Effect of cannabinoids and Cannabis extracts on the proliferation of some of the cell lines investigated in this study. MCF-7 cells (A), C6 cells (B), and DU-145 cells (C and D) were treated with increasing concentrations of cannabidiol, cannabinoids (C), and cannabidiol-rich extracts (daily added with each change of medium for 4 days). Effect on cell proliferation was measured by Crystal Violet vital staining. After staining, cells were lysated in 0.01% acetic acid and analyzed by spectrophotometric analysis (PerkinElmer Lambda 12, λ = 595 nm). Results are reported as percentage of inhibition of proliferation where optical density value from vehicle-treated cells was considered as 100% of proliferation and represent the mean ± S.E. of three different experiments. *, p < 0.05 versus cannabidiol pure by ANOVA followed by Bonferroni's test. E and F, effect of cannabidiol on nontumoral cell lines. Cells were treated with two different concentrations of cannabidiol for 4 (E) or 3 days (F), and vitality was evaluated by using trypan blue dye exclusion assay (see Materials and Methods). In cells treated with vehicle, mortality was always lower than 2%. Data are expressed as percentage of control and represent the mean ± S.E. of three different experiments. Statistical analysis was carried out by ANOVA followed by Bonferroni's test (**, p < 0.01; ***, p < 0.001 versus the same concentration of cannabidiol on MDA-MB-231 cells). CBG, cannabigerol; CBC, cannabichromene; CBD-A, cannabidiol acid; THC-A, THC acid; CBD-rich, cannabidiol-enriched cannabis extract; THC-rich, THC-enriched cannabis extract.
In vivo actions of cannabidiol on tumor growth and metastasis. A and B, effect of cannabidiol (5 mg/kg) and cannabidiol-rich extract (6.5 mg/kg) on two different xenograft tumor models in athymic mice. KiMol cells (A) or MBA-MD-231 cells (B) were injected s.c. (day 0 of treatment) into the dorsal right side of athymic mice, and the intratumor treatments were administered twice per week. Results represent mean ± S.E. (*, p < 0.05 versus vehicle; n = 6 by ANOVA followed by Bonferroni's test). C, effect of cannabidiol and cannabidiol-rich extract on breast cancer cell metastasis. MDA-MB-231 cells were injected into the left paw of 30-day-old BalB/c male mice. Animals were divided into three groups (n = 11 for vehicle; n = 14 for treated) and treated with vehicle (CTR), cannabidiol (5 mg/kg/dose), or cannabidiol-rich extract (6.5 mg/kg/dose). The drugs were injected i.p. every 72 h. Lung metastatic nodules were evaluated 21 days after the injection. Data represent mean ± S.E. of number of nodules (*, p < 0.05; **, p < 0.01 versus CTR). Statistical analysis was performed by ANOVA followed by Bonferroni's test. CBD-rich, cannabidiol-enriched cannabis extract.
A to D, representative fluorescence-activated cell sorter analyses showing the effect of 2 days of treatment of 10 μM cannabidiol (CBD) on apoptosis rate in various cell lines calculated as the percentage of cells showing a subdiploid DNA peak (subG1). Graphs are representative of three independent experiments with similar results. Graphs on the left are from cells treated with vehicle and those on the right from cells treated with cannabidiol. Line bar shows where the subdiploid DNA peak is calculated. CTR, vehicle-treated cells. E, effect of cannabidiol on caspase 3 release from procaspase. Western immunoblotting analysis was performed to detect the levels of caspase-3 expression. Proteins were extracted from DU-145 cells (lanes 1 and 2) or MDA-MB-231 cells (lanes 3 and 4) treated with vehicle (CTR, lanes 1 and 3) or 10 μM cannabidiol (cannabidiol, lanes 2 and 4) for 48 h. Determination of relative band intensity was carried out using a GS700 densitometer, and the results are presented in arbitrary scanning units. DU-145, CTR = 5.7 ± 0.81; cannabidiol = 4.2 ± 0.74; MDA-MB-231, CTR = 3.11 ± 0.67; cannabidiol = 2.64 ± 0.26 (Procaspase), 2.89 ± 0.51 (Caspase), mean ± S.E. of n = 3 separate experiments.
Effect of plant cannabinoids and Cannabis extracts on vanilloid TRPV1 receptor activation. HEK293 cells overexpressing the human recombinant TRPV1 receptor were loaded with a selective fluorescent probe (see Materials and Methods). The TRPV1-mediated effect on [Ca2+]i was determined by measuring cell fluorescence before and after the addition of the test compounds at increasing concentrations. Data are reported as percentage of the maximal effect obtained with Ionomycin 4 μM and are means of n = 3 separate experiments. Error bars are not shown for the sake of clarity and were never higher than 5% of the means. CBG, cannabigerol; CBC, cannabichromene; CBD-A, cannabidiol-acid; THC-A, THC acid; CBD-rich, cannabidiol-enriched cannabis extract; THC-rich, THC-enriched cannabis extract.
Influence of selective receptor antagonists on CBD antiproliferative action. MDA-MB-231 cells were treated with 10 μM cannabidiol in presence or in absence of selective antagonist for CB2 receptors [0.5 μM SR144528 (SR2)], TRPV1 receptors [100 nM 5′-I-resiniferatoxin (I-RTX)], or a mixture of both compounds (mix). Data are shown as percent inhibition of proliferation. Cells vehicle-treated were used as 100% of proliferation. *, p < 0.05 versus cannabidiol only, by ANOVA followed by Bonferroni's test.
Representative photomicrographs demonstrating localization of CB1, CB2, and TRPV1 receptors in human breast adenocarcinoma (MDA-MB-231) cells as determined by the immunofluorescence technique described under Materials and Methods. A, CB1 receptor immunoreactivity. B, CB2 receptor immunoreactivity. C, TRPV1-immunoreactivity was performed using rabbit polyclonal anti-CB1, anti-CB2 (both diluted 1:50), and Alexa Fluor 488-conjugated secondary antibody (1: 100) and goat polyclonal anti-TRPV1 diluted 1:100 and Alexa Fluor 546-conjugated secondary antibody (1:200). Magnification, 63×. Scale bar, 40 μm. Immunofluorescence was almost undetectable when preincubating antibodies with the corresponding blocking peptides (data not shown).
Effect of CBD on intracellular Ca2+ in MDA-MB-231 cells. A, dose-related effect of cannabidiol in the presence of extracellular Ca2+, as determined with Fluo-4. Data are mean ± S.E. of n = 4 experiments and are expressed as percentage of the effect obtained with 4 μM ionomycin. B, time-related effect of cannabidiol (10 μM) in the presence of extracellular Ca2+. Trace is representative of n = 4 experiments. C, dose-related effect of cannabidiol in the absence of extracellular Ca2+, as determined with Fura-2. Data are mean ± S.E. of n = 4 experiments. Maximal Δfluorescence was 0.235 ± 0.031 at 10 μM cannabidiol and was usually attained after 200 s (D). Effect of various antagonists (the CB1 antagonist AM251, 1 μM; the CB2 antagonist AM630, 1 μM; the TRPV1 antagonist I-RTX, 0.1 μM; 5-min pretreatment) and the intracellular calcium chelating agent BAPTA-AM (20 μM, loaded onto the cells before stimulation) on cannabidiol (1 μM) action on intracellular Ca2+. Similar results were obtained with SR141716A and SR144528.
Study of the involvement of oxidative stress in the effect of CBD. A, antiproliferative effect of 10 μM cannabidiol on MDA-MB-231 cells was measured after 4 days of treatment in absence or in presence of increasing concentrations of α-tocopherol. Data represent mean ± S.E. of percent inhibition of proliferation (*, p < 0.05 by ANOVA followed by Bonferroni's test). B, time course of ROS production by MDA-MB-231 cells (16 × 103 cells/well) as measured by spectrofluorometric analysis. Cells were loaded 1 h with 10 μM fluorescent probe in the presence of 0.05% Pluronic; fluorescence was measured in a 96-well microplate reader (PerkinElmer LS50B, λEx, 495 nm; λEm, 521 nm). Fluorescence detection was carried out after the incubation of either 100 μMH2O2 or increasing concentrations of cannabidiol at different times (0-30-60-120 min). 100 μMH2O2 was used as a positive control in these experiments. The fluorescence measured at time 0 was considered as basal ROS production and subtracted from the fluorescence at different times (Δ1). Data are reported as Δ2, i.e., Δ1 values at different doses subtracted of the Δ1 values of cells incubated with vehicle, and are mean ± S.E. of n = 3 experiments. The effects of H2O2 and of all doses of cannabidiol were significantly different from control values as determined by ANOVA followed by Bonferroni's test. Inset, lack of effect of cannabidiol 10 μM on ROS production (after 60 min) in the absence of both extracellular and intracellular Ca2+ is shown. ***, p < 0.005 by ANOVA, n = 5.
