Acute anti-proliferative and anti-migratory effects of cannabidiol on C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 mouse hippocampal neuronal cell cultures
Source / Quelle: https://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2026.1727831/full
Abstract
Background:
The treatment of central nervous system tumors remains challenging owing to their highly proliferative nature, aggressiveness, and poor prognosis. Additionally, existing treatment methods have several problems, including high risk of complications, systemic side effects, and impact on patients’ quality of life. Recently, cannabidiol (CBD), a non-psychoactive cannabinoid found in Cannabis sativa, has emerged as an alternative therapeutic medication because of its potential antitumor activity with fewer side effects.
Methods:
We evaluated the cell viability, clonogenicity, migration, apoptotic nuclear morphology, and cell cycle phases of C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 immortalized mouse hippocampus neuronal cultures treated with CBD ranged between 0 and 10 μg/mL.
Results:
CBD concentrations exceeding 5 μg/mL induced significant reductions in cell viability in C6 glioma and SH-SY5Y neuroblastoma cultures, accompanied by decreased clonogenicity in both cultures at 10 μg/mL. A scratch assay for cell migration revealed that 5 μg/mL CBD suppressed C6 glioma cell migration. Additionally, late apoptotic nuclear morphology was observed in C6 glioma cultures treated with 10 μg/mL cannabidiol. Similarly, HT22 hippocampal neuronal cultures exhibited decreased cell viability and clonogenicity, with apparent nuclear signs of apoptosis at CBD concentrations over 5 μg/mL. Notably, CBD disrupted HT22 cell migration at concentrations of 2.5 and 5 μg/mL. Proteomic profiling of C6 glioma revealed upregulation of ribosomal proteins, molecular chaperones, and modulators of cytoskeletal dynamics upon treatment with 1 μg/mL CBD. In comparison, treatment with 2.5 μg/mL CBD led to marked downregulation of endoplasmic reticulum chaperones, mitochondrial ATP synthase, and cytoskeletal regulators.
Conclusion:
Our findings confirm the sensitivity of glioma, neuroblastoma, and hippocampal neuronal cultures to CBD, providing valuable insights for further research into its therapeutic potential against glioma, neuroblastoma, and neuronal disorders.
1 Introduction
Brain and central nervous system (CNS) tumors remain among the most lethal cancer types globally despite their relatively low incidence. According to the latest GLOBOCAN 2022 estimates, approximately 322,000 new cases of brain and CNS tumors were diagnosed worldwide in 2022, with an age-standardized incidence rate of approximately 3.5 per 100,000 and nearly 248,000 associated deaths (Kim et al., 2025). These figures reflect a continued global burden and underscore the urgent need for improved therapeutic approaches, particularly for aggressive subtypes such as glioblastoma. Recent trends also suggest notable regional and demographic variations in incidence and outcomes, with higher rates observed in parts of North America and Western Europe (Filho et al., 2025). The most common type of primary malignant tumor of the CNS is a glioma, which arises from glial cells. Nearly fifty percent of all newly diagnosed gliomas in the US are glioblastomas, the most severe type of glioma with a less than 5% 5-year survival rate (Low et al., 2022; Miller et al., 2021; Ostrom et al., 2014). This poor prognosis may be due to multiple cellular factors, including high proliferation and invasion, angiogenesis induction, and the presence of glioma stem-like cells within the tumor mass (Dumitru et al., 2018; Liebelt et al., 2016), which contribute to the frequent recurrence of malignant tumor cells and their resistance to conventional therapeutic methods. Another well-known tumor of CNS is neuroblastoma, which is the most frequent type of brain and central nervous system tumor in infants as well as young children. Even though neuroblastoma has a better prognosis than glioblastoma, the co-administration of chemotherapy and radiotherapy usually leaves patients with neurological conditions such as weakness or prolonged pain sensation (Matthay et al., 2016; Park et al., 2010). Neurosurgical intervention, chemotherapy, and radiation therapy are the main therapeutic approaches for CNS malignancies currently (Omuro and DeAngelis, 2013; Wen and Kesari, 2008). In recent years, phytochemicals have gained substantial attention as alternative or integrative therapeutic agents due to their ability to modulate multiple oncogenic pathways while generally exhibiting lower systemic toxicity compared with conventional chemotherapeutic agents. Several comprehensive reviews have highlighted the potential of plant-derived compounds in hepatocellular carcinoma, breast cancer, and head and neck cancer, where phytochemicals demonstrate anti-proliferative, pro-apoptotic, anti-angiogenic, and anti-metastatic effects through regulation of oxidative stress, cell cycle checkpoints, and survival signaling pathways (Rodriguez et al., 2021; Santiago et al., 2025; Wali et al., 2025). Unlike many cytotoxic chemotherapies that are associated with severe adverse effects such as myelosuppression, gastrointestinal toxicity, neurotoxicity, and off-target tissue damage, phytochemicals are often reported to exert more selective biological activity with comparatively fewer undesirable systemic complications, although dose optimization and safety evaluation remain essential.
