Beta‐Caryophyllene Augments Radiotherapy Efficacy in GBM by Modulating Cell Apoptosis and DNA Damage Repair via PPARγ and NF‐κB Pathways
Department of Biomedical Imaging and Radiological Sciences National Yang Ming Chiao Tung University Taipei Taiwan
Department of Radiology Cathay General Hospital Taipei City Taiwan
School of Medicine Fu‐Jen Catholic University New Taipei City Taiwan
Division of Radiation Oncology, Department of Radiology Far Eastern Memorial Hospital New Taipei City Taiwan
* Correspondence:Hui‐Yen Chuang (huiyen@nycu.edu.tw)
ABSTRACT
Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy with limited treatment options. Radiotherapy (RT) is often used for treating unresectable GBM; however, the outcomes are often limited due to the radioresistance of GBM. Therefore, the discovery of potential radiosensitizers to enhance GBM responses to RT is crucial. Beta‐caryophyllene (BCP), a natural cannabinoid, promotes cancer apoptosis by upregulating the PPARγ signaling pathway and can cross the blood–brain barrier due to its lipophilic nature. This study aimed to evaluate the radiosensitizing potential of BCP in GBM cells. U87MG and GL261 cells and a GL261 tumor‐bearing model were treated with RT, BCP, or both. Treatment efficacy was assessed using the MTT assay and tumor growth tracking, and the underlying mechanisms were investigated using western blotting, immunofluorescence staining, and other analyses. BCP synergistically enhanced the efficacy of RT in cell culture, as evidenced by the combination index determined through the MTT assay. This enhancement was mediated by the BCP‐induced deceleration of DNA damage repair, as demonstrated by sustained γH2AX signal, upregulated PPARγ levels, and reduced expression of pAKT, pERK, and NF‐κB, indicating apoptosis induction and inhibition of survival pathways. BCP significantly inhibited tumor growth in GL261 tumor‐bearing mice with no discernible side effects. These findings indicate that BCP may serve as a potential radiosensitizer for improving RT outcomes in GBM by inhibiting DNA repair, inducing apoptosis, and suppressing anti‐apoptotic and survival pathways.
Graphical
The Importance of the Current Study: Radiotherapy (RT) is a standard treatment for brain tumors, but the prognosis for aggressive glioblastoma multiform (GBM) remains poor, largely due to radioresistance. Thus, the identification of effective radiosensitization strategies is urgently needed to improve GBM outcomes. Beta‐caryophyllene (BCP), a cannabinoid with antitumor properties, presents a promising avenue for enhancing RT effectiveness by activating the PPARγ signaling pathway and inducing apoptosis. Moreover, given the expanding medical applications of cannabinoids, this study investigated the potential of BCP as a radiosensitizer for GBM and elucidated its underlying therapeutic mechanisms. This study can significantly impact GBM treatment paradigms by introducing innovative therapeutic approaches to combat GBM and improve outcomes.
Boxed Text
Article notes
Footnote Group
- BCP
- Beta‐Caryophyllene
- CB1
- Cannabinoid receptors type 1
- CB2
- Cannabinoid receptors type 2
- CI
- Combination index
- GBM
- Glioblastoma multiform
- RT
- Radiotherapy
1Introduction
Glioblastoma multiform (GBM), a grade IV astrocytoma (Ostrom et al. 2014), is the most common and aggressive primary malignant brain tumor, with a 5‐year survival rate of approximately 5% (Chang et al. 2016). Radiotherapy (RT) primarily serves as an adjuvant treatment for brain tumors, yet it is the standard approach for unresectable GBM (Fernandes et al. 2017). However, current medical interventions only extend the median survival of patients with GBM by 15 months (Stupp et al. 2005). The resistance of GBM to therapies is attributable to various factors, including the presence of cancer stem cells, tumor heterogeneity, hypoxic microenvironment, and apoptosis evasion, which often cause radioresistance and treatment failure post‐RT (Ali et al. 2020; Valdes‐Rives et al. 2017). Given the importance of RT in GBM treatment and the poor prognosis of patients with GBM, the discovery of potential radiosensitizers augmenting the efficacy of RT in managing unresectable GBM is crucial.
Recently, the use of cannabinoids for cancer treatment has garnered attention recently. Cannabinoids have been used to treat various cancer types, including colon cancer (Romano et al. 2014), breast cancer (Caffarel et al. 2010), pancreatic cancer (Sakarin et al. 2022; Sharafi, He, and Nikfarjam 2019), and GBM (Gurley et al. 2012; Lopez‐Valero, Torres, et al. 2018; McAllister et al. 2005). Cannabinoids interact with cannabinoid receptors type 1 (CB1) and type 2 (CB2), modulating signaling pathways by acting as either agonists or antagonists. CB1 is mainly expressed in the central nervous system, whereas CB2 is expressed by immune and cancer cells. Recent results obtained from the Xena database revealed a strong correlation between higher CB2 expression and poorer overall survival in patients with GBM (Goldman et al. 2020). Furthermore, studies have shown elevated CB2 expression and unchanged CB1 expression in GBM cells compared with surrounding normal cells (Sanchez et al. 2001). Therefore, targeting CB2 has emerged as a potential solution for treating GBM while causing minimal side effects (Dumitru, Sandalcioglu, and Karsak 2018; Schley et al. 2009).