Study of the involvement of oxidative stress in the effect of CBD. A and B, antiproliferative effect of cannabidiol (24-h incubation) was evaluated in standard growth conditions or after 12 h of serum deprivation to induce oxidative stress. Poststarvation, cells were treated with 0.5 or 10 μM cannabidiol for 24 h, and the effect on proliferation was evaluated by Crystal Violet staining. Data are reported as mean ± S.E. of percent inhibition of proliferation, n = 3. C, ROS production after 2 h of incubations with cannabidiol or H2O2 was measured in MDA-MB-231 cells (16 × 103 cells/well) by spectrofluorometric analysis. The effect of cannabidiol per se (0.5 and 10 μM) is reported in Fig. 9. Cells were loaded 1 h with 10 μM fluorescent probe in presence of 0.05% Pluronic, and fluorescence was measured in a 96-well microplate reader (PerkinElmer LS50B, λEx, 495 nm; λEm, 521 nm). Fluorescence was measured at t = 0 and after 2 h of incubation with H2O2 in the presence or absence of increasing concentration of cannabidiol. Data are expressed as explained in the legend to Fig. 9. Cannabidiol inhibited ROS production by H2O2 only at the lowest concentration (0.5 μM, p < 0.05 by ANOVA followed by Bonferroni's test).
Effect of cannabinoids and Cannabis extracts on cancer cell growth
Various epithelial cell lines of various tumoral origin were treated with different concentrations of drugs, and after 4 days, the cell number was measured with Crystal Violet Vital staining (see Materials and Methods). Data are reported as mean ± S.E. of IC50 values (micromolar) calculated from three independent experiments. CBG, cannabigerol; CBC, cannabichromene; CBD-A, cannabidiol-acid; THC-A, THC-acid; CBD-rich, cannabidiol-enriched cannabis extract; THC-rich, THC-enriched cannabis extract.
Determination of cell cycle arrest, apoptosis, and mortality in the various cell lines exposed for 48 h to 10 μM of cannabidiol before flow cytometry analysis (see Fig. 4 and Materials and Methods); each experiment was repeated three times
Schematic and qualitative representation of the results of the RT-PCR analyses of mRNAs for cannabinoid and vanilloid receptors in the cell lines under study
Total RNA from cells was extracted, and its integrity was verified. RNA was further treated with RNase-free DNase I (Ambion DNA-free kit) to digest contaminating genomic DNA and to subsequently remove the DNase and divalent cations. The expression of mRNAs was examined by RT-PCR. Transcripts for fatty acid amide hydrolase (FAAH) and CB1 and CB2 receptors were analyzed and are classified as: a, abundant; m, medium; w, weak; and nd, not detected, based on the intensity of the band normalized to the band corresponding to glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene and on the number of cycles necessary to obtain a visible band. Results are based on n = 3 separate determinations.
Effect of plant cannabinoids on anandamide inactivation
Membranes from N18TG2 cells were incubated with [14C]anandamide in the presence of compounds for 30 min at 37°C (see Material and Methods) to determine the effect on the enzymatic hydrolysis by fatty acid amide hydrolase (FAAH). Intact RBL-2H3 cells were incubated with [14C]anandamide in the presence of compounds for 5 min at 37°C (see Material and Methods) to determine the effect on anandamide cellular uptake (ACU). Data represent mean ± S.E. of three different experiments.