer, Cupertino, CA, USA) and control software (Pulse + PulseFit, HEKA Elektronik, Lambrecht/Pfatz, Germany).

The intracellular Ca²⁺ concentration, [Ca²⁺]_I, in drug-treated OUMS-27 cells, was measured by Fura-2 AM, a calcium-sensitive dye (Dojindo, Kumamoto, Japan). Briefly, OUMS-27 cells were loaded with 5 μM Fura-2 AM in loading buffer containing 0.01% pluronic F-127 (Dojindo) for 30 min at 37 °C. After washing Fura-2 AM out of the loading buffer, the relative transient calcium concentration (OD_340nm/OD_380nm excitation ratio) was recorded before and after the addition of 10 mM GABA (Sigma-Aldrich) in a perfusion chamber using the AQUACOSMOS/RATIO, C7773 (Hamamatsu photonics, Hamamatsu, Japan). Recording was continued after washing the 10 mM GABA out of the buffer. [Ca²⁺]_I after pretreatment with 1 μM CGP and application of 10 mM GABA was recorded using a similar method.

The results are shown as the mean ± standard deviation (SD) and P values less than 0.05^*, 0.01**, or 0.001*** were considered statistically significant using Student’s t-tests. Each experiment was performed at least three times under identical conditions.

We detected specific mRNA expression of GAD65, but not GAD67, in OUMS-27 cells. The mRNA expression of GABA_A receptor subunits α1, α2, α3, α5, β1, β3, γ1–3, δ, θ, and ε and the GABA_B receptor subunits R1 and R2, were also detected (Fig. 1a). In addition, immunohistochemistry revealed that GABA, GAD65, α2, α3, β1, and γ3 subunits of the GABA_A receptor, and the R1 and R2 subunits of the GABA_B receptor were expressed in the OUMS-27 cells (Fig. 1b).

BrdU incorporation into OUMS-27 cells treated with 100 μM GABA, the GABA_A receptor agonist, 50 μM MUS and the GABA_B receptor agonists, 100 μM BFN and 10 μM SKF were significantly increased. However, the proliferation of the OUMS-27 cells treated with 100 μM GABA was significantly inhibited by the GABA_A receptor antagonist, 100 μM BMC and the GABA_B receptor antagonist, 1 μM CGP (Fig. 1c).

We performed flow cytometric analysis to quantitatively assess apoptosis in the OUMS-27 cells treated with CGP. The percentage of apoptotic (TUNEL- positive) cells significantly increased in response to CGP treatment in a dose-dependent manner (Fig. 2a).

To clarify the mechanism by which apoptosis was induced by CGP, we examined the activities of caspase 3, caspase 8, and caspase 9 in CGP-treated OUMS-27 cells using a cell viability assay and caspase luminescent assay. The activities of caspase 3 and caspase 9 were significantly elevated in cells treated with CGP for 24 h compared to in control cells. The activity of caspase 8 did not differ significantly between control and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells (Fig. 2b). These results indicate that the GABA_B receptor antagonist inhibited cell proliferation via apoptotic pathways.

We performed flow cytometric analysis to determine the percentage of OUMS-27 cells in each phase of the cell cycle after CGP treatment. The percentage of cells in G1 phase increased and those in S phase decreased significantly in a CGP dose-dependent manner (Fig. 2c). These results indicate that the GABA_B receptor antagonist induced G1 phase arrest and S phase suppression in the cell cycle.

The activation of MAPKs such as ERK, p38, and JNK, and PI3K/AKT/mTOR was examined by western blot analysis using antibodies that specifically recognize the phosphorylated forms of these proteins. The activities of ERK and phospho-ERK did not change after CGP or BFN treatment (Fig. 3a). However, MAPKs such as p38 and JNK were activated after CGP treatment (Fig. 3b). There were no apparent differences in total Akt levels between CGP-treated and control cells. The activities of both phospho-Akt-Thr308 and phospho-Akt-Ser473 in CGP-treated OUMS-27 cells were suppressed compared to in control cells (Fig. 3c).

We investigated the mRNA levels of cell cycle-regulatory genes in OUMS-27 cells treated with CGP and control cells, as CGP treatment induced cell cycle arrest in the previous experiment. Real-time analysis revealed that the expression of p21 and p53 was significantly higher and that of cyclin–dependent kinase (CDK)6 and CDK2 was significantly lower in OUMS-27 cells treated with CGP compared to in control cells (Fig. 4).

We performed whole-cell patch-clamp recordings of membrane Ca²⁺ channel currents in drug-treated OUMS-27 cells. The application of 100 μM GABA to the bath inhibited the Ca²⁺ currents. The Ca²⁺ currents were reproducibly regained after washing away the GABA. Additionally, the inhibitory effect of GABA on Ca²⁺ currents was attenuated by 1 μM CGP, which is a GABA_B receptor antagonist (Fig. 5).

[Ca²⁺]_I decreased immediately after inhibition of Ca²⁺ influx in response to 10 mM GABA in OUMS-27 cells. However, the [Ca²⁺]_I level increased with Ca²⁺ influx after washing. [Ca²⁺]_I did not change following application of 10 mM GABA after pretreatment with 1 μM CGP (Fig. 6).