The human glioma cell lines LN-229 and SW1088 and the human melanoma cell line A375 (American Type Culture Collection (ATCC), Manassas, VA, USA) were maintained in Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech, Inc., Herndon, VA, USA) and the human melanoma cell line WM35 (ATCC) was maintained in Eagle's Minimum Essential Medium (MEM) (Mediatech, Inc.). All cells were supplemented with 10% fetal bovine serum (FBS) (Gemini Biological Products, Calabasas, CA, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) and cultured in a humidified incubator with 5% CO₂ at 37°C. For cell viability assays, 2.5 × 10⁴ cells were plated onto 12-well tissue-culture treated plates (Fisher Scientific, Pittsburgh, PA, USA) using media supplemented with 1% FBS. A-192621 (Abbott Laboratories, Abbott Park, IL, USA), BQ788 (EMD Chemicals Inc., San Diego, CA, USA) and/or BQ123 (EMD) were added 24 h after plating and viable cell number was assessed using the Live/Dead Viability/Cytotoxicity Kit for mammalian cells (Invitrogen) according to the manufacturer's instructions. Fluorescent intensity was measured on an FLx800 multi-detection microplate reader (BioTek, Winooski, VT, USA) and values represent the mean of a 25-point well scan.

LN-229 and SW1088 cells were plated at 5 × 10⁵ cells per 100 mm dish in DMEM with 1% FBS, and A-192621 was added 24 h later. Cell cycle analysis was performed with a BrdU/propidium iodide double stain using the Absolute-S Cell Proliferation Kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions allowing 40 minutes to pulse-label cells with BrdU. Fluorescent intensity was measured using the BD FACSCalibur System (Becton Dickinson, Franklin Lakes, NJ, USA). The rate of cell proliferation was assessed using the CellTrace CFSE Cell Proliferation Kit (Invitrogen) according to the manufacturer's instructions. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) at the time of plating and fluorescent intensity was analyzed at 9, 24, 48 and 72 h after the addition of A-192621 using the BD FACSCalibur System. Cell death was quantified by staining cells with propidium iodide (Invitrogen) following treatment with A-192621. Fluorescent intensity was analyzed using the BD FACSCalibur System. All FACS data was analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA). Caspase 3/7 activity was measured using EnzChek Caspase-3 Assay Kit #2 (Invitrogen) according to the manufacturer's instructions and values adjusted for total cell number. Fluorescent intensity was measured using the FLx800 multi-detection microplate reader.

LN-229 and SW1088 cells were treated with vehicle, 10 nM or 100 μM A-192621 for 12 h and total RNA was prepared using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). The quality of the samples was checked using the RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara, CA, USA). RNA samples were then processed according to the Affymetrix Eukaryotic Sample and Array Processing protocol. Hybridization of the in vitro amplified RNA to Affymetrix Human Genome U133Plus 2.0 chips (Affymetrix, Inc., Santa Clara, CA, USA), washing and scanning of the arrays were performed following standard Affymetrix protocols using a Hybridization Oven 640, a Fluidics Station 450, and a GeneChip^® Scanner 3000 7G. The raw data (*.cel files) from the Affymetrix hybridizations were processed and analyzed using Resolver (Rosetta Biosoftware, Seattle, WA, USA). Genes were identified using cutoffs of fold-change > 3 and p < 0.0001.

