Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells

Abstract

In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

Keywords: Cannabis sativa L.; anticancer activity; cannabidiol; cannabinoids; leukaemia.

FIGURE 1

Representative HPLC‐UV overlapped chromatograms of R1 (blue) and D1 (red) extracts at 220 nm. The main neutral and acid cannabinoids are shown

FIGURE 2

Viability of HT29 (A,B), U87MG (C,D) and K562 (E,F) cells treated at 24–48 hr with raw (R1) and decarboxylated (D1) CBD‐type hemp extracts at 5–100 μg/ml. Data in the graphs are shown as mean ± SD (n = 4 per group)

FIGURE 3

(a) Dose/response curves of decarboxylated D1, D2 and D3 hemp extracts at 5–100 μg/ml on K562 cells after 48 hr of treatments. Data in the graphs are shown as mean ± SD (n = 4 per group). (b) Effect of pure CBD on K562 cells after 48 hr of treatment compared with the extract D1. The dose is shown as μg/ml for D1 extract and μM for CBD, respectively. Data in the graphs are shown as mean ± SD (n = 4 per group)

FIGURE 4

Cell cycle profile and percentage of cells in G0/G1, S and/or G2/M phase

FIGURE 5

(a) Fluorescence microscopy analysis of K562 cells treated with 10 and 20 μg/ml of the D1 extract at 16 hr and stained with calcein (green dots) and PI (red dots). (b) Flow cytometric analysis of K562 cells stained with Annexin‐V and propidium iodide (PI). Representative dot plots of K562 cells after a 16 hr treatment with increasing concentrations of D1. (Q1) Cells undergoing apoptosis. (Q2, Q4) Late apoptosis or necrosis. (Q3) Living cells without signs of apoptosis

FIGURE 6

Effect of the D1 extract on p53, bcl‐2, bcl‐xl protein expression in K562 cells and on cytochrome c release. (a) Representative western blots at different concentration/time treatments. (b) Densitometric analyses of protein levels of p53, bcl‐2, and bcl‐xl of K562 cells lysate after incubation with 20, 30 and 40 μg/ml of D1 for 16 hr; densitometric analyses of cytosolic and mitochondrial cytochrome c in K562 cells lysate after 16 and 24 hr incubation with 20 μg/ml of D1. Densitometry values were normalized to the protein loading control GAPDH. The values are expressed as mean ± SD of three independent experiments (n = 3 per group)

FIGURE 7

(a) Live imaging microphotographs taken with a 20× objective. Caspase – activated cells (green) shown at different time points (0, 18 and 24 hr) in the control (Ctrl) and in the cells treated with D1. Scale bar (red) 200 μm. (b) Histogram of caspase – activated positive cells for each condition, *p < 0.05 compared to control. For all panels, data are expressed as mean ± SD (n = 4 per group)

FIGURE 8

(a) Live imaging microphotographs taken with a 10x objective. Ratiometric view ranging from violet (totally depolarized mitochondria) to bright green (polarized mitochondria) shown at different time points (0, 2.30, 5 and 10 hr) in the control (Ctrl) and in the cells treated with D1 cells. Each image composing the panel shows an area of 1770 μm × 1770 μm. (b) Histogram showing the number of polarized mitochondria positive cells for each condition at 0, 2.30, 5 and 10 hr, *p <  0.05 compared to control. For all panels, data are expressed as mean ± SD (n = 6 per group)