Antitumor activity of (R,R’)-4-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse

(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell-type specific modulation of β2-adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ∼1 nmol/L) was refractory to pharmacological inhibition of β2-adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT-PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline-treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF-dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.


Supplemental Methods
Determination of MNF levels in C6 glioma tumours. The accumulation of MNF in vivo in C6 tumour xenografts in athymic mice was assessed in comparison with vehicle-treated tumour-bearing animals. The frozen tumour samples were thawed, and 300-400 mg tumour tissue was placed in 300 µl of water supplemented with 9 µl acetonitrile containing 500 ng/ml of methoxyfenoterol as the internal standard.
After thorough homogeneization of the tumour tissue on ice using a Polytron micro homogenizer (Pro200 Bio-gen series, Proscientific , Oxford, CT, USA), 600 µl acetonitrile was added and the mixture was vortexed for 10 s. The solution was centrifuged at 20,800 rcf for 10 min at 4ºC. The pellet was discarded and the supernatant was centrifuged again for an additional 10 min before being transferred to a Shimadzu SIL-20A autosampler for analysis. The samples were maintained in the autosampler tray at 4ºC, and injections of 20 µl aliquots were made to an analytical HPLC column as followed: The separation of MNF was accomplished by HPLC (Shimadzu Prominence HPLC system; Shimadzu, Columbia, MD, USA) followed by LC-MS/MS. In brief, the assays were conducted using an Eclipse XDB-C 18 guard column (4.6 mm x 12.5 mm) and an Atlantis HILIC analytical column (150 x 2.1 mm ID, 5 µm). The mobile phase consisted of water containing 0.1% formic acid as component A and acetonitrile as component B. A linear gradient was run as follows: 0 min 95% B; 5 min 60% B; 6 min 80% B; 10 min 95% B at a flow rate of 1.0 ml/min. The total run time was 20 min per sample. The calibration curve was prepared in a similar fashion as the samples, with 300-400 mg of tumor tissue spiked with a 0.5 serial dilution of MNF (300 to 9.375 ng/ml) and the addition of 9 µl acetonitrile containing 500 ng/ml of methoxyfenoterol.
Identification and quantification of the analytes was accomplished using a triple quadrupole API-4000 LC-MS/MS system equipped with Turbo Ion Spray® (TIS) (Applied Biosystems, Foster City, CA, USA). The data, which was acquired in positive electrospray ionization mode and multiple reaction monitoring (MRM), was analyzed with Analyst v.1.4.2 (Applied Biosystems). The standards were characterized using the following MRM transitions: MNF (369-200) and methoxyfenoterol (318-149) as the internal standard. Tumor tissues from vehicle-injected mice were used as negative controls. The TIS instrumental source settings for temperature, curtain gas, ion source gas 1 (nebulizer), ion source gas 2 (turbo ion spray), entrance potential and ion spray voltage were 500 °C, 10 psi, 60 psi, 60 psi, 10V and 5500 V, respectively. The TIS compound parameter settings for declustering potential, collision energy, and collision cell exit potential were 70V, 30V, 12V for MNF; 95V, 26V and 12V for methoxyfenoterol Analysis of gene expression in rat C6 glioma xenografts. Total cellular RNA from rat C6 glioma xenografts was extracted using an RNeasy plus mini kit (QIAGEN, Valencia, CA, USA), and its quality was assessed using an Agilent BioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, CA, USA). Transcriptional profiling was determined using Illumina Sentrix BeadChips (Illumina, San Diego, CA, USA). Total RNA was used to generate biotin-labeled cRNA with the Illumina TotalPrep RNA Amplification Kit. In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP. A total of 0.75µg of biotinlabeled cRNA was hybridized at 58 °C for 16 h to Illumina's Sentrix Rat Ref-12 Expression BeadChips.
The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3.
Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina's GenomeStudio software, version 1.6.1. For statistical analysis, the expression data were filtered to include only probes with a consistent signal on each chip and an Illumina detecton p value < 0.02.