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

ABSTRACT: (R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice. Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (2mg/kg) for five days per week for16 days. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p<0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative RT-PCR and immunoblot analyses. Significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (Bub1b), cyclin-dependent kinase inhibitor 3 (Cdkn3) and cyclin A2 (Ccna2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (Olig1) and SRY-box 4 (Sox4) was observed in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cancer in vivo was accompanied by marked reduction in the expression of cell cycle regulator proteins, including cyclin A and cyclin D1, in tumor extracts. This study is the first demonstration of MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and may offer a potential mechanism for its anticancer action in vivo. Keywords: fenoterol derivative; C6 glioma; tumor xenografts; microarray analysis;

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE45307 | GEO | 2015/08/06

SECONDARY ACCESSION(S): PRJNA193439

REPOSITORIES: GEO

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Project description:(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice. Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (2mg/kg) for five days per week for16 days. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p<0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative RT-PCR and immunoblot analyses. Significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (Bub1b), cyclin-dependent kinase inhibitor 3 (Cdkn3) and cyclin A2 (Ccna2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (Olig1) and SRY-box 4 (Sox4) was observed in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cancer in vivo was accompanied by marked reduction in the expression of cell cycle regulator proteins, including cyclin A and cyclin D1, in tumor extracts. This study is the first demonstration of MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and may offer a potential mechanism for its anticancer action in vivo. Keywords: fenoterol derivative; C6 glioma; tumor xenografts; microarray analysis; Total RNA was isolated from rat C6 glioma xenografts harvested from vehicle and MNF-treated nude mice (n = 3 per group, cohort 1). This analysis was repeated in a second cohort of animals (n = 3 per group, cohort 2). Total cellular RNA was extracted using an RNeasy plus mini kit (QIAGEN, Valencia, CA), and its quality was assessed using an Agilent BioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, CA). Transcriptional profiling was determined using Illumina Sentrix BeadChips (Illumina, San Diego, CA). 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 biotin-labeled 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 detection p value < 0.02.

Project description:Murine BV2 microglia cells were transfected either with siRNA negative control or siRNA against caspase-3 for 48h. Later on some the of the BV2 transfected cells were co-cultured with C6 glioma cells during 6h. We used the SA Biosciences Mouse Wound Healing PCR Array (PAMM-121Z) to quantitate gene expression of relevant genes related to the wound healing process Glioma cells recruit and exploit microglia, resident immune cells of the brain, for their proliferation and invasion capability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype remains elusive. Here, we report that glioma-induced microglia conversion is coupled to a reduction of basal microglial caspase-3 activity, increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and demonstrate that caspase-3 inhibition regulates microglial tumor-supporting function. Further, we identified nitric oxide synthase-2 (NOS2) activity originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to reduction in both microglia recruitment and tumor expansion, whereas depletion of the microglial caspase-3 gene promoted tumor growth. This study provides evidence that the inhibition of Trx2-mediated denitrosylation of SNO-procaspase-3 is part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells. qPCR gene expression profiling. Three independent experiments of siControl BV2 monoculture, siCaspase3 BV2 monoculture, siControl BV2 cocultured 6h with C6 glioma cells and siCaspase3 BV2 cocultured 6h with C6 glioma cells. Equal amount total RNA from each culture was used for the gene expression analysis. Please note that the raw data for three independent experiments (prior to averaging the data) is provided in the 'Raw_Data_File_with_the_Ct_values_for_3_indep_experiments.txt'.

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

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