Serum deprivation or modification of serum content differently affects the effect of cannabinoids (CBD, CBG, CBC) on the viability of human PCCs in the MTT assay. (A) Cells were grown in presence of 10% FBS in six-well dishes. After adhesion, cells were serum-deprived (with or without 0.5%BSA) for 16 h and subsequently treated with compounds for 24 h. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity, calculated from three independent experiments. ***P < 0.001, FBS-free vs. plus 0.5% BSA; anova followed by Bonferroni's test. (B) Cells grown in presence of 10% FBS were incubated, after adhesion, under different conditions (a–c) in six-well dishes for 72 h: (a) cells were incubated in presence of 10% FBS; (b) cells were incubated with 10% FBS that had been protein-depleted; (c) cells were incubated again in 10% FBS that had been protein-depleted, but supplemented with 0.5%BSA. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity calculated from three independent experiments. ###P < 0.001 protein-depleted serum [b] vs. 10% FBS [a] and protein-depleted serum plus 0.5% BSA [c]; $$P < 0.01 protein-depleted serum plus 0.5% BSA [c] vs. 10% FBS [a]; anova followed by Bonferroni's test. (C) Cells were grown in 10% FBS in six-well dishes. After adhesion, cells were serum-deprived for 16 h and then treated with compounds for 24 h. Cell viability was assessed by the MTT assay. Data shown are means ± SEM of % inhibition of MTT-reducing activity calculated from three independent experiments.

Effect of CBD-BDS on the growth of xenograft tumours from LNCaP and DU-145 cells in athymic mice, per se or co-administered with docetaxel (taxotere) or bicalutamide. Effect of increasing doses of CBD-BDS (i.p.) or docetaxel (i.v.) or combinations thereof on the growth of LNCaP (A) and DU-145 (B) cell xenografts. N= 10 mice were used for each group. In panel A, *P= 0.0008, **P < 0.0001 versus vehicle; ***P < 0.0001 versus both vehicle and CBD–BDS + docetaxel; in panel B, *P < 0.0001 versus vehicle; #P < 0.0001 versus docetaxel alone; two-way anova. (C) Effect of increasing doses of CBD-BDS (i.p.) or bicalutamide (p.o.) or in combination, on the growth of LNCaP cell xenografts. A higher dose of bicalutamide (50 mg·kg⁻¹) was studied, but the effect was not different from that of the 25 mg·kg⁻¹ dose. *P= 0.0005 (group 5 vs. group 1); #P = 0.001 (group 5 vs. group 2); two-way anova. (D) Kaplan–Meier survival plots for the study described in (C). Statistical significance was assessed by the Log rank-Wilcoxon analysis.

Effect of cannabinoids and on the release of caspase 3/7 from various PCC lines. Cells (10 000 per data point) were treated under the conditions shown, and caspase 3/7 activity was assessed with the luminescence assay. Other compounds tested that exhibited no activity are not shown. Effect of various compounds, at various concentrations, in LNCaP (A) and DU-145 (DU) (B) cells incubated in the presence of serum for 24 h, and of the positive control, anti-FAS + camptothecin (campto + AFAS). Note how the TRPM8 channel antagonist OMDM233 stimulates caspase 3/7 activity and how the TRPM8 channel agonist icilin inhibits the effect of CBD in LNCaP cells. (C) Effect of varying duration of SDP before and after treatment (12 h + 8 h treatment, 12 h + 12 h treatment, 24 h + 12 h treatment) with varying doses of the TRPM8 antagonist OMDM233, or of the TRPM8 agonist icilin, or of CBD, with and without icilin, on caspase 3/7 activity in LNCaP cells. (D) Effect of various durations of SDP before and after treatment (12 h + 12 h treatment, 12 h + 24 h of treatment or 24 h + 12 h treatment) with varying doses of CBG and CBC on caspase 3/7 activity in LNCaP cells. (E) Effect of BSA (0.5%), or testosterone (T, 50 µM), or subsequent addition of FBS, or of the use of charcoal-stripped FBS (strip) on the effect of CBD on caspase 3/7 activity in LNCaP cells. These experiments were carried out under different conditions of pre-treatment serum deprivation (12 or 24 h), whereas the treatment with CBD (10 µM) was always carried out for 12 h, thus leading to total durations of experiments of 24 or 36 h. (F) Effect of CBD on caspase 3/7 activity in SDP DU-145 cells. Experiments were carried out with either varying doses of CBD for 18 h, after a previous serum deprivation of 6 h, or with 10 µM CBD for a total of 24 h with different combinations of previous serum deprivation (0, 2 and 4 h) and treatment (24, 22 and 20 h). Finally, the effect of CBC and CBG were studied in 22RV1 (G) and PC3 (H) cells. In both cases, the experiment lasted for 24 h, with a previous serum deprivation of 4 h in panel G and 6 h in panel H. Data are means ± SEM of at least n= 3 experiments. Means were compared by anova followed by Bonferroni's test. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective control (first bar in each panel, which represents the baseline level of caspase 3/7, which, for a given cell line, did not vary significantly regardless of the duration of the experiment and the presence of serum in the 12–36 h range). In panels C and E, ^#P < 0.01 versus SDP 24 + 12 CBD 10 µM.

