Cannabinoid receptor expression in nonsmall cell lung cancer. Effectiveness of tetrahydrocannabinol and cannabidiol inhibiting cell proliferation and epithelialmesenchymal transition in vitro, Lara Milian et al., 2020

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

Lara Milian, Manuel Mata, Javier Alcacer, Marıa Oliver, Marıa Sancho-Tello, Jose Javier Martın de Llano, Carlos Camps, Jose Galbis, Julian Carretero, Carmen Carda

PLoS ONE, 2020, 15, (2): e0228909.

Doi : 10.1371/journal.pone.0228909



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 tetrahydro-cannabinol (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.



Lung cancer is the leading cause of cancer-related death; more than 1 million patients are diagnosed annually. In many cases, life expectancy is only a few months and the 5-year survival rate is < 15% [1–2]. Non-small cell lung cancer (NSCLC) represents 85% of all lung cancers and the most common subtypes are adenocarcinoma and squamous cell carcinoma [3]. Both subtypes are characterized by genetic abnormalities, which lead to alterations in signaling pathways that are targets for drug therapies [4]. All patients with NSCLC eventually develop resistance to antitumor agents, including endothelial growth factor receptor (EGFR) inhibitors and chemotherapeutics, possibly due to abnormal signal transduction and EGFR overexpression [5–7]. This necessitates the development of new complementary pharmacological agents.

The endocannabinoid system is composed of the G-protein–coupled receptors CB1 and CB2, their endogenous ligands anandamide and 2-araquidonoglicerol, and their synthetic and degradative enzymes [8]. CB1 receptor is expressed not only in the central nervous system, but also in other tissues and organs, where its activation exerts both central and peripheral effects [9]. CB2 is expressed in immune cells, microglia, vascular smooth muscle cells, hepatic stellate cells, and endothelial cells. CB2 modulates Ca2+ channels, mitogen-activated protein kinase activation, and cAMP production [9]. According to reports, both receptors are expressed in, for example, breast and prostate cancer, glioblastoma, rhabdomyosarcoma, and colorectal cancer cells [10–14].

Although CB1 and CB2 are expressed in a variety of cancer cell lines and types of tumors, including adenocarcinomas [15], the relationships of their expression levels with lesion characteristics and disease progression have not been investigated. We thus assessed the correlation between the expression levels of the two receptors and the disease and clinical characteristics of a cohort of patients with NSCLC.

Cannabinoid-receptor agonists have potential as complementary pharmacological agents for NSCLC due to their analgesic, antianorexic, antiemetic and antineoplastic properties. For example, cannabinoid receptor agonists modulate key signaling pathways—including the extracellular signal-related kinase (ERK), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (p38 MAPK), and ceramide pathways—in vitro and in vivo, inducing apoptosis and inhibiting cancer dissemination [16–18]. Cannabinoids act on cannabinoid receptors and include endocannabinoids (produced naturally in the body of animals), phytocannabinoids (found in cannabis and some other plants), and synthetic cannabinoids (manufactured artificially). Cannabis sativa contains more than 150 cannabinoid agonists, including Δ9-tetrahydricannabinol (THC), cannabidiol (CBD), cannabinol, cannabichroemene, and cannabigerol [19]. Among these, THC and CBD have demonstrated antitumor efficacy against glioblastoma, leukemia, and melanoma, as well as cervical, breast, and prostate cancer [20]. THC is a partial agonist of CB1 and CB2 receptors, and induces analgesia and muscle relaxation, suppresses emesis and stimulates appetite; however, the psychotropic activity of THC limits its clinical use [21]. CBD has greater affinity for CB2 than CB1 [22]. It also stimulates vanilloid pain receptors (VR1) and inhibits the uptake of anandamide [23]. CBD has anti-inflammatory, neuroprotective, anticonvulsant, muscle-relaxant, and anti-psychotropic effects [22]. Combined used of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. Thereby, in rats, CBD administered with THC ameliorate adversely effect (e.g. dysphoria) often associated with THC alone and
did not alter the discriminative stimulus effect of THC [24]. Moreover, this combination enhances anticancer activity compared with THC alone and reduces the doses of THC that are needed to inhibit tumor growth [25–27]. CBD has also been shown to alleviate some of the undesired effects of THC administration, such as convulsions, discoordination and psychotic events, and, therefore, improves the tolerance of cannabis-based medicines [25]. Moreover, the combined used of THC and CBD reduces cell viability and migration, and induces apoptosis in human glioblastoma [28]; however, its effect on NSCLC is unclear. We thus investigated the influence of CB1 on the antineoplastic effects of THC in an in vitro model of lung cancer.

The epithelial-to-mesenchymal transition (EMT) involves complex phenotypic changes of tumor cells [29]. During the EMT, epithelial markers (including E-cadherin) are downregulated and mesenchymal markers (such as vimentin [VIM], N-cadherin, and smooth muscle alpha actin [ASMA]) are upregulated by the transcription factors snail, ZEB1, and ZEB2 in a manner involving transforming growth factor-β1 (TGF-β1) [30–32]. Ravi et al. assessed the effect of the CB2 agonist JWH-015 on the EMT in A549 cells exposed to TGF-β1, and in an in vivo model of tumorigenesis [4]. They founded that JWH-015 inhibited EMTE in A549 cells and also reversed the mesenchymal nature of CALU-1 cells by downregulating EGFR signaling. JWH-015 decreased also migratory and invasiveness of A549 cells. In the present study,
we also evaluated the effect of the non-selective cannabinoids agonists THC and CBD by separate or in combination on the EMT in three lung cancer cell lines and we explored the additive effect of CBD in combination with THC.

In this study, we investigated the correlation of the expression levels of CB1 and CB2 with the clinical and pathological features of 157 samples of well-characterized lung tumors. We determined the effect of the non-selective cannabinoid agonists CBD and THC by separate or in combination on cell proliferation, the expression of EGFR, the EMT and migration of A549, H460 and H1792 cells exposed to TGF-β or EGF. We found an additive effect of THC/CBD, which support the use of this combination in order to minimize the dose of THC and its psychotropic effects. The results emphasize the importance of the endocannabinoid system, as well as the potential use of CB1 and CB2 as biomarkers of survival in patients with NSCLC.