Article Figures & Data
Figures
- Figure 1.
CBD is the most effective inhibitor of invasiveness and Id-1 expression in MDA-MD231 cells. A, the Boyden chamber invasion assay was used to determine the effects of cannabinoids on the invasiveness of aggressive human breast cancer MDA-MB231 cells. Compounds were added at concentrations of 0.1, 1.0, or 1.5 μmol/L. Data are presented as relative invasiveness of the cells through the Matrigel, where the respective controls are set as 100%. B, proteins from MDA-MB231 cells treated with vehicle (control), 0.1, 1.0, or 1.5 μmol/L of CBD for 3 d were extracted and analyzed for Id-1 by Western blot analysis as described in Materials and Methods. C, proteins from MDA-MB231 cells treated with additional cannabinoids for 3 d were extracted and analyzed for Id-1 by Western blot analysis. Normalization was carried out by stripping the blots and reprobing with a monoclonal antitubulin antibody. Densitometry readings of the blots were taken and the percentage of relative expression was calculated as the expression of Id-1 in the treated cells / vehicle cells × 100. D, the inhibitory effect of 1.5 μmol/L of CBD on Id-1 expression was compared over a time course of 1, 2, and 3 d. Columns, mean of at least three replicates; bars, SE. Data were compared using a one-way ANOVA with a corresponding Dunnett's post hoc test. *, P < 0.05, statistically significant differences from control.
- Figure 2.
CBD reduces invasion as well as Id-1 expression in MDA-MD436 cells. A, the Boyden chamber invasion assay was used to determine the effects of CBD on the invasiveness of human breast cancer MDA-MB436 cells. Data are presented as relative invasiveness of the cells through the Matrigel, where the respective controls are set as 100%. B, proteins from MDA-MB436 cells treated with vehicle (control) or 2.0 μmol/L of CBD for 3 d were extracted and analyzed for Id-1 by Western blot analysis. Normalization was carried out by stripping the blots and reprobing with a monoclonal antitubulin antibody. C, densitometry readings of the blots were taken from three independent experiments and the percentage of relative expression was calculated as the expression of Id-1 in the treated cells / vehicle cells × 100. *, P < 0.05, statistically significant differences from control.
- Figure 3.
CBD inhibits the expression of Id-1 gene at the mRNA and promoter levels in MDA-MB231 and MDA-MB436 cells. A, the inhibition of the Id-1 gene product (434 bp) by CBD was investigated in MDA-MB231 cells using reverse transcription-PCR. Expression of the β-actin gene product (232 bp) was used as a control. B, luciferase activity in MDA-MB231 cells transiently transfected with Id-1-sbsluc was determined in the presence of vehicle (control) or 1.5 μmol/L of CBD. Cells were treated for 2 d and luciferase activity was measured. C, cells were treated for 3 d. For both B and C, all values were normalized for the amount of β-gal activity present in the cell extracts. Columns, mean of at least three replicates; bars, SE. The data are represented as percentage of activity of the treated cells / vehicle cells × 100. Data were compared using the unpaired Student's t test. *, P < 0.05, statistically significant differences from control. D, the inhibition of the Id-1 gene product by CBD was investigated in MDA-MB436 cells using reverse transcription-PCR. Expression of the β-actin gene product was used as a control. E, luciferase activity in MDA-MB436 cells transiently transfected with Id-1-sbsluc was determined in the presence of vehicle (control) or 2 μmol/L of CBD. Cells were treated for 2 d and luciferase activity was measured.
- Figure 4.
Ectopic expression of Id-1 blocks the effect of CBD on MDA-MB231 invasiveness. A, data are presented as relative invasiveness of control MDA-MB231 cells (−Id-1) and of MDA-MB231 cells that ectopically expressed Id-1 (+Id-1) after a 2-d treatment with vehicle (control) or 1.5 μmol/L of CBD (CBD), and then an overnight invasion assay. The respective controls are set as 100%; columns, mean of at least three replicates; bars, SE. Data were compared using the unpaired Student's t test. *, P < 0.05, statistically significant differences from control. B, the inhibitory effect of CBD on Id-1 expression in −Id-1 and +Id-1 MDA-MB231 cells was compared using Western analysis. Equal loading was confirmed by stripping the blots and reprobing with a monoclonal antitubulin antibody.
Tables
- Table 1.
Antiproliferative potencies of cannabinoids during a 3-d treatment of MDA-MB231 and MDA-MB436 breast cancer cells
Compound MDA-MB231 MDA-MB436 
Δ9-THC 1.2 (1.0–1.4) 2.5 (1.8–3.4) CBN 1.2 (0.9–1.5) 2.6 (1.8–3.7) WIN55,212-2 1.7 (1.5–2.2) 2.4 (1.6–3.4) CP55,940 2.5 (1.5–4.1) 1.3 (0.7–1.6) CBD 1.3 (1.0–1.9) 1.6 (1.1–2.2) CBG 2.3 (2.1–2.5) 2.1 (1.5–3.0) NOTE: Cells were treated with cannabinoid compounds for 3 d and the IC50 values for the antiproliferative effects of the compounds were calculated. Data are the means and corresponding 95% confidence limits of at least three experiments. IC50 values are reported in μmol/L.
Volume 6, Issue 11
