Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

Figure 1 Hypoxia induced regulation of N-Myc down-regulated gene 1 in human brain cancer hypoxia-inducible factor-1α induced regulation of hypoxia induced N-Myc down-regulated gene 1 expression in human tumour cells. A: Under normoxic oxygenation conditions in the tumor cell microenvironment, hypoxia-inducible factor (HIF)-1α is rapidly degraded via the von Hippel-Lindau tumour suppressor gene product (pVHL)-mediated ubiquitin proteasome pathway; B: When the tumor environment aeration conditions shifts from normoxic to hypoxic aeration conditions, HIF-1α subunit becomes stable, translocates into the cellular nucleus and interacts with co-activators of which its transcription machinery is consisted such as p300/CBP to modulate the transcriptional activity of numerous hypoxia inducible genes, like N-Myc down-regulated gene 1 (NDRG1) in the case and about 61 other hypoxia induced genes[28,40]. HRE: Hypoxia response element.

Figure 2 Detection and experimental monitoring of hypoxia induced N-Myc down-regulated gene 1 in specimens of human brain cancer. The experimental monitoring approach includes (A) experimental approach detection of the experimental results. Here, the tumor cell lines are first cultivated in vitro and subsequently transfected with the N-Myc down-regulated gene 1 (NDRG1) short dsRNA oligonucleotides (siRNA) construct and treated with fixed O₂ concentration in the hypoxia chamber followed by the specimens extraction, quantification, quality control and molecular seperation of tumor cells specimens. Further protein or mRNA blotting and Hybridization with subsequent NDRG1 expression image detection and documentation take place. The experimental approach with these different stages are repeated at least three times to have statistical significant results that are necessary for evaluation of the experimental results (B), where first the films with the detected results are scanned followed by the detection of the genes expression (which is in the case NDRG1 gene and the house keeping genes or loading controls (β-actin and 18s RNA, respectively) measurement of intensities in the analyzed specimens, statistical analysis and evaluation of the obtained results.

Figure 3 Inhibition of N-Myc down-regulated gene 1 protein and mRNA expression in U373 human glioblastoma cell line in vitro via short dsRNA oligonucleotides- and iodoacetate-mediated interference into human tumor cellular glycolysis process. Upper panel: Western blotting analysis result diagram related to the specific inhibition of hypoxia induced expression in human brain tumor cells (U373 Glioblastoma cell line as an example) on protein level via Western blotting analysis. Clear complete inhibition of N-Myc down-regulated gene 1 (NDRG1) induced by extreme hypoxic conditions in the tumor microenviroment (0.1% O₂/for 24 h) was present upon transfection with the one of the two variants of NDRG1 short dsRNA oligonucleotides (siRNA) construct applied in these experiments when compared to the nearly complete inhibition via inhibitive interaction with the tumor cell glycolysis pathway. β-actin served as a loading control. Figure shows one representative experiment out of three experiments; Lower pannel: Northern blotting analysis displaying the specific inhibition of hypoxia induced NDRG1 mRNA expression in human brain tumor cells (U373 Glioblastoma cell line as an example) on mRNA level. Complete inhibition of NDRG1 induced by extreme hypoxic conditions in the tumor microenviroment (0.1% O₂/for 24 h) was could be achieved upon transfection with the one of the two variants of NDRG1 siRNA construct applied in these experiments. Inhibitive interaction into the glycolysis pathway due to the parallel treatment with 50 μmol/L with iodoacetate for 24 h showed a similar functional effect. The 18s RNA fragment with a molecular weight of 1.9 kb served as a loading control. This is one representative experiment out of three experiments.

Figure 4 Expression of N-Myc down-regulated gene 1 mRNA in human brain cancer tissue in vivo. NDRG1: N-Myc down-regulated gene 1; NB: Nonneoplastic brain; LGA: Low-grade astrocytoma; GBM: Glioblastoma.