Figure 1.
Dexamethasone promotes glioma cell death and growth inhibition.
Different rodent and murine glioma cells (F98; RG2 and GL261) were treated with various concentrations of dexamethasone (DEXA) and proliferation was determined. (A), DEXA treatment of F98 glioma cells for 24 hs (left) and 48 hs (right). After the indicated time points, cell growth was determined. (B), DEXA treatment for duration of 48 hs in rodent RG2 glioma cells (left) and in murine GL261 glioma cells (right). Note, that low DEXA levels from 1 μg/ml onwards already reduce glioma proliferation in RG2 and GL261 cells. (C) Representative images of glioma cells treated with DEXA. Propidium iodide (PI) staining reveals dead cells (white dots, upper panel). Note the increase in PI-positive cells and reduced cell number under white light (WL) view in the groups treated with low or high dexamethasone. Scale bar represents 200 μm. (D) Dexamethasone does not alter the proliferation of rat primary astrocytes. DEXA was tested at concentrations ranging from 1 μg/ml to 200 μg/ml. Determination of cell death assays was repeated at least three times. Values are given as mean ± S.D. Quantification is given for n = 12. Statistical analysis was performed with Student's t-test (two-sided), asterisks indicate p-values <0.05 which were considered significantly different from control groups (set as 100%). Abbreviation: DEXA, Dexamethasone; PI, propidium iodide; WL, white light.
Figure 2.
Dexamethasone acts differentially on human glioma cells.
(A), Dexamethasone (DEXA) promotes human T98G and U251 glioma growth. Human T98G and U251 glioma cells were cultured at various concentrations of DEXA and cell viability was determined with the MTT assay. (B), Dexamethasone reduces human U87 glioma cell growth solely at high concentrations. Right, representative images of U87 glioma cells treated with low and high dosage of DEXA. Scale bar represents 200 μm. (C), Human embryonic kidney cells are growth sensitive to increasing DEXA concentration. (D), Human primary astrocytes are resistant to various DEXA concentrations. Cell growth is given in relation to untreated controls. Quantification is given for n = 12. Values are given as mean ± S.D. with controls set as 100%. Differences were considered statistically significant with p<0.05 (asterisks, two-sided Student's t-test).
Figure 3.
Dexamethasone does not affect cell viability of primary neurons.
(A), Dexamethasone (DEXA) was applied to rodent hippocampal neurons for 48 hs and cell death was determined by PI staining (PI, propidium iodide, left column, Scale bar represents 200 μm). Right, white light microscopy indicates morphological integrity of neurons following DEXA treatment. Scale bar 100 μm (B), Neurons were cultured at various concentrations of DEXA for two days and cell viability was determined by measuring the PI signal intensity.
Figure 4.
Dexamethasone alters the cell cycle profile of glioma cells.
(A), Dexamethasone (DEXA) was applied to DEXA-sensitive F98 glioma cells for 48 hs and cell death was determined by PI staining and apoptosis was evaluated by HOECHST staining. Arrows indicate apoptotic nuclei defined as fragmented or irregular shaped. Scale bar represents 200 μm (B), Cell cycle analysis in rat glioma cells (F98) and following DEXA treatment at various concentrations. Representative profile graphs are shown on the left. Pie charts on the right show percentage of cells in different cell cycle phases (n = 3).
Figure 5.
Dexamethasone differentially impacts stress responses in rat and human gliomas.
