Cannabinoid receptor expression in non-small cell lung cancer. Effectiveness of tetrahydrocannabinol and cannabidiol inhibiting cell proliferation and epithelial-mesenchymal transition in vitro

Abstract

Background/objective: Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines.

Methods: Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10-100 μM), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF).

Results: The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increased survival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used.

Conclusions: The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.

Fig 1. Cannabinoid receptors (CB1 and CB2) expression in 157 patients with non-small cell lung cancer (NSCLC).

Analysis of the survival of patients with NSCLC according to whether their CB1 (A), CB2 (B), or CB1/CB2 (C) ΔCt values were higher or lower than the mean ΔCt value of the entire cohort.

Fig 2. Tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit the proliferation and epithelial growth factor receptor (EGFR) expression in lung cancer cells.

Proliferation of A549 (A), H460 (B) or H1792 (C) cells treated with 10–100 μM THC, CBD, or CBD/THC. PT (100 μM THC in the presence of PTX 100 ng/ml). PC (100 μM CBD in the presence of PTX 100 ng/ml). PTC (100 μM THC/CBD in the presence of PTX 100 ng/ml). EGFR expression in cells treated with 30 μM THC or CBD, or 10 μM THC/CBD in the presence or absence of 100 ng/mL PTX (D). The results are expressed as mean ± SD of three independent experiments. Each condition was evaluated in six replicates from three independent wells. # p < 0.05 versus the control group.

Fig 3. THC/CBD inhibits the epithelial-to-mesenchymal transition (EMT) in cancer cells.

A549 (A-H), H460 (I-P) and H1792 (Q-X) cells were cultured in the presence or absence of 10 μM THC/CBD, and/or 15 ng/mL TGF-β. Representative images of cell morphology and fluorescence images of F-actin are shown. All experiments were performed in six replicates and five fields were assessed per condition. Scale bars equal to 25 μm.

Fig 4. Tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit gene expression of EMT-related genes.

A549, H460 and H1792 cells were treated with 30 μM THC or CBD, or 10 μM THC/CBD and/or 15 ng/ml TGF-β. Relative gene expression levels of CDH1 (A), VIM (B) and CDH2 (C) were calculated by real-time RT-PCR using GAPDH as the endogenous control. The results are means ± SD of three independent experiments. Each condition was evaluated in six replicates. The comparative ΔΔCt method was used to analyze the data. #p < 0.05 versus the control group.

Fig 5. Tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit EGF-induced cell migration in cancer cells.

A549, H460 and H1792 cells were cultured until confluence. Scratching was performed and the cells were cultured in the presence or absence of 30 μM THC or CBD, or 10 μM THC/CBD. Twenty ng/ml EGF was used to induce cell migration. Cells were cultured for 48 hours. Phase-contrast images were collected of 5 fields from each well. Representative images are shown. Total wound areas were measured using Scion Image software, and the percentage of wound recolonization was calculated. The results are means ± SD of three independent experiments. #p < 0.05 versus the control group. Scale bars equal to 50 μm.