PMC

A dose-response curve of A549 cells after 24 h incubation with or without (control) 2-10 μg/ml of CAN1-CAN12 calculated from at least 5 independent experiments.

The matrix of the ESI-LC/MS phytocannabinoid analysis Z-scores representing the set of associations was scaled by column to range from -8 to 8. Negative values (dark blue) indicate that the extract contained very low levels of the phytocannabinoid, and positive values (red) indicate that this extract was composed of high levels of the phytocannabinoid. Dendrograms indicating the clustering relationships are shown to the left and above the heat map. The 124 extracts segregate into five major clusters comprised of phytocannabinoids that associate with: (1) larger amounts of CBG-type; (2) larger amounts of CBD-type.; (3) larger amounts of CBDA-type; (4) larger amounts of Δ⁹-THC-type; (5) larger amounts of Δ⁹-THCA-type.

A549 cells were treated with 1-2 μg/ml CAN5, CAN9 or CAN10 extracts or control (DMSO) for 48 h. Cells were stained with anti-proliferation marker Ki67 antibody and counterstained with Hoechst. Percentage proliferation was calculated as % of Ki67-positive cells out of total cells. (A) Representative images of control and CAN9 treatment (1-2 μg/ml). Blue - Hoechst, green - Ki67, Turquoise - overlay of Hoechst and Ki67. (B) Bar chart presents % proliferation following incubation with CAN5, CAN9 or CAN10. Data are presented as mean ± SE (N=5). Asterisks indicate statistically significant differences between extract treatments vs. control. (*P < 0.05, **P < 0.005, ***P < 0.0005; two-way ANOVA with Bonferroni's post hoc multiple comparisons test).

A549 cells were incubated for 24 hours with 4 or 8 μg/mL CAN5, CAN9, CAN10 extracts or with DMSO (control). Apoptosis (early and late) was assessed by Annexin V/PI staining using flow cytometry. Results were calculated as % of positive annexin V-FITC cells out of total cells counted. (A) Representative dot plots of cells treated with 4 or 8 μg/ml CAN5, CAN9 and CAN10 extracts. (B) Bar chart of total apoptosis following incubation with extracts. Data are presented as mean ± SE (N=5). Asterisks indicate statistically significant differences compared to control (***P < 0.0001; two-way ANOVA with Bonferroni's post hoc multiple comparisons test). (C) Cells were lysed and resolved on 15% SDS-PAGE followed by western blotting with anti-cleaved caspase 3 and Anti β-Tubulin antibodies.

(A) A549 cells were treated for 24 h with 4 µg/ml of 14 Δ⁹-THC-rich Cannabis extracts or pure Δ⁹-THC. Data are reported as mean ± SE of % dead cells out of total cells (N=5). Extracts are ordered on the x-axis by increasing Δ⁹-THC content in each extract (µg/ml). Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's multiple comparisons test. Bars labelled with different letters are significantly different (P ≤ 0.05) from one another, according to the post-hoc ANOVA statistical analysis. (B) Principal Component Analysis (PCA) of 14 Δ⁹-THC-rich extracts according to the phytocannabinoid content. Criteria for cannabinoid inclusion in the PCA were detection in at least three extracts and a minimum concentration of 0.5 % w/w in one of the included extracts. (C) Linear regression of the percentage of dead A549 cells in response to 14 Δ⁹-THC-rich extracts according to their PC1 score.

Cancer cell lines of various tumor origins were treated with 4 µg/ml of 12 different Cannabis extracts for 24 h. Data are reported as mean ± SE of % dead cells out of total cells (N=7). (A) The effect of different Cannabis extracts on cell lines A549, NCI-H460, A375, A-431, SW480, HT-29, MCF7, MDA-MB-231, LNCaP, PC-3, U-87 MG and T98G. Asterisks represent statistically significant differences compared to control (*P < 0.05, **P < 0.005, ***P < 0.0005; one-way ANOVA). (B) A comparison between the effect of Cannabis extracts on PC-3 and LNCaP prostate carcinoma cell lines. Asterisks indicate statistically significant differences between LNCaP and PC-3 cell lines (*P < 0.05, **P <0.005, *P < 0.0005; two-way ANOVA with Bonferroni's post hoc multiple comparisons test) (C) Representative fluorescent images overlaid onto transmitted light images of LNCaP and PC-3 prostate cancer cells treated with or without (control) 4 µg/ml of CAN6 (blue- Hoechst- all cells, red- PI- dead cells). (D-E) A comparison between the effect of neutral (white columns) and heat-decarboxylated (black columns) phytocannabinoid contents of Cannabis extracts on A549 and LNCaP cells. Asterisks indicate statistically significant differences between extracts (*P < 0.05, **P < 0.005, ***P < 0.0005; two-way ANOVA with Bonferroni's post hoc multiple comparisons test).

(A) A549 and NCI-H460 lung carcinoma cells and normal bronchial epithelial cells (Normal AECs) were incubated with 4 µg/ml of CAN1-12 or control (ctl). Data are reported as mean ± SE of percentage of dead cells out of total cells relative to control treatment (N=3). Asterisks indicate statistical differences between cancer vs. normal AECs treated with Cannabis extracts (*P < 0.05, **P < 0.005, ***P < 0.0005; two-way ANOVA with Bonferroni's post hoc multiple comparisons test). (B, D) Representative brightfield images of normal AECs and lung carcinoma (NCI-H460, A549) (B) or HT-29 colon carcinoma (D) cell lines treated with control or CAN5. (C) Dose-response curve of HT-29 cells after 24 hours incubation with or without (control) 2-10 μg/ml of CAN1-CAN12. Data are presented as mean ± SE (N=9). (E) Screening results of 4 μg/ml of 43 different Cannabis extracts, or control on the survival of HT-29 cells (N=1). (F) Cannabimimetic receptors CNR1 (CB1), CNR2 (CB2), GPR55, TRPV1, TRPA1, TRPV2, TRPM8 mRNA levels were evaluated by qPCR. Expression levels were represented as ΔCT levels of the receptors. *Jurkat cell line was added as a control for CNR2 expression. Results are presented as mean expression of N=3 and normalized to the GUSB housekeeping gene. Values were color-coded according to the magnitude of expression. UD - Under Detectable Level. The higher the ΔCT values are the lower receptor expression.