RT induces cell death and tumor suppression through various mechanisms, including PPARγ activation (Kaur et al. 2019). Conversely, RT‐triggered PI3K/AKT and MAPK signaling pathways, along with subsequent NF‐𝛋B activation, are often observed in surviving cells post‐RT, leading to enhanced cell survival and radioresistance (Pordanjani and Hosseinimehr 2016; Singh, Gupta, and Arora 2015). NF‐𝛋B functions as a critical pathway hub, regulating immunity, cellular survival/apoptosis, and angiogenesis, significantly impacting treatment outcomes (Antonangeli et al. 2020; Puliyappadamba et al. 2014).
Beta‐caryophyllene (BCP), a hydrophobic cannabinoid with a bicyclic sesquiterpene structure (logP = 6.3), has been explored in various intracranial studies (Mallmann et al. 2022; Serra et al. 2022), demonstrating its potential in the treatment of brain‐related diseases. Notably, BCP‐mediated PPARγ activation and NF‐𝛋B suppression have been demonstrated in GBM cell lines (Irrera et al. 2020).
Based on these observations, we hypothesized that BCP could serve as a potential candidate for enhancing RT efficacy in GBM. Although the anticancer properties of BCP have been demonstrated across several cancer types (Arul et al. 2020; Dahham et al. 2021; Irrera et al. 2020), its radiosensitization potential remains unexplored. In this study, we validated the radiosensitization potential of BCP using U87MG and GL261 cells. Our results suggest that BCP‐mediated radiosensitization is related to PPARγ‐mediated apoptosis and DNA repair impairment. Furthermore, combined treatment with BCP and RT significantly slowed tumor growth in subcutaneous GL261‐bearing mice compared with the other treatment groups.
2Materials and Methods
2.1Survival Analysis of Patients With GBM
The effects of CB2 expression on overall survival in patients with GBM were investigated by analyzing data from the TCGA Glioblastoma dataset accessed through the UCSC Xena platform (Goldman et al. 2020). Using the Xena platform, we conducted a survival analysis focusing on the 1‐year survival of patients with high and low expression levels of CB2 encoded by the CNR2 gene. From the Xena database, we obtained 166 GBM gene expression profiles. These profiles were further divided into two groups: those with CNR2 expression levels < 0.575 (indicating low expression) and those with CNR2 expression levels ≥ 0.575 (indicating high expression). Subsequently, a 1‐year clinical survival curve was generated based on this stratification, and the outcomes were visualized using Kaplan–Meier curves.
2.2Cell Lines
The human glioblastoma cell line U87MG and murine glioma cell line GL261 were used in this study. U87MG and GL261 cells were grown in Minimum Essential medium (MEM, #61100061; Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) and Dulbecco's modified Eagle's medium (DMEM, #12100046, Gibco; Thermo Fisher Scientific), respectively. All media were supplemented with 10% fetal bovine serum (#35‐010‐CV; Corning, Glendale, AZ, USA), 100 IU/mL penicillin, and 0.1 mg/mL streptomycin (Penicillin–Streptomycin Solution, #30‐002‐CI; Corning). Both cell lines were obtained from Prof. Hsin‐Ell Wang (Dept. Biomedical Imaging and Radiological Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan). All the cells were maintained at the exponential growth phase in a humidified CO2 incubator at 37°C.
2.3Drug Preparation
A fresh stock solution of BCP (#22075, ≥ 80%, FCC, FG, Sigma‐Aldrich, Burlington, MA, USA) was prepared for cell treatment due to its susceptibility to oxidation. The BCP solution was prepared by dissolving 1 μL of the purchased BCP (4.4 M) in 139.8 μL of DMSO to obtain a final BCP concentration of 31.25 mM.
2.4Cytotoxicity Assay
We conducted the MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay to assess BCP‐induced cytotoxicity. Various concentrations of the BCP solution were prepared by diluting the stock solution with the medium while maintaining a constant DMSO volume concentration of 0.8% across all wells to ensure that the observed differences in cell viability were solely due to BCP. U87MG or GL261 cells were seeded at a density of 1 × 104 cells per well in a 96‐well plate 1 day before being treated with BCP for 24 h. Cell viability was determined using the MTT assay. Dose–response curves for both cell lines were plotted to determine the IC50 of BCP using GraphPad Prism9 (version 9.5.0, GraphPad Software Inc., San Diego, CA, USA).
2.5Radiation Survival Curve
The cells were irradiated with various radiation doses (0, 2, 4, 6, and 8 Gy) using a cabinet irradiator (RS2000, Rad Source, Buford, GA, USA) at a dose rate of 2.9 Gy/min. Based on the receiving radiation dose, different numbers of cells were seeded in 6‐cm dishes and incubated for 14 days. The colonies were then fixed, