A Comparative Analysis of the Action Mechanisms of Cannabidiol, Cannabigerol, and Cannabinol

A Comparative Analysis of the Action Mechanisms of Cannabidiol, Cannabigerol, and Cannabinol in Human Cholangiocarcinoma Cell Lines

Source / Quelle: https://www.mdpi.com/1420-3049/31/14/2446

Abstract

Background: Chemoresistance remains a major obstacle in managing cholangiocarcinoma (CCA). The cannabis plant contains several phytocannabinoids, including cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN), which exhibit anticancer properties. However, to the best of our knowledge, their effects on CCA have not been previously investigated. This study aimed to explore the molecular mechanisms underlying the anticancer effects of CBD, CBG, and CBN in CCA cells. Methods: KKU-100 and KKU-452 cells were treated with varying concentrations of CBD, CBG, and CBN for 24 and 48 h. Cytotoxicity was assessed using the MTT assay, and half maximal inhibitory concentration (IC50) values were calculated. KKU 452 cells were further analyzed for apoptosis, mitochondrial membrane potential (MMP), and Ki67 expression using flow cytometry. Proteomics profiling was performed to compare the effect of these cannabinoids with those of gefitinib and cisplatin. Results: Monotherapy with CBD, CBG, or CBN induced dose-dependent cytotoxicity at 24 and 48 h with lower IC50 values than those of cisplatin and comparable efficacy to that of gefitinib. At low doses, CBD, CBG, and CBN induced early apoptosis, while higher doses triggered late apoptosis. MMP loss increased by 2.5-, 4.9-, and 1.7-fold, respectively, after 6 h. Ki67, highly expressed in KKU-452 cells (Ki67-positive ratio = 3.16 ± 0.16), was significantly reduced after the cannabinoid treatment, with Ki67-positive ratios of 0.38 ± 0.22, 0.38 ± 0.13, and 0.32 ± 0.23 for CBD, CBG, and CBN, respectively. Proteomics analysis identified 2781 proteins affected by CBD, CBG, CBN, cisplatin, and gefitinib. All three cannabinoids downregulated key upstream regulatory proteins (LARP1, TFEB, and BCR). Similar patterns of LARP1 and TFEB downregulation were also observed with cisplatin and gefitinib. CBN showed the closest similarity to cisplatin, followed by gefitinib, by targeting CDK4/6 and PCGEM1 proteins. CBD and CBG exhibited the greatest similarity to each other, also influencing MASTL expression. Conclusions: CBD, CBG, and CBN exhibit potential anticancer activity in CCA by suppressing proliferation, reducing Ki67 expression, and inducing apoptosis through MMP disruption. The identification of shared molecular targets, including LARP1 and TFEB, provides new mechanistic insight and supports the potential development of cannabinoid-based therapeutic strategies for cholangiocarcinoma.

Keywords:

 cannabidiol; cannabigerol; cannabinol; cholangiocarcinoma; anticancer; apoptosis; Ki67; proteomics; cisplatin; gefitinib

1. Introduction

Cholangiocarcinoma (CCA) is an aggressive adenocarcinoma of the hepatobiliary system, classified into intrahepatic, perihilar, and distal subtypes [1]. The incidence and mortality rate of intrahepatic CCA have been rising, with ~50% of cases diagnosed in patients without identifiable risk factors [2]. The highest incidence of CCA has been reported in northeastern Thailand [3], where it is associated with infection by the liver fluke Opisthorchis viverrini [4,5], which the International Agency for Research on Cancer has classified as a group 1 carcinogen [6]. Nevertheless, the incidence of CCA in this region has decreased since 2002 [7]. In most other countries, where liver fluke infection is not endemic, CCA remains a rare malignancy. Non-fluke-related risk factors for CCA include cholangitis, cirrhosis, choledochal cyst (particularly type V), and Caroli’s disease [2]. For patients with unresectable CCA and poor prognosis, the current first-line treatment is a combination of gemcitabine and a platinum derivative [8,9,10], most notably cisplatin, which serves as a critical clinical backbone for systemic chemotherapy. Second-line chemotherapy regimens typically comprise fluoropyrimidine-based monotherapy or combinations of folinic acid, fluorouracil, and oxaliplatin (FOLFOX) [9,11,12,13]. Molecular targeted therapies have also emerged as treatment options for unresectable disease, including inhibitors of fibroblast growth factor receptor (FGFR) [14] and epidermal growth factor receptor (EGFR) [15]. Among these, gefitinib, an EGFR inhibitor, represents a key benchmark for evaluating molecular targeted interventions against aberrant receptor tyrosine kinase signaling. However, long-term treatment is frequently complicated by chemoresistance and high recurrence rates. Several studies indicate that median overall survival with first-line chemotherapy does not exceed 12.2 months [8,9,16]. Therefore, new therapeutic strategies for CCA are currently being explored both in vitro and in clinical trials. Among these, plant-derived anticancer agents represent a promising avenue for combating CCA.