(A), qRT-PCR analysis of F98 glioma cells treated with dexamethasone (DEXA) for 6 hs. DEXA induces upregulation of xCT and VEGFA expression in rat F98 gliomas. Quantification of RT-PCR results is given showing relative xCT mRNA (left) and VEGFA mRNA (right) values after DEXA treatment [0, 10, 100 and 200 μg/ml] in F98 glioma cells. (B) qRT-PCR analysis of DEXA-resistant U251 glioma cells. Note, that DEXA does not affect xCT mRNA expression in human U251 glioma cells. Quantification of the qRT-PCR results is depicted showing relative xCT mRNA level after DEXA treatment [0, 10, 100 and 200 μg/ml]. (C), Western blot analysis of xCT after DEXA treatment for 48 hs. Left, top, representative blot is given. Bottom, quantification of western blot (xCT/actin) showing that xCT is up-regulated in F98 glioma cells after DEXA treatment for 48 hs. Right, DEXA does not affect xCT expression in xCT silenced glioma cells. Top, Representative immunoblot of xCT in rat xCT knockdown gliomas after DEXA treatment. Bottom, quantification of western blot ratios (xCT/actin) after DEXA treatment for 48 hs. (D), Short-duration DEXA treatment enhances glutamate secretion in rat glioma cells. Glutamate concentration in condition medium 24 hs after DEXA application measured by ion-exchange chromatography with an amino acid analyzer reveals extracellular glutamate elevation after dexamethasone treatment. (E), siRNA mediated xCT knockdown increases glioma susceptibility to DEXA. DEXA treatment of control (GFP) transfected F98 glioma cells and xCT knockdown (si xCT) F98 cells. Two days after DEXA treatment, cell growth was determined. Means are given with quantification out of n≥3. Values are given as mean ± S.D. Control groups set as 100%. Differences were considered statistically significant with p<0.05 (asterisks, two-sided Student's t-test). Abbreviation: qRT-PCR, quantitative real-time reverse transcriptase PCR.
Figure 6.
Dexamethasone induces tumor cell death in organotypic brain tissue.
(A) Dexamethasone (DEXA) treatment induces tumor cell death in glioma cell implanted brain slices and reduces tumor-induced cell death. Top, F98 glioma cells were implanted in brain slices and after 8 days cell death was evaluated (propidium iodide positive cells are depicted in red). Bottom, Cell death quantification in untreated tumor-implanted brain tissue (control) reveals increased peritumoral cell death (peritumoral zone), whereas the tumor zone is spared of cell death. DEXA treatment reduces brain damage as revealed by reduced cell death in control cortical areas apart from the tumor, and in the peritumoral zones, while DEXA induces massive tumor cell death within the tumor core zone. Cell death intensity was quantified with NIH-Image J and for statistical analysis the t-test was applied. Means ± S.D. are given. *P<0.05, Student's t-test (two sided) (n = 12). The dashed circle marks the tumor core zone. Scale bar represents 1 mm. (B), DEXA treatment in native brain slices. Cell death quantification in untreated brain slices (control), and native brain slices with DEXA treatment at a concentration of 10 μg/ml, 100 μg/ml and 200 μg/ml. Scale bar represents 1 mm. (C), Assessment of cell death in native brain slices treated with DEXA. Cell death intensity was quantified with NIH-Image J and for statistical analysis the two tailed t-test was applied. Means ± S.D. are given. *P<0.05, Student's t-test (n≥3). (D); Glioma cells induced neuronal cell death. Tumor-implanted brain slices were stained for the neuronal markers NeuN (top, given in green) and NeuroTrace (bottom, depicted in blue) and cell death assessed by PI staining (red). Arrows showing that the dead cells (PI+) are mostly NeuN+ (green) or NeuroTrace+ (blue) neurons. Scale bar, 50 μm.
Figure 7.
Dexamethasone inhibits specifically tumor angiogenesis.
(A) DEXA treatment reduces tumor-induced vascular density. Top, tumor-implanted brain slices were analysed for vessels and morphological differences between tumor-implanted control groups (control) and dexamethasone treated groups were investigated. The dashed line designates the tumor core (top) from the peritumoral area (bottom). Vessel density in peritumoral area is reduced after 100 and 200 μg/ml DEXA treatment. Scale bar represents 100 μm. Bottom, quantitative analysis of vessel density in untreated controls and DEXA treated brain slices. Means are given with ± S.D. For statistical analysis the two tailed Student's t-test was applied and revealed *P<0.05 and n≥3. (B) DEXA treatment reduces newly formed tumor-induced vessels. Peritumoral area of tumor-implanted brain slices were analysed for vessels and differences in CD105 expression between tumor-implanted control groups (control) and dexamethasone treated groups were investigated. Laminin positive vessel structure is given in red (Left). CD105 positive endothelial cells are depicted in blue (middle). Merged images are shown on the right. Arrows indicate CD105 positive proliferating or newly formed endothelial cells. Scale bar represents 100 μm. (C) DEXA does not inhibit normal vessel growth. Native brain slices were treated with DEXA and vascular density was investigated. Quantitative analysis of vessel density in untreated brain slices (control) and DEXA treated brain slices. Scale bar, 50 μm. Means ± S.D. are given. For statistical analysis the two tailed Student's t-test was applied with *P<0.05 and n≥3.