Time-dependent inhibition of U87 and U373 cell proliferation induced by CBD. Cells were cultured in serum-free medium in the absence (□) or presence (•) of 25 μM CBD, added at a day “0,” for the time indicated. A and B, MTT and trypan blue tests on U87 glioma cell line; C and D, MTT and trypan blue tests on U373 glioma cell line. Results correspond to three different experiments and values are expressed as mean (O.D. or number of cells) ± S.E.M. ★, p < 0.05; ★★, p < 0.01; ★★★, p < 0.001 versus untreated cells (□), Dunnett's t test.
Effect of concomitant treatment of U87 and U373 glioma cells cultured in serum-free medium with a combination of the selective antagonists 0.5 μM SR1, 0.5 μM SR2, and/or 0.625 μM CPZ upon the sensitivity to the antiproliferative effects of CBD (MTT test). Cells were treated for 24 h or 4 days with 25 μM CBD and the indicated antagonist compounds as reported in the figure. Results correspond to at least three different experiments and values are expressed as mean O.D. ± S.E.M ★★★, p < 0.001; ★★, p < 0.01, versus untreated cells (control); †, p < 0.05 versus 25 μM CBD, Dunnett's t test.
Absence of an effect of PTX on CBD-induced antiproliferation. U87 and U373 cells were preincubated for 18 h with 100 ng/ml PTX and treated with different concentrations of CBD for 24 h. Viability was determined by MTT test. Results correspond to three different experiments and values (O.D.) are expressed as mean ± S.E.M.
CBD-induced apoptosis in U87 glioma cells. Cultures were grown in either serum-free medium alone (control) or in a medium containing 10 μM or 25 μM CBD. After 24 h of exposure, cells were detached, centrifuged, resuspended, and incubated with PI solution, and apoptosis was quantified as reduced fluorescence by flow cytometry on the total cell population (adhering + detached cells). A representative experiment is shown in the figure. Y values represent the relative cell number and X values represent the DNA content (PI fluorescence). Numbers on the graphs represent the percentage of apoptotic cells. Histograms represent the mean values ± S.E.M. of the percentage of apoptotic cells obtained in four independent experiments. ★★★, p < 0.001 versus control (C), Student's t test. Similar results were obtained with U373 glioma cells (data not shown).
ELISA-ssDNA monoclonal antibody detection of apoptosis induced by CBD on glioma cells. Cells were untreated (C) or exposed to CBD (10 μM and 25 μM) for 24 h, as previously described (see Materials and Methods). Cells treated with hyperthermia (HT; 56°C for 1 h, followed by an incubation at 37°C for 1 h) were used as necrotic cells-negative control of apoptosis. Data are expressed as O.D. (405 nm) and represent the mean ± S.E.M of at least three experiments. ★★, p < 0.01; ★★★, p < 0.001 versus control (C), Student's t test.
Effect of increasing concentrations of α-tocopherol upon the antiproliferative effect of 25 μM CBD in U87 glioma cells. Cell viability was determined by MTT assay after 24 h of treatment. Results correspond to three different experiments, and values are expressed as mean O.D ± S.E.M. ★, p < 0.05; ★★, p < 0.01, versus untreated cells (control); OO, p < 0.01 versus CBD alone, Dunnett's t test.
CBD effect on subcutaneous U87 glioma cell growth. CBD (0.5 mg/mouse) administration began 7 days after U87 cell inoculation into the left flank of the athymic nude mice (day 0 of the treatment). CBD was injected in the peritumoral area once a day, 5 days per week. Tumor diameters were measured twice a week. Results represent the mean of seven mice in each group and are expressed as mean volume ± S.E.M. ★, p < 0.05; ★★, p < 0.01, versus control mice; Mann-Whitney nonparametric test. On the right of the graph is reported an example of s.c. gliomas after dissection. Tumors were grown in the presence of vehicle (control) or CBD after 23 days of treatment.