In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

Abstract

Background: Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects.

Methods: We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells.

Results: Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts.

Conclusions: Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

Keywords: Neuroblastoma; apoptosis; cannabidiol; non-psychoactive cannabinoids; tumour xenograft models; Δ9-tetrahydrocannabinol.

FIGURE 1

Δ⁹-Tetrahydrocannabinol (THC) and cannabidiol (CBD) reduce viability of neuroblastoma (NBL) cell lines in vitro, with CBD having a better effect. (A) Cell lines SK-N-SH (open squares), NUB-6 (open circles), IMR-32 (open triangles), and LAN-1 (crosses) were incubated with increasing concentrations (0–50 μg/mL) of THC or CBD for 24 hours and 48 hours. Viability was measured by MTT assay. (B) Mean ± standard deviation of SK-N-SH cell viability after incubation with 10 μg/mL THC or CBD for 24 and 48 hours. *** Denotes a significant change relative to control (p = 0.0004). Data are expressed as a percentage of the vehicle control and are the mean of pooled results from experiments performed in triplicate.

FIGURE 2

Alteration of SK-N-SH cell cycle progression induced by cannabidiol (CBD). (A) Cell cycle analysis in untreated SK-N-SH cells and in cells treated with increasing concentrations of CBD for 48 hours. (B) Change in cell accumulation percentages during cell cycle progression after incubation with CBD for 48 hours. UT = untreated.

FIGURE 3

Apoptotic effects of cannabidiol (CBD) on SK-N-SH cells. (A) Changes in SK-N-SH cell morphology: untreated cells compared with cells treated with 10 μg/mL CBD for 48 hours. (B) Apoptotic effects of CBD on SK-N-SH cells analyzed by annexin-V assay. Cells were treated with CBD in a dose- and time-dependent manner (7.5 μg/mL, 10 μg/mL; 24 hours, 48 hours) and were stained with annexin-V and 7-amino actinomycin D (7AAD). Q1 = percentage of dead cells; Q2 = percentage of cells in late apoptosis; Q3 = percentage of cells in early apoptosis; Q4 = percentage of live cells. (C) Apoptotic effects of CBD on SK-N-SH cells analyzed by caspase-3 assay. Cells were treated with increasing doses of CBD (7.5 μg/mL, 10 μg/mL) for 24 hours.

FIGURE 4

Anti-invasiveness effect of cannabidiol (CBD) on SK-N-SH cells. The invasion assays were performed using cell cultures (2×10⁵ cells/well) treated with CBD (15 μg/mL, 20 μg/mL) for 24 hours; results were compared with those for untreated cells (2×10⁵ cells/well). For each well (treated or untreated cells), 10 fields were examined by light microscopy.

FIGURE 5

Cannabidiol (CBD) suppresses tumour growth in a mouse xenograft model and increases cleaved caspase-3 staining in treated xenografts. (A) Growth rate of SK-N-SH cell–derived tumour xenografts treated for 14 days with intraperitoneal injections of ethanol-vehicle (n = 12, closed triangles), 20 mg/kg Δ⁹-tetrahydrocannabinol (n = 12, closed squares), 20 mg/kg CBD (n = 12, closed circles) and untreated controls (n = 12, open squares). Data represents tumour volume during 14 days of treatment. ^a p < 0.05 and ^b p < 0.01 for CBD compared with ethanol treatment (Mann–Whitney U-test). (B) Activated caspase-3 immunostaining in SK-NS-H cell–derived tumour xenografts treated with CBD 20 mg/kg or ethanol vehicle for 14 days. (C) Counts of cleaved caspase-3 immunoreactive cells in 18×10 lens fields from xenografts of CBD- and ethanol-treated mice. ^a p < 0.0001 compared with ethanol.