Molecular mechanisms of the pro-apoptotic effect of CBD in PCC lines. (A) Effect of CBD (10 µM) on the mRNA expression of the PUMA in the four cell lines examined in this study. Cells were cultured in presence of serum (dark grey bars), in serum-deprived medium for 24 h (striped bars) and in presence of 10 µM CBD in the optimal conditions evaluated by caspase 3/7 assay (light grey bars); that is, 12 h (LNCaP), 18 h (DU-145 and PC-3) and 20 h (22RV1) of CBD treatment after 12, 6 and 4 h of previous serum deprivation respectively. The expression levels, normalized respect to reference genes, were scaled for each cell line to the expression value of the cells cultured in presence of serum, put as 1. The means of the quantitative cycles (cq) for the these conditions were 28.68 (LNCaP), 28.62 (DU-145), 27.32 (PC-3) and 25.63 (22RV1). The reaction background was 37.30 cq. (B) Representative Western blots for the stimulatory effect of CBD on the expression and phosphorylation of p53 in LNCaP, DU-145 and 22RV1 cells, serum-deprived for 12 h and then treated with vehicle or CBD (10 µM) for a further 12 h. Histograms show the quantitative determination of the chemoluminescence in Western blots from two separate experiments, normalized to β-actin and expressed as fold amounts relative to the corresponding serum-deprived, vehicle-treated control cells: LNCaP (black bars), 22RV1 (dark grey bars) and DU-145 (light grey bars) cells. (C) Transcriptional expression of CHOP in prostatic cell lines: CHOP mRNA levels in LNCaP, DU-145, PC3 and 22RV1 prostatic cancer cell lines. Cells were cultured in presence of serum (first bar in each group), in serum deprived medium for 24 h (second bar in each group) and in presence of 10 µM CBD in the optimal conditions evaluated by caspase 3/7 assay (third bar in each group) (see panel A). qRT-PCR was performed, using 20 ng of cDNA per assay. The expression levels, normalized respect to reference genes, were scaled for each cell-line to the expression value of the cells cultured in presence of serum, put as 1. The means of quantitative cycles (cq) for the these conditions were 24.88cq (LNCaP), 20.73cq (DU-145), 21,79cq (PC3) and 21,87cq (22RV1). The reaction background was 35.30 cq. Standard deviations were calculated by the gene expression module of iQ5 real-time PCR. All differences indicated in the graph with (*) were statistically significant (P < 0.05) as evaluated according to (see Supporting information). A typical experiment (R.I.N. > 8.5) is shown. (D) Transcriptional expression of androgen receptor in LNCaP and (E) 22RV1 cells. The cells were cultured in presence of serum (CTR), in serum-deprived medium for 24 h (SDP) and in presence of 10 µM CBD for 12 (LNCaP cells) or 20 (22RV1) h during 24 h total growth in serum-deprived medium (SDP + CBD). LNCaP cells (D) were also growth for 24 h in serum-deprived medium containing testosterone 50 µM in absence (SDP + T) or in presence (SDP +T + CBD) of CBD, following the conditions described above. The expression levels normalized respect to the reference gene were scaled to the lowest expression value condition [i.e. SDP +T + CBD, 26.76 cq vs. background >40 cq for (D) and CTR, 26.76 cq vs. background >40 cq, for (E)], considered as 1. (F) Transcriptional expression levels of TRPM8 in LNCaP cells cultured in presence of serum (CTR), in serum-deprived medium for 24 h (SDP) and in the presence of 10 µM CBD for 12 h during 24 h total growth in serum-deprived 50 µM in absence (SDP + T) or in presence (SDP +T + CBD) of CBD, following the conditions described above. The expression levels normalized respect to the reference gene were scaled to the lowest expression value condition (SDP, 27.30 cq vs. background at 35.01 cq), considered as 1. In panels A, C, D, E, a representative experiment (R.I.N. > 8.5) is shown and qRT-PCR was performed as described in Methods, using 20 ng of cDNA per assay. Standard deviations were calculated by the gene expression module of iQ5 real-time PCR. All differences indicated in the graph (*) were significant (P < 0.05 vs. values in dark grey bars) as evaluated according to (see Supporting information). In panel F, # denotes P < 0.05 vs. SDP, and § denotes P < 0.05 vs. SDP + T.

