Article Figures & Data
Figures
- Figure 1.
CB1 expression in tposRMS cells. A, gene expression values of CB1 are shown in arbitrary units. Samples analyzed by microarray gene expression profiling were translocation-negative (tnegRMS) versus translocation-positive (tposRMS) biopsy samples. B, quantitative and normal RT-PCR with primers for CB1 and for GAPDH were done with cDNA of cell lines MRC-5 (fibroblast); RD (tnegRMS); and Rh4, Rh28, and RMS13 (all tposRMS) cells. Quantitative results are indicated in arbitrary units. C, CB1 protein levels of MRC-5, RD, and Rh4 cells were determined by Western blotting. D, confocal images of immunofluorescence stainings with anti-CB1 antibody (red fluorescence) on RD and Rh4 cells (scale bar, 100 μm).
- Figure 2.
Cannabinoids reduce viability of tposRMS cells. A, cell lines Rh4, Rh28 (tposRMS), RD (tnegRMS), and MRC-5 (fibroblasts) were incubated with increasing concentrations of HU210 for 72 h. Subsequent viability measurements by means of MTT are shown (n = 3, ±SE; significance at 1.25 μmol/L: P < 0.005). B, viability measurements are shown for Rh4 cells preincubated with vehicle or with 0.5 μmol/L AM251 before undergoing subsequent treatment with 1 μmol/L HU210 for 24 h (n = 3, ±SE, significance: P < 0.05). C and D, dose-dependent viability of Rh4, RD, and MRC-5 cells after treatment with THC for 24 h or Met-F-AEA for 48 h was measured (n = 3, ±SE, significance at 5 μmol/L THC and 10 μmol/L Met-F-AEA: P < 0.05).
- Figure 3.
Cannabinoids induce apoptosis in tposRMS cells. A, Rh4 cells were treated for 6, 16, 24, and 48 h with either 1.25 μmol/L HU210 or DMSO. Rh28 and Rh4 cell were incubated with increasing concentrations of HU210 for 24 h (bottom). Subsequently, Western blotting was done with an anti-PARP antibody. B, after preincubation of Rh4 cells with 0.5 μmol/L of CB1 antagonist AM251, HU210 was added at concentrations of 1 and 1.25 μmol/L HU210 for 20 h. Cell lysates were probed with anti-PARP (top) and anti-actin (bottom) by immunoblotting. C, densitometric quantification of the ratio of cleaved to uncleaved PARP product (values ± SE, n = 2). D, percentage of cells staining positively for proapoptotic caspase-3 is shown as evaluated after 20 h of HU210 treatment of Rh4 cells (values ± SE, n = 3, significance P < 0.005). Rh4 cells were either treated with 2 and 4 μmol/L of THC (E) or 5 and 10 μmol/L of Met-F-AEA (F) for 24 h. Protein extract was analyzed for PARP and actin by Western blotting (here shown a representative blot).
- Figure 4.
Cannabinoid receptor agonists affect AKT and ERK signaling in tposRMS cells. A, Rh4 cells were incubated with 1.25 μmol/L HU210 for 30 min, 1 h, and 2 h, and cell lysates were prepared. Then, Western blotting was done with antibodies against phospho-AKT (Ser473; top left) and against phospho-ERK (bottom left). Anti–phospho-GSK (bottom right) and anti–phospho-AKT (Thr308; top right) were probed on extracts of cells treated for 2h with 1.25 μmol/L of HU210. B, phosphorylation status of AKT at Ser473 was analyzed by Western blotting after treatment of Rh4 cells with 2 and 4 μmol/L THC (top) or 5 and 10 μmol/L Met-F-AEA (bottom) for 24 h.
- Figure 5.
Induction of proapoptotic p8. A, Rh4 cells were treated with 0.5 and 1 μmol/L of HU210, 2.5 and 3.5 μmol/L of THC (B), or 5 and 10 μmol/L of Met-F-AEA for 16 h. RNA was extracted and analyzed for p8 transcripts with quantitative RT-PCR (n = 3, ±SE, P < 0.05). Values were normalized to GAPDH. B, p8 was down-regulated by means of siRNA. Top, a representative RT-PCR and quantitative values (in arbitrary units, normalized to scrambled siRNA transfected control cells). Viability after HU210 (1.25 μmol/L) treatment was assessed at 48 h with MTT (n = 3, ±SE, P < 0.05).
- Figure 6.
HU210 reduces tumor growth in vivo. NOG mice were injected with 7.5 × 106 tposRMS (Rh4) cells s.c. into the flank. After reaching a tumor size of 100 to 150 mm3, animals were assigned randomly to either the vehicle (n = 7) or the HU210 (n = 6) group. Treatment was given daily by injecting either 0.2 mg/kg HU210 or DMSO in PBS peritumorally for 13 d, whereas tumor growth was monitored daily and mice were sacrificed on the day after the last treatment. A, tumor growth over time is shown for HU210-treated compared with vehicle-treated animals (±SE, P < 0.001). B and C, representative sections of tumors from vehicle- and HU210-treated animals were stained with H&E. Original magnification, 100×.
Volume 8, Issue 7
