Article Figures & Data
Figures
- Figure 1.
Cannabidiol (CBD) enhances the inhibitory effects of Δ9-THC on glioblastoma cell growth. To test for positive and negative interactions, a 2 × 2 factorial design using specific μmol/L concentrations of drug was used as described in Materials and Methods. Cell proliferation was measured using the MTT assay. SF126 (A) and U251 (B) cells were treated for 3 d with vehicle/no drug, Δ9-THC, cannabidiol, or a combination of Δ9-THC and cannabidiol. Concentrations of Δ9-THC and cannabidiol that produce only minimal effects on cell proliferation were also tested in 2 × 2 factorial design in SF126 (C) and U251 (D) cells. Percent control was calculated as the MTT product absorbance in the treated cells/control cells ×100. Data are the mean of at least three independent experiments; bars, ± SE. Data were analyzed using two-way ANOVA (GraphPad Prism). *, statistically significant interaction (P < 0.01). D, inset, representative light microscope image of the effects of the combination treatment on U251 cells (×40).
- Figure 2.
Δ9-THC in combination with cannabidiol does not produce a greater overall inhibition of glioma invasiveness. To test for positive and negative interactions, a 2 × 2 factorial design was used as described in Materials and Methods. The Boyden chamber invasion assay was used to determine the effects of treatment on the invasiveness of U251 cells. U251 cells were treated for 3 d with Δ9-THC (0.1 μmol/L), cannabidiol (0.1 μmol/L), or a combination of Δ9-THC (0.1 μmol/L) and cannabidiol (0.1 μmol/L). Data are presented as relative invasiveness of the cells through the Matrigel, where the respective controls are set as 100%. Data are the mean of at least three independent experiments; bars, ± SE. *, statistically significant differences from control (P < 0.05).
- Figure 3.
The combination treatment of Δ9-THC and cannabidiol specifically inhibits ERK activity. The effects of cannabinoids on mitogen-activated protein kinases were analyzed using Western analysis. A, U251 cells were treated with vehicle or a combination of Δ9-THC (1.7 μmol/L) and cannabidiol (0.4 μmol/L) for 3 d. Proteins were then extracted and analyzed for pERK, total ERK, pJNK1/2, and pP38 MAPK. B, U251 cells were treated with Δ9-THC (1.7 μmol/L) or cannabidiol (0.4 μmol/L) alone for 3 d and analyzed for pERK and total ERK. C, U251 cells were treated with vehicle or a combination of Δ9-THC (1.7 μmol/L) and cannabidiol (0.4 μmol/L) for 1 and 2 d. D, SF126 cells were treated with vehicle or a combination of Δ9-THC (1.6 μmol/L) and cannabidiol (1.1 μmol/L) for 12 h or 1 d. Either α-tubulin or β-actin was used as a loading control (LC). Blots are representative of at least three independent experiments.
- Figure 4.
The effects of the combination treatment are the result of CB2 receptoractivation. The number of U251 cells positive for annexin (apoptosis) staining after 3 d treatment was measured using FACS analysis. Cells were treated with A, Δ9-THC (1.7 μmol/L), cannabidiol (0.4 μmol/L), or a combination of Δ9-THC (1.7 μmol/L) and cannabidiol (0.4 μmol/L) denoted as THC/CBD; B, a combination of Δ9-THC (1.7 μmol/L) and cannabidiol (0.4 μmol/L) denoted as THC/CBD; C, 2.5 μmol/L Δ9-THC; and D, 2.0 μmol/L cannabidiol. In B, C, and D, cells were also treated in the presence of 0.5 μmol/L of the CB1 antagonist SR141716A (SR1), 0.5 μmol/L of the CB2 antagonist SR144528 (SR2), or 20 μmol/L TCP. Percent control was calculated as positive annexin staining of the treated cells minus control cells. Data are the mean of at least three independent experiments; bars, ± SE. Data were compared using one-way ANOVA with Bonferroni's multiple comparison posthoc analyses. *, statistically significant differences from control (P < 0.05); #, statistically significant differences from the combination treatment of THC/CBD (P < 0.05).
- Figure 5.
When combined, Δ9-THC and cannabidiol produce an increase in activation of p8 and multiple caspases. The effects of cannabinoids on p8 and caspase expression were analyzed using semiquantitative reverse transcriptase-PCR and Western analysis, respectively. RNA and protein were collected from U251 cells treated for 3 d with cannabidiol (0.4 μmol/L), Δ9-THC (1.7 μmol/L), or a combination of Δ9-THC (1.7 μmol/L) and cannabidiol (0.4 μmol/L). A, reverse transcriptase-PCR was run on RNA extracted from control-treated and Δ9-THC/cannabidiol-treated samples. Expression of the β-actin gene product was used as a control for equal loading. B, data are represented as percentage p8 expression of the treated cells/control cells ×100, and all values were normalized against β-actin. Blots and PCR reactions are representative of at least three independent experiments. Data were compared using one-way ANOVA with Dunnett's multiple comparison posthoc analyses. *, statistically significant differences from control (P < 0.05). C, proteins were extracted from treated cells and analyzed for cleaved caspase 3, 7, 9, and PARP expression.
Tables
- Table 1.
Cannabinoid modulation of cell cycle
Treatment, μmol/L Mean (G0-G1) Mean (S) Mean (G2-GM) CBD 0.4 1.08* ± 0.03 0.90 ± 0.05 0.97 ± 0.28 Δ9-THC 1.7 1.12* ± 0.04 0.78 ± 0.11 1.28 ± 0.37 Δ9-THC 1.7/CBD 0.4 1.23*,† ± 0.02 0.49*,† ± 0.08 2.69*,† ± 0.56 NOTE: Cell cycle was measured using PI staining and FACS analysis, and Modfit was used to determine the percentage of cells in G0-G1, S, and G2-GM phase. U251 cells were treated for 3 d with CBD (0.4 μmol/L), Δ9-THC (1.7 μmol/L), or a combination of CBD (0.4 μmol/L) and Δ9-THC (1.7 μmol/L). The percentage of cells in each compartment was standardized by dividing it by the average percentage for the vehicle. This procedure was carried out for data from each experiment on each day. Statistical analysis was done as described in the Material and Methods.
Abbreviation: CBD, cannabidiol.
Supplementary Data, Marcu et al.,
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