Fig 1.
Glutamate secreted by LM7 cells was measured in the media from day 0 to day 7. U87 cells were used a positive control represented by triangles and HeLa cells were used as a negative control, shown as circles. LM7 cells are shown as squares. The experiment was repeated three times and the average concentrations are shown.
Table 1.
Cell Viability.
Fig 2.
Riluzole inhibits growth of LM7 and OS482 cells both in 0.5% and 10% serum.
A. Proliferation of LM7 cells was measured in the presence of 0.5% serum containing media. Riluzole was added at various concentrations as indicated for 72 hours. The blue bars represent total number of DAPI positive cells and the green bars represent total number of Ki-67 positive cells (P value <0.001 = ***). B. Proliferation of LM7 cells was measured in the presence of 10% serum containing media. Riluzole was added at various concentrations as indicated. The blue bars represent total number of DAPI positive cells and the green bars represent total number of Ki-67 positive cells (P value <0.001 = ***). C. Representative images of cells from each sample in 10% serum are shown. D. Proliferation of mouse osteosarcoma cells, OS482, was measured in the presence of 0.5% serum containing media. Riluzole was added at various concentrations as indicated for 72 hours. The blue bars represent total number of DAPI positive cells and the green bars represent total number of Ki-67 positive cells (P value <0.001 = ***). E. Proliferation of OS482 cells was measured in the presence of 10% serum containing media. Riluzole was added at various concentrations as indicated. The blue bars represent total number of DAPI positive cells and the green bars represent total number of Ki-67 positive cells (P value <0.001 = ***).
Fig 3.
Riluzole induces apoptosis in LM7 and OS482 cells.
LM7 cells were seeded and treated with Riluzole at various concentrations or DMSO for control for 72 hours. The cells were fixed and the TUNEL assay was performed. Images were captured and analyzed. A. Blue bars represent DAPI positive cells and red bars represent TUNEL positive cells. B. Percentage of TUNEL positive cells in each sample was calculated based in total number of DAPI positive cells for that sample (P value <0.001 = ***). C. Representative images of the TUNEL assay. D. OS482 were treated with various concentrations of Riluzole or DMSO for control and TUNEL assay was performed as in A. E Percentage of TUNEL positive cells in each sample was calculated for OS482 cells based in total number of DAPI positive cells for that sample (P value <0.001 = ***).
Fig 4.
Riluzole prevents migration of LM7 cells.
Scratch assay was performed to measure migration of LM7 cells. Cells were either treated with vehicle for control or treated with 100 μM Riluzole, scratched and images of the gap created were captured at 0 time, 8 and 24 hours using phase contrast inverted scope. A. Representative images of the scratch in samples at 0 h and 24 hr. B. The gap distance was measured from 3 scratches in each image at 0, 8 and 24 hours. C. The distance travelled by the cells in the images was measured at 8 and 24 hrs.
Fig 5.
Blocking metabotropic receptors inhibits LM7 growth.
A. Specific inhibitor of mGluR1, CPCCOEt (CCPD) 40 μM or mGluR5 inhibitor, Fenobam 300 μM, were used to measure total number of cell by DAPI staining. B. Proliferation was measured using Ki-67 staining after blocking with specific mGluR inhibitor. C. Apoptosis was performed using TUNEL in LM7 cells in the presence of specific mGluR1 or mGluR5 inhibitors. D. Percent apoptosis was calculated based on DAPI numbers for samples in Fig 5A. E. LM7 cells express mGluR5. Whole cell extracts from LM7 cells were analyzed by Western blot using mGluR5 antibodies. P value <0.01 = **, P value < 0.001 = ****.
Fig 6.
Riluzole alters phosphorylation status of AKT, ERK1/2 and JNK.
A. Western blotting was carried out using antibodies against Phospho AKT T308 or phospho AKT S473 or total AKT or phospho P70S6 Kinase or total P70S6 kinase. Relative densities of the western blots are shown in the right panel. P value<0.0001 = *** and NS = not significant. B Antibodies against Phospho ERK1/2, total ERK1/2, Phospho JNK1/2, and total JNK1/2 were tested. Tubulin was used as a loading control. Relative densities of the western blots are shown in the right panel. P value<0.0001 = *** and NS = not significant.
Fig 7.
Knockdown of mGluR5 slows growth of LM7 cells.
Colonies stained by Cresyl Violet are shown from WT, control ShRNA and mGluR5 KO samples. B. The number of colonies in each sample from three independent experiments. C. Western blot of whole cells extracts from WT, control ShRNA and mGluR5 KO using anti mGluR5 antibody. μ-Tubulin is used as a loading control.
Fig 8.
Schematic of mechanism of action of Riluzole.
Glutamate released from LM7 cells binds and activates mGluR receptor signaling that in turn induces the activation of genes involved in proliferation. Riluzole treatment inhibits glutamate release thereby preventing the activation of glutamate receptors. Similarly, blocking of mGluR receptors with Fenobam also prevents mGluR5 activation and signaling thereby blocking activation of proliferation genes.