Cannabis plants belong to the Cannabaceae family and include the species Cannabis sativa, Cannabis ruderalis, and Cannabis indica. For example, Cannabis sativa L. has been used as an herbal remedy since the Han Dynasty in Ancient China and in Indian Ayurvedic medicine [17]. In Thailand, cannabis has been legal since February 2022. Cannabis is the primary source of several phytocannabinoids [18,19]. Notable cannabinoids such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN) have been used as antiemetics in patients receiving chemotherapy [20,21]. THC and non-THC cannabinoids have demonstrated potential anticancer effects in vitro and in vivo across a variety of cancer models, including lung, breast, prostate, hepatocellular, pancreatic, lymphatic, and skin cancers, as well as CCA [17,22,23,24]. CBD and CBG are non-psychoactive cannabinoids, with CBD present in relatively high amounts in cannabis, while CBG and CBN are considered minor cannabinoids. Previous studies have shown that CBD and CBG induce cytotoxicity in CCA cells by stimulating autophagic, apoptotic and senescence signaling pathways and inhibiting cell migration and invasion [24,25,26]. CBN, which exhibits only mild psychoactive effects, has also been shown to exert antiproliferative activity [27] and enhance chemosensitization by inhibiting the multidrug transporter ABCG2 [28,29]. The anticancer effects of these compounds are often attributed to their ability to activate cannabinoid receptors or transient receptor potential (TRP) vanilloid type-1 (TRPV1) channels, leading to promotion of cell death, inhibition of cell proliferation, and disruption of tumor-related processes including angiogenesis, migration, invasion, adhesion, and metastasis [30,31,32,33,34,35,36,37,38,39,40,41,42,43,44]. However, the molecular mechanisms underlying the anticancer effects of CBD, CBG, and CBN in CCA have not yet been elucidated. To resolve these mechanisms, we investigated their effects through apoptosis analysis, mitochondrial membrane potential assessment, Ki67 proliferation analysis, and quantitative high-throughput proteomic profiling. We hypothesized that the non-psychoactive cannabinoids CBD, CBG, and CBN induce cytotoxicity and apoptosis in CCA cells by disrupting mitochondrial membrane potential and suppressing cell proliferation, mediated through the modulation of candidate upstream regulators (such as LARP1 and TFEB) that converge with pathways targeted by standard clinical chemotherapeutic benchmarks.

2. Results

2.1. Cytotoxic Activity of CBD, CBG, and CBN

The cytotoxic effects of cannabinoids (CBD, CBG, and CBN), cisplatin, and gefitinib on cholangiocarcinoma cells were evaluated using the MTT assay. All three cannabinoids suppressed CCA cell proliferation in a concentration and time-dependent manner. CBD, CBG, CBN, cisplatin, and gefitinib induced cytotoxicity both in KKU-452 (Figure 1A–E) and KKU-100 (Figure 1F–J) cells after 24 and 48 h of treatment. At 16 μM, CBD, CBG, and CBN produced maximal growth inhibition that persisted through 48 h. In KKU-452 cells, the half-maximal inhibitory concentration (IC50) values for CBD, CBG, and CBN were 4.49 ± 1.34, 3.51 ± 1.40, and 4.12 ± 1.32 μM at 24 h, and 0.58 ± 0.18, 0.54 ± 0.07, and 0.50 ± 0.10 μM at 48 h, respectively (Table 1). Similar dose- and time-dependent response patterns were observed in KKU-100 cells, with IC50 values of 6.11 ± 3.83, 4.60 ± 2.06, and 7.20 ± 4.50 μM at 24 h, and 2.34 ± 0.97, 2.14 ± 0.96, and 3.70 ± 1.90 μM at 48 h, respectively (Table 1). These data indicate that KKU-452 cells are more sensitive to CBD, CBG, and CBN than KKU-100 cells. The results also demonstrate that CBD, CBG, and CBN exert potent cytotoxic effects that surpass those of cisplatin (a conventional chemotherapy drug) and gefitinib (an EGFR-targeted therapy) in both CCA cell lines. Moreover, CCA cells displayed resistance to cisplatin, whereas gefitinib showed a good response, especially in KKU-100 cells at 48 h.

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