Fig 1.
R. graveolens extract induces cell death of U87MG human glioma cells.
(A) MTT assay on proliferating U87MG human glioma cells treated with vehicle (♦) or with 1mg/ml R. graveolens a.e. (■) for 24, 48, 72, 96h, *p<0,01 vs control conditions. (B) Trypan blue exclusion test on U87MG glioma cells treated (light gray) or not (dark gray) with 1mg/ml R. graveolens a.e. for 24, 48 and 72h; *<0,01 vs control conditions.
Fig 2.
R. graveolens a.e. induces cell death in proliferating but not in differentiated A1 cells.
(A) Microphotographs of the mouse mesencephalic embryonic cell line A1 mes c-myc (A1). They are proliferating/undifferentiated in the presence of serum (left panel) but acquire a neuronal phenotype upon serum withdrawal and stimulation with cAMP (right panel). (B) MTT assay on proliferating A1 cells in control conditions (♦) or treated with 1mg/ml R. graveolens a.e. (■) for 24, 48, 72 and 96 hours. *p<0.01 vs controls. (C) MTT assay on differentiated A1 cells in control conditions (♦) or treated with 1mg/ml R. graveolens a.e. (■) for 48 and 72 hours *p<0.01 vs controls. (D) Trypan blue exclusion test on proliferating A1 cells treated (light grey) or not (dark grey) with 1mg/ml R. graveolens a.e. for 24, 48 and 72h; *p<0.01 vs controls. (E) Trypan blue exclusion test on differentiated A1 cells treated (light gray) or not (dark gray) with 1mg/ml R. graveolens a.e. for 48 and 72h; *p<0.01 vs controls.
Fig 3.
Temozolomide and cisplatin affect viability of proliferating and differentiated A1.
(A-B) MTT assay on proliferating A1 cells treated with increasing concentrations of temozolomide (A) and cisplatin (B) for 48 (♦) and 72 (■) hours; *p<0.01 vs controls; (C-D) MTT assay on differentiated A1 cells treated with increasing concentrations of temozolomide (C) and cisplatin (D) for 48 (♦) and 72 (■) hours; *p<0.01 vs controls.
Fig 4.
R. graveolens a.e. is able to induce ERK1/2 phosphorylation in glioma and in A1 proliferating neural cells.
Western blotting detection of p-ERK1/2 and ERK1/2 proteins in C6 glioma cells (A), in U138 glioma cells (B), in A1 cells (C) and in U87MG (D) treated with 1mg/ml R. graveolens a.e. for 5, 10, 30 and 60 minutes. Two specific bands are observed respectively at 44 and 42 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-ERK1/2 and ERK1/2 in the different cell lines. Data are expressed as ratios of p-ERK/ERK. Asterisks represent p<0.05 vs controls. (G-H) MTT assay in U87MG cells (E), A1 cells (F), C6 glioma cells (G) and U138 cells (H) treated for 48 hours with 1mg/ml R. graveolens a.e. (R48), 10μM PD98059 (PD) or in combination (PD+R48); *p<0.01 vs control conditions.°p<0.05 vs R48.
Fig 5.
R. graveolens a.e. is able to induce AKT phosphorylation in glioma and proliferating neural cells.
(A) Western blotting detection of p-AKT and AKT proteins in U87MG (A), in C6 glioma cells (B) and in A1 cells (C) treated with 1mg/ml R. graveolens a.e. for 5, 10, 30 and 60 minutes. A specific band is observed at 60 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-AKT and AKT in the different cell lines. Data are expressed as ratios of p-AKT/AKT. Asterisks represent p<0.05 vs controls. (D-E) MTT assay in U87MG cells (D), C6 glioma cells (E) and A1 cells (F) treated for 48 hours with 1mg/ml R. graveolens a.e. (R48) or with 1μM wortmannin (W), or in combination (W+R48); *p<0.01 vs control conditions.°p<0,05 vs R48.
Fig 6.
R. graveolens a.e. induces apoptosis in A1 cells.
(A) Cell cycle was analyzed by means of Tali image-based cytometry on proliferating A1 cells in control conditions (dark grey) and 48h after 1mg/ml R. graveolens a.e. treatment (light grey) *p<0.01 vs controls (B) Number of apoptotic nuclei/100 cells treated (R48) or not (CTRL) with 1 mg/ml R. graveolens a.e. for 48 hours. *p<0.01 vs controls. (C-E) Caspase 3 activity expressed as absorbance at 400 nm in A1 cells (C), U87MG cells (D) and C6 cells (E) treated with vehicle (CTRL), 1mg/ml R. graveolens a.e. for 24 (R24) or 48 (R48) hours, 10μM PD98059 in combination with ml R. graveolens a.e. for 48 hours (PD+R48), 1μM wortmannin in combination with ml R. graveolens a.e. for 48 hours (W+R48) or the combination of the two inhibitors (PD+W+R48) for 48 hours. *p<0.01 vs control conditions.
Fig 7.
Rutin does not influence proliferating A1 cells viability.
Trypan blue exclusion test on proliferating A1 cells treated with or without (control) increasing concentrations of rutin (3μg/ml, 30μg/ml and 300μg/ml) for 24, 48 and 72h.