Total RNA was prepared using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). The cDNA was prepared from 1 μg total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturers instructions. Primer/probe design was accomplished using the Universal ProbeLibrary (UPL) Assay Design Center (Roche Applied Science). The primer sets (Integrated DNA Technologies, Coralville, IA, USA) were as follows: 5'-GGC AGA AGC TGA AAG GTC TC-3' and 5'-CAT CGA AGC ACT GTC TCA GAG T-3' (DR5), 5'-GGA GAG CAG AAG ACC GAA AG-3' and 5'-AGT GAT CGT GCG CTG ACT C-3' (GADD45A), 5'-GCT TCT GGC AGA CCG AAC-3' and 5'-GTA GCC TGA TGG GGT GCT T-3' (GADD34), 5 '-ACT GCG TCT TTG GCA TCA G-3' and 5'-GTA GCA GGC CAC TGT CTT GA-3' (Sestrin 2), 5'-AAG GCA CTG AGC GTA TCA TGT-3' and 5'-TGA AGA TAC ACT TCC TTC TTG AAC AC-3' (GADD153), 5'-TTC ATC CCG TTC AGA AGA CA-3' and 5'-CCA ATG GCA AGC AGA AAT AGA-3' (ETRB) and 5'-TGA CCT TGA TTT ATT TTG CAT ACC-3' and 5'-CGA GCA AGA CGT TCA GTC CT-3' (HPRT). The corresponding probes were UPL probe #63 (DR5), #65 (GADD45A), #28 (GADD34), #17 (Sestrin 2), #21 (GADD153), #83 (ETRB) and #73 (HPRT) (Roche Applied Science). PCR was performed and analyzed on a LightCycler 480 System (Roche Applied Science) using LightCycler 480 Probes Master (Roche Applied Science). The PCR was done under the following conditions: pre-incubation at 95°C for 5 minutes, 45 cycles of amplification with melting at 95°C for 8 seconds, annealing at 60°C for 15 seconds and extension at 72°C for 2 seconds, and 1 cycle of cooling at 40°C for 10 seconds. All gene expression was quantified relative to HPRT expression.

Following the reverse transfection protocol, Lipofectamine RNAiMAX (Invitrogen) was diluted in Opti-MEM I Medium (Invitrogen) and ON-TARGETplus SMARTpool ETRB siRNA or ON-TARGETplus siCONTROL non-targeting siRNA (Dharmacon, Lafayette, CO, USA) was diluted in Opti-MEM I Medium without serum. These solutions were then combined and incubated together at room temperature for 10–20 minutes. The siRNA duplex-Lipofectamine RNAiMAX complexes were then plated and overlaid with 2.5 × 10⁴ cells per ml in media with 1% FBS. Media was changed 4–6 h after plating and cells were treated as described above. ETRB gene expression was assessed by real-time PCR and was reduced by 70 – 93%.

Values are expressed as mean ± SEM. Statistical analysis was done using one-way ANOVA with Tukey posthoc unless otherwise noted. P < 0.05 was considered statistically significant.

To test the effectiveness of A-192621 in reducing viable glioma cells we employed two human glioma cell lines, LN-229 and SW1088. LN-229 was originally derived from a grade IV glioblastoma and SW1088 was derived from an anaplastic astrocytoma. Twenty-four hours after plating, A-192621 was added at concentrations from 1 to 100 μM to non-confluent cells and incubated for 24 to 72 h. Viable cells were quantified by the capacity of their intracellular esterases to convert non-fluorescent calcein AM into green-fluorescent calcein. This assay reveals a decrease in viable cells with increasing A-192621 concentration (Fig. 1A). This decrease is enhanced at longer incubation times (Fig. 1A). In addition, A-192621 is effective in reducing viable cells in the human melanoma cell lines A375 and WM35 in a dose- and time-dependent manner (Fig. 1A). This finding with melanoma cells is consistent with previously published data reporting that A-192621 inhibits melanoma growth in nude mice 13. We also tested the ability of ETRB-specific antagonist BQ788 to reduce the viability of the glioma cells. BQ788 was added to non-confluent cells in the same manner as A-192621 at concentrations from 1 to 100 μM. This inhibitor causes a significant decrease in viable LN-229 cells after 48 or 72 h of treatment but no significant change in SW1088 cells (Fig. 1B). BQ788 also reduces cell viability in both melanoma cell lines in a dose- and time-dependent manner (Fig. 1B). This last result is consistent with previous findings of melanoma cells in our laboratory 22. To assess the involvement of ETRA in viability, cells were treated with BQ123, an ETRA-specific antagonist. BQ123 was added to cells at 1 to 100 μM and incubated for 24 to 72 h. No significant changes in cell viability were observed in any of the four cell lines, at any concentration or time point (Additional file 1). Thus, potential cross-reaction of A-192621 or BQ788 with ETRA does not play a role in their effects on cell viability.