Effect of cannabinoids on intracellular Ca²⁺ and ROS in prostate carcinoma cells. (A) Typical dose-dependent effects for cannabinoids on intracellular Ca²⁺ in PCCs, with either efficacy or potency being higher in cells serum-deprived (SDP) for 24 h. The effect of CBG in LNCaP and DU-145 cells is shown. See for the full data in the four PCCs with CBD, CBG and CBC. (B,C) Involvement of ROS in the effect of CBD on different PCCs. Time course of ROS production by PCC cells as measured by spectrofluorometric analysis as described in Methods. Fluorescence detection was carried out after the incubation of either 100 µM H₂O₂ or CBD (10 µM) at different times (0–30–60–120 min) in cells grown in normal medium (B) or in cells kept without serum prior to treatment (C). 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 minus the Δ1 values of cells incubated with vehicle), and are mean ± SEM of at least n= 3 experiments. Note how the effect of both CBD and H₂O₂ becomes significant only in the absence of serum (C) and only in LNCaP cells.

Effects of CBD on neuroendocrine-like LNCaP cells. Cells were differentiated with db-cAMP + IBMX for 36 h in serum-deprived medium, in the presence or absence of CBD and various other compounds. (A) Effect on caspase 3/7 activity of just serum deprivation for 36 h, alone or with CBD (10 µM) for 12 h, or with db-cAMP + IBMX for 36 h, or with db-cAMP + IBMX for 24 h followed by 12 h CBD, or with db-cAMP + IBMX followed by 12 h icilin (1 µM) or OMDM233 (2 µM), or with db-cAMP + IBMX followed by 12 h CBD + icilin. *, **, ***P < 0.05, 0.01, 0.001 versus SDP 36. (B) NSE mRNA in differentiated LNCaP cells. Cells were cultured in the presence of serum (CTR), in serum-deprived medium for 36 h in the presence of db-cAMP and IBMX (SDP) and in the presence of 10 µM CBD for 12 h during db-cAMP + IBMX treatment (SDP + CBD). qRT-PCR was performed using 20 ng of cDNA per assay. The expression levels of NSE mRNA, normalized respect to the reference gene, were scaled to the lowest expression value condition, considered as 1; i.e., SDP + CBD (28.67 cq vs. background >40 cq). PUMA (C), p27^kip (D), AR (E) and TRPM8 (F) mRNA levels in LNCaP cells following various treatments in serum-deprived (SDP) cells. Cell were cultured in presence of serum (CTR), in serum-deprived medium for 36 h in presence of db-cAMP + IBMX (dbcAMP) and in presence of 10 µM CBD for 12 h during db-cAMP + IBMX treatment (dbcAMP + CBD). For all the targets, the expression levels normalized respect to the reference gene were scaled to the lowest expression value condition, considered as 1; i.e. CTR (28.68 cq vs. background at 37.53) for PUMA and p27^kip (24.75 cq vs. background at 38.40cq); and db-cAMP + CBD for AR (29.04 vs. background >40 cq) and TRPM8 (29.50 vs. background at 35.80 cq). In panels B–F, qRT-PCR was performed using 20 ng of cDNA per assay, and a typical experiment (R.I.N. > 8.5) is shown. Standard deviations were calculated by the gene expression module of iQ5 real-time PCR. All differences indicated in the graph (*) were significant (P < 0.05) versus CTR as evaluated according to (see Supporting information). # denotes P < 0.05 versus CTR.