Synergistic cytotoxic activity of cannabinoids from cannabis sativa against cutaneous T-cell lymphoma (CTCL) in-vitro and ex-vivo

Abstract

Cannabis sativa produces hundreds of phytocannabinoids and terpenes. Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma (CTCL), characterized by patches, plaques and tumors. Sézary is a leukemic stage of CTCL presenting with erythroderma and the presence of neoplastic Sézary T-cells in peripheral blood. This study aimed to identify active compounds from whole cannabis extracts and their synergistic mixtures, and to assess respective cytotoxic activity against CTCL cells. Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Cytotoxic activity was determined using the XTT assay on My-La and HuT-78 cell lines as well as peripheral blood lymphocytes from Sézary patients (SPBL). Annexin V assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle. RNA sequencing and quantitative PCR were used to determine gene expression. Active cannabis compounds presenting high cytotoxic activity on My-La and HuT-78 cell lines were identified in crude extract fractions designated S4 and S5, and their synergistic mixture was specified. This mixture induced cell cycle arrest and cell apoptosis; a relatively selective apoptosis was also recorded on the malignant CD4⁺CD26^- SPBL cells. Significant cytotoxic activity of the corresponding mixture of pure phytocannabinoids further verified genuine interaction between S4 and S5. The gene expression profile was distinct in My-La and HuT-78 cells treated with the S4 and S5 synergistic mixture. We suggest that specifying formulations of synergistic active cannabis compounds and unraveling their modes of action may lead to new cannabis-based therapies.

Keywords: CTCL cell lines; cannabinoids; cannabis; cutaneous T-cell lymphoma; peripheral blood lymphocytes.

Figure 1

(A) HPLC profile of fractions of C. sativa SCBD extract. HPLC profile was obtained from preparative HPLC. Fractions were collected as indicated in the Figure. S2, S4, S5, S6 and S7 represent the five fractions into which the peaks were divided. (B) Cell viability of My-La cells treated with different fractions of C. sativa SCBD extracts. Cell viability was determined by XTT assay as a function of live cell number. Cells were seeded and treated with C. sativa ethanol extracts of SCBD (crude), S2, S4, S5, S6 and S7 at a concentration of 40 μg/mL for 48 h. Doxorubicin (DOXO, 300 nM) served as a positive control. Methanol (control) treatment served as solvent control. Values were calculated as the percentage of live cells relative to the solvent control after reducing the absorbance without cells. Error bars indicate ± SE (n = 3). Levels with different letters are significantly different from all combinations of pairs by Tukey–Kramer honest significant difference (HSD; P ≤ 0.05). ^*indicates significantly different mean from the control based on Student T-test (P ≤ 0.05). (C) Dose-effect curves of fractions S4 or S5 of C. sativa SCBD extract on the viability of the My-La cell line. Data points were connected by non-linear regression lines of the sigmoidal dose-response relation. GraphPad Prism was used to produce dose-response curve and IC50 doses for S4 and S5 fractions.

Figure 2

Cell viability of My-La (A) and HuT-78 (B) cells treated with synergistic concentrations of S4, S5, S4+S5 fractions and with pure CBD, CBG, THC and CBC. Determination of cell viability using XTT assay as a function of live cell number. Cells were seeded and treated with S4 (5 μg/mL), S5 (6 μg/mL) or S4 (5 μg/mL) + S5 (6 μg/mL), CBD (7.3 μg/mL), CBG (3.5 μg/mL), THC (0.044 μg/mL) and CBC (0.027 μg/mL), or a combination of these pure compounds, for 48 h. Methanol (control) treatment served as solvent control. Doxorubicin (DOXO, 300 nM) served as positive control for cell cytotoxicity. Error bars indicate ± SE (n = 3). Levels with different letters are significantly different from all combinations of pairs treated by a certain S4+S5 combinations or untreated, according to Tukey-Kramer honest significant difference (HSD; P ≤ 0.05).

Figure 3

Determination of stages of cell cycle arrest following treatment with S4, S5, or S4+S5 on My-La (A) or HuT-78 (B) cell lines. Starved cells were treated with S4 (5 μg/mL), S5 (6 μg/mL), S4 (5 μg/mL) + S5 (6 μg/mL) and methanol (control) for 48 h. The treated cells were harvested, fixed, and analyzed in FACS following PI staining. The percentage of cells in G0/G1, G2/M and S phase were analyzed from 10,000 events per treatment. Error bars indicate ± SE (2 biological replicates were done, in each n = 3). Levels with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P ≤ 0.05). Determination of proportion of viable, apoptotic or necrotic cells following treatment with S4, S5, or S4+S5 on My-La (C) or HuT-78 (D) cell lines. Cells were treated with S4 (5 μg/mL), S5 (6 μg/mL), S4 (5 μg/mL) + S5 (6 μg/mL) and methanol (control) for 48 h. The treated cells were harvested and analyzed in FACS following Annexin V-FITC and PI staining. Shown are the percentages of viable, necrotic, and apoptotic cells, analyzed from 10,000 cells per treatment. FACS, fluorescence-activated cell sorting; PI, propidium iodide. Error bars indicate ± SE (2 biological replicates were done, in each n = 3). Levels with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P ≤ 0.05).

Figure 4. Apoptosis induction in PBL from Sézary patients following treatment with S4, S5, and S4+S5.

PBL were isolated from blood samples of Sézary patients (n = 6), and were treated with S4 (5 μg/mL), S5 (6 μg/mL), S4 (5 μg/mL) +S5 (6 μg/mL) and control (methanol; Vehicle treatment) for 48 h. Cells were harvested and analyzed by FACS following CD4-APC, CD26-alexa 405, Annexin V-FITC and PI staining. Apoptotic-induced cells (Annexin positive cells) were determined in the CD4⁺CD26^- cell population and in non-CD4⁺CD26^- cells of treated cells minus control. (A) The percent of apoptotic-induced CD4⁺CD26^- cells is presented for single treatment compared to combined treatment; (n = 4); ^***denote significant difference between means (one way ANOVA; p < 0.001). (B) The percent of apoptotic-induced cells following the combined treatment was compared between CD4⁺CD26^- cells and non-CD4⁺CD26^- cells of SPBL; (n = 6); ** denote significant difference between means (paired student T test; 0.001 < P < 0.05).

Figure 5. Hierarchical clustering and Venn diagram of genes significantly differentially expressed genes in My-La and HuT-78 cells treated with S4, S5 or the S4+S5 synergistic combination.

(A) Hierarchical clustering using Pearson correlations among the four conditions based on the genes expression (average fragments per kilobase of transcript per million fragments mapped [FPKM] of the three replications) followed by a log2 transform. Pearson correlations were calculated with the R software package. (B) Venn diagrams illustrating the relationships between significantly differentially expressed genes (padj < 0.05) in the three treatments against the control. Venn diagrams were generated using the online tool at bioinformatics.psb.ugent.be/webtools/Venn/.