To investigate how A-192621 reduces glioma cell number we assessed the rate of cell proliferation over time. The human glioma cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). This non-fluorescent reagent passively diffuses into cells, where the acetate groups are cleaved by intracellular esterases, producing green-fluorescent carboxyfluorescein succinimidyl ester. The ester groups react with intracellular amines and fluorescent intensity is exponentially diluted as cells divide. Fluorescent intensity was measured by FACS at 9 to 72 h. We tested two doses of A-192621, 10 nM, a concentration slightly above the IC₅₀ that is calculated from radio-labeled ET-1 binding displacement studies 23, and 100 μM, a concentration that dramatically reduces the number of viable cells in both the LN-229 and SW1088 lines (Fig. 1A). We find that 100 μM, but not 10 nM, significantly reduces fluorescent carboxyfluorescein succinimidyl ester dilution in both LN-229 and SW1088 cells at 24 h, and this effect is sustained through later time points, indicating that A-192621 reduces the rate of cell division (Fig. 2A).

We also examined the cell cycle status following A-192621 treatment using BrdU/propidium iodide (PI) double staining. Following treatment with A-192621, cultures were labeled with BrdU for 40 min to identify cells undergoing DNA synthesis and fixed immediately afterwards. Cells were analyzed by FACS, and BrdU intensity was plotted against DNA content. In both glioma cell lines, 100 μM A-192621 significantly increases the percentage of cells in the G2/M phase compared to vehicle, or to 10 nM treatment after 24 h (data not shown). This accumulation of cells in the G2/M phase continues through 72 h and is coupled with a concomitant decrease in the G1/G0 population (Fig. 2B), indicating that A-192621 induces a G2/M phase arrest. In addition to effects on proliferation, we investigated whether A-192621 treatment also affects cell death, as measured by the percentage of total cells that stain positively with PI. A-192621 significantly increases PI staining at 48 h in both LN-229 and SW1088 cells, and at 72 h in SW1088 cells (Fig. 3A). Moreover, as a measure of apoptotic cell death, caspase 3/7 activity is increased at 72 and 48 h in LN-229 and SW1088 cells, respectively (Fig 3B).

In order to further understand the effects of A-192621 on glioma cells, we assessed changes in gene expression by microarray analysis following a 12 h treatment with vehicle, 10 nM or 100 μM A-192621 in glioma cells. Changes in gene expression were identified using the criteria of > 3-fold change and P < 0.0001. A striking finding is that, in both cell lines, 100 μM A-192621 up-regulates several genes that are known to be induced by DNA damage (Table 1). These genes were not significantly up-regulated by 10 nM A-192621. These genes include growth arrest and DNA-damage-inducible (GADD) 153, GADD45A, GADD34, sestrin 2 and death receptor 5 (DR5). The up-regulation of these genes was confirmed by real-time PCR in the human glioma cell lines, and also in the human melanoma cell lines (Table 1). A-192621 increases GADD153 expression the most dramatically, with a 10- to 66-fold increase, depending on the cell line treated. A-192621 increases GADD45A and GADD34 expression similarly by 5- to 23- and 3- to 21-fold, respectively. Expression of sestrin 2 and DR5 is up-regulated 12- to 19- and 4- to 12-fold, respectively.

The concentrations A-192621 and BQ788 required to reduce cell viability in vitro are far above the IC₅₀ concentrations required to displace ET-1 2324. Moreover, when we examined expression levels by real-time PCR, we could not detect ETRB in the SW1088 cell line after 45 amplification cycles (Fig 4A). These findings led us to question the involvement of ETRB in the growth inhibition by ETRB antagonists. To address this question directly, we reduced the expression level of ETRB 69–93%, using small interfering RNA (siRNA) (Fig 4B). All cell lines were transfected with siRNAs targeting ETRB or a non-targeting, scrambled siRNA, and then treated 24 h later with 100 μM A-192621, BQ788 or their respective vehicles. The number of viable cells was assessed at 24 to 72 h. First, reduced ETRB expression alone does not affect cell viability. Second, both A-192621 and BQ788 decrease the number of viable cells equivalently in cells transfected with ETRB-targeting siRNA or scrambled siRNA at all time points (Fig. 4C; 24 and 48 hours not shown). The lack of effect of ETRB knockdown on A-192621- and BQ788-mediated reduction in cell number is further evidence that antagonism of this receptor is not the primary mechanism for the viability effects of these two drugs in vitro.