In vitro anticancer activity and oxidative molecular damage by cannabidiol administered alone and in combination with epirubicin

Figures & data

Figure 1. Dose-response curves of human cell lines HL-60, HL-60/Dox and MCF-7 cells after 72 h exposure to CBD.

Note: Data are expressed as means in three independent experiments; SD have been omitted for clarity; CBD, cannabidiol.

Figure 2. Cytotoxicity of CBD + EPI in leukemic cell lines HL-60 (a), HL-60/Dox (b), and breast cancer cells MCF-7: applied simultaneously (c) and in a drug sequence: CBD 24 h followed by EPI 48 h (d).

Note: Data are presented as tumor inhibition (T/C,%) ean ± SD *Legend: [

] EPI; [

] CBD; [

] EPI + CBD; EPI, epirubicin; CBD, cannabidiol.

Table 1. Combination index (CI) values of epirubicin in combination with varying concentrations of cannabidiol on three tumor cell lines*.

EPI, µmol/L	CBD, µmol/L	HL-60 CI	HL-60/Dox CI	MCF-7 CI
0.625	6.25	1.29	1.38	10.24
0.625	12.5	1.46	2.31	1.77
0.625	25	1.21	1.09	0.97
1.25	6.25	1.28	1.24	2.40
1.25	12.5	1.35	1.88	1.63
1.25	25	1.46	1.09	1.18
2.5	6.25	1.86	0.88	1.47
2.5	12.5	2.02	0.97	1.39
2.5	25	2.28	0.92	1.53

* CIs are calculated on the basis of experimental data after 72 h exposure to CBD and EPI, applied simultaneously. CI < 0.9 denotes synergism; CI = 0.9–1.10 denotes additive effect; CI > 1 denotes antagonism. CI, combination index; EPI, epirubicin; CBD, cannabidiol.

Table 2. Combination index (CI) values of epirubicin plus cannabidiol at fixed-ratio concentrations of 1:10 on three tumor cell lines*.

Cell line	HL-60		HL-60/Dox		MCF-7
Fa	Total dose	CI	Total dose	CI	Total dose	CI
0.30	3.39	0.83	11.46	1.41	13.17	2.91
0.40	4.44	0.93	13.27	1.35	14.41	2.58
0.50	5.70	1.04	15.70	1.30	15.66	2.32
0.60	7.31	1.16	17.35	1.25	17.00	2.08
0.75	11.20	1.41	21.82	1.17	19.59	1.76
0.90	22.00	1.90	31.40	1.06	24.52	1.37
0.95	34.82	2.34	40.20	0.98	28.55	1.17

* On the basis of computer-simulated dependencies between dose and median effect (Fa). Total dose (µmol/L), the sum of epirubicin and cannabidiol concentrations. Values in boldface are indicative of synergism or additive effect.

Table 3. Dose reduction index (DRI) values of EPI and CBD at fixed-ratio concentrations of 1:10 on HL-60 and MCF-7 cells*.

HL-60				Fa	MCF-7
EPI, µmol/L	CBD, µmol/L	DRIEPI	DRICBD		EPI, µmol/L	CBD, µmol/L	DRIEPI	DRICBD
0.91	10.97	1.75	2.11	0.50	1.01	15.62	0.71	1.10
1.03	12.83	1.54	1.93	0.60	1.28	17.61	0.83	1.14
1.17	15.23	1.34	1.75	0.70	1.64	20.08	0.97	1.19
1.27	16.78	1.25	1.65	0.75	1.90	21.63	1.07	1.21
1.39	18.76	1.14	1.54	0.80	2.24	23.55	1.19	1.25
1.78	25.67	0.88	1.28	0.90	3.57	29.95	1.60	1.34
2.24	34.27	0.71	1.08	0.95	5.49	37.37	2.11	1.44

* Values were calculated on the basis of computer-simulated dependencies between total dose and median effect (Fa) by CompuSyn software; EPI, epirubicin; CBD, cannabidiol.

Figure 3. Degree of 2-deoxyribose (DR) oxidative damage in the absence or presence of different concentrations of EPI and CBD, applied by themselves or simultaneously at fixed-ratio concentrations of 1:10.

Note: Data are presented as percentage of molecular damage, mean ± SD. Groups were compared by one-way ANOVA with Bonferroni post hoc test. Asterisks indicate statistically significant differences versus the control: **p < 0.01, ***p < 0.001 [

] EPI; [

] CBD; [

] EPI + CBD; EPI, epirubicin; CBD, cannabidiol.

Figure 4. Impact of the investigated combinations CBD + EPI on the luminol-dependent chemiluminescence in the presence of xanthine/xanthine oxidase-generated superoxide.

Note: Data are presented as means ± SD. Three parallel measurements were performed for each composition (n = 3), each representing an individual data point. The baseline was subtracted from both control and sample measurements: (1) Control measurement – 0.1 mL xanthine solution, 0.05 mL xanthine oxidase solution (2.9 mU), 0.1 mL luminol, 0.75 mL PBS; (2) Sample measurement – 0.1 mL xanthine solution, 0.05 mL xanthine oxidase solution (2.9 mU), 0.1 mL luminol, the necessary volumes of stock CBD and EPI solutions, PBS up to 1.0 mL; (3) Baseline sample measurement – 0.1 mL xanthine, 0.1 mL luminol, 0.8 mL PBS; [

] EPI + CBD; EPI, epirubicin; CBD, cannabidiol.

Table 4. Equieffective concentrations of EPI, CBD and their combination at fixed-ratio concentrations of 1:10 after 72 h exposure of HL-60 and MCF-7 cells, calculated using computer-simulated dose-response relationship*.

Cell line	Drug	ED50	ED75	ED90
HL-60	EPI	0.91	1.27	1.78
	CBD	10.97	16.78	25.67
	EPI + CBD = 1:10	[0.52 + 5.18]	[1.02 + 10.18]	[2.00 + 20.00]
MCF-7	EPI	1.01	1.90	3.57
	CBD	15.62	21.63	29.95
	EPI + CBD = 1:10	[1.42 + 14.23]	[1.78 + 17.81]	[2.23 + 22.29]

* EPI, epirubicin; CBD, cannabidiol.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.