Owing to its antiproliferative and anti-invasive effects reported in both in vitro culture models and animal studies, cannabidiol (CBD) has emerged as a potential anticancer medication (Kis et al., 2019; Velasco et al., 2012; Wang and Multhoff, 2021). CBD application resulted in apoptotic cell death in the A549 and H460 lung cancer cell lines (Ramer et al., 2013) and reduced cell division and migration in diverse types of breast cancer cell lines (Elbaz et al., 2015). In recent years, increasing preclinical and translational studies have investigated the anti-proliferative, pro-apoptotic, and anti-invasive effects of cannabidiol (CBD) in glioma and other CNS malignancies. Recent systematic and translational reviews (Feng et al., 2024; Javid et al., 2025) highlight the growing interest in CBD as a potential adjunct or alternative therapeutic agent in glioma management. CBD and THC synergistically reduce glioma cell proliferation and modulate cell-cycle progression (Marcu et al., 2010), while recent studies in the SH-SY5Y neuroblastoma cell line have indicated that extracts isolated from Cannabis sativa strains with high CBD content can lead to apoptotic cell death (Sanchez-Sanchez et al., 2023) and modulate the expression of genes involved in cell adhesion and mitochondrial activities (Abyadeh et al., 2023). Although accumulating evidence supports the anti-tumor potential of CBD, recent analyses emphasize that its dose-dependent effects and cell-type specificity remain incompletely characterized, particularly in the context of balancing tumor inhibition with neuronal safety (Javid et al., 2025).
Despite increasing mechanistic insights, few studies have directly compared tumor-derived and non-malignant neuronal cell models under identical experimental conditions. Moreover, global proteomic responses to sublethal CBD exposure in glioma cells remain insufficiently characterized. Therefore, the present study aimed to compare the CBD-treated neuronal and glial tumor cell lines in terms of their cell viability, clonal formation, migration, and apoptotic morphology. In addition, we performed label-free quantitative proteomic analysis in C6 glioma cells exposed to sublethal CBD concentrations to identify early molecular alterations associated with cellular stress and anti-migratory effects. An immortalized hippocampal neuronal cell line (HT22) was included to comparatively evaluate the potential cytotoxic effects of CBD on non-malignant neuronal cells. This approach allows comparative evaluation of tumor and neuronal sensitivity to CBD exposure and provides preliminary insight into whether a potential therapeutic window may exist. The findings of this study can provide novel insights into the safe and effective administration of CBD in in vivo models and clinical trials.
2 Materials and methods
2.1 Cell culture and cannabidiol
The glioma cell line C6 derived from Wistar rat brain (CCL107) and human neuroblastoma cell line SH-SY5Y (CRL-2266) were acquired from the American Type Culture Collection (ATCC) and chosen in this study as archetypal tumor cell lines of CNS. The immortalized neuronal cell line HT22 (SCC129) derived from the mouse hippocampus was sourced from EMD Millipore and was used as a model of immortalized neurons. Dulbecco’s modified Eagle’s medium (DMEM) (ATCC, 30-2002) was used as a culture medium for maintaining the C6 and HT22 cell lines, while DMEM/F-12 Ham mixture (Millipore, DF-042-B) was used for maintaining SH-SY5Y cells. Both types of culture media were completed with 10% fetal bovine serum (FBS) (Sigma-Aldrich, F7524) and 10 mL/L of penicillin/streptomycin (Sigma-Aldrich, P4333). Cultures were maintained in a humidified incubator at the conditions of 5% CO2 at 37 °C, and the culture media were replaced with new media every 2-3 days. Dr. CBD Co., Ltd. (Bangkok, Thailand) provided the powdered CBD that we reconstituted and used in all experiments. We dissolved powdered CBD in dimethyl sulfoxide (DMSO) to create a 3 mg/mL stock solution, which we subsequently aliquoted, kept at −20 °C, and protected from light until use. In all experiments, vehicle control groups received an equivalent concentration of DMSO (0.1% v/v), which did not affect cellular outcomes.
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