The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

Abstract

The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ⁹-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds. We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro. Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays. Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells. In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death. In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.

Keywords: Cannabis sativa; Apoptosis; Autophagy; Breast cancer; Cannabinoids; Metastasis.

Fig. 1

Cytotoxicity assay of MDA-MB-231 (A) and MCF-7 (B) cells treated with CBD/THC mixture (10, 12.5, 15, 17.5, 20 and 25 μg/mL) for 24 and 48 h. Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.

Fig. 2

Cell cycle analysis of MDA-MB-231 (A–D) and MCF-7 (E–H) cells treated with CBD/THC (12.5, 15, 17.5 μg/mL) for 24 h and 48 h. Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.

Fig. 3

Apoptosis assay using Annexin V / PI dual staining in MDA-MB-231 (A–B) and MCF-7 (C–D) after 24 h and 48 h of treatment with CBD/THC mixture (12.5, 15, 17.5 μg/mL). Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** Corresponds to p-value < 0.01, and ^*** Corresponds to p-value < 0.001.

Fig. 4

DNA fragmentation detection in MDA-MB-231 (A) and MCF-7 (B) via cell death detection ELISA after 24 h of treatment (12.5, 15, 17.5 μg/mL). Western blot analysis of PARP proteins in both cell lines (C), along with the quantification in MDA-MB-231 (D) and MCF-7 (E) cells. Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.

Fig. 5

Molecular evaluation of apoptosis via western blot analysis of apoptotic regulatory markers in MDA-MB-231 (A–B) and MCF-7 (A & C) cells treated for 24 h with CBD/THC (12.5, 15, 17.5 μg/mL). Cell viability assay using MTS reagent following CBD/THC treatment alone or in combination with caspases inhibitor, Z-VAD-FMK (50 μM) for 24 and 48 h in MDA-MB-231(D) and MCF-7 (E) cells. ROS production evaluation in MDA-MB-231 and MCF-7 (F) cells after 2 and 24 h of treatment with CBD/THC. Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.

Fig. 6

Molecular evaluation of autophagy via western blot analysis of autophagy related markers in MDA-MB-231 (A–C) and MCF-7 (A & G–H) cells treated for 12 (C and H) and 24 h (B and G) with CBD/THC. Cell viability assay using MTS reagent following CBD/THC treatment alone or in combination with autophagy inhibitors, CQ (10 μM) (E) and Wort (50 nM) (F) for 24 h in MDA-MB-231 and MCF-7 cells. Immunofluorescence staining of MDA-MB-231 with LC3B antibody following treatment for 24 h with CBD/THC (F). Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.

Fig. 7

Evaluation of the anti-metastatic properties of CBD/THC on TNBC cells. Wound healing assay (A), trans-well migration (B) and invasion (C) assays. Molecular evaluation of motility regulatory proteins following 24 h of treatment with CBD/THC (3.12 and 6.25 μg/mL) (D). Data represents mean ± SD from three independent experiments. Statistical significance reported as ^* corresponds to p-value < 0.05, ^** corresponds to p-value < 0.01, and ^*** corresponds to p-value < 0.001.