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. 2017 Mar 24;8:144.
doi: 10.3389/fphar.2017.00144. eCollection 2017.

Cannabidiol Reduces Leukemic Cell Size - But Is It Important?

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Free PMC article

Cannabidiol Reduces Leukemic Cell Size - But Is It Important?

Nikoletta Kalenderoglou et al. Front Pharmacol. .
Free PMC article

Abstract

The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.

Keywords: Jurkat; cannabidiol; cell size; leukaemia; physiological normoxia; protein kinase B; ribosomal protein S6.

Figures

FIGURE 1
FIGURE 1
Cannabidiol affects Jurkat cell viability and cell cycle progression. (A) Cells were treated with CBD (0–10-5 M) for 72 h in RPMI with 10% serum (formula image), 5% serum (formula image), and 1% serum (formula image). Cell viability was measured using the PrestoBlue® assay. Results are expressed as average percentage viability (±SD) relative to untreated controls, n = 3. (B) Cell cycle histograms obtained by population-based DNA content analysis using flow cytometry, see Table 1.
FIGURE 2
FIGURE 2
Cannabidiol-induced mTOR and S6 dephosphorylation. (A) Representative blots of CBD dose response (10-5–10-8 M) on PKB, p42/44, and S6 phosphorylation, top panel. CTL denotes cells treated with vehicle alone. (B) Time course up to 8 h of S6 and mTOR phosphorylation with and without CBD (10-5 M), top panel. Bottom panels show the intensity of protein bands normalized to Rab11, calculated as fold difference from controls (mean ± SEM), n = 3 (p < 0.05).
FIGURE 3
FIGURE 3
Recovery from cannabidiol treatment. 106 cells/mL were treated with or without CBD (10-5 M) for 24 h in serum-free medium, resuspended in complete medium without CBD at a density of 106 cells/mL for 24 h. This was repeated at 48 h for a further 24 h. Cells were counted daily (A) and viability calculated (B), UC denotes untreated control. In one set of experiments, CBD (10-5 M) was reapplied at 48 h in serum-free medium and allowed to recover for 24 h in complete medium without CBD (C). Viability is expressed as the percentage of live cells in a total cell count, n = 3.
FIGURE 4
FIGURE 4
Cell size distribution. Cells in routine culture at different densities were counted and size-gated (A). Cells in complete medium (control +), or serum-free medium in the absence (control –) or presence of CBD, were incubated for 24 h (B–D). (B) Representative phase contrast microscope images of cells stained with Trypan Blue, with arrows indicating small viable cells as seen by eye. Mean FSC-H histograms (C) with quantitation (D), (p < 0.05) n = 4. (E) Cells from recovery experiments (see Figure 3B) were counted and sized daily. Data is depicted as the percentage of viable small cells in the viable parent population, (p < 0.05) n = 3.
FIGURE 5
FIGURE 5
Physiological normoxia impacts on Jurkat cell sensitivity to cannabidiol. Cells from either AtmosO2 (A) or PhysO2 (B) cultures were seeded into 96-well plates (105 cells/well) in medium (with 5% serum) with or without CBD (5 or 10 μM, indicated as CBD5 or CBD10) and/or DOX (10-5–10-8 M) for 72 h. Cell viability was measured using the PrestoBlue® assay. Results are expressed as average percentage viability (±SD) relative to untreated controls. Area under the curve (AUC) analysis (inset in A) (p < 0.05), n = 3. An increased dose response was performed with CBD up to 300 μM on both activated and non-activated cells from PhysO2 conditions in medium with 5% serum (C) or 1% serum (D), n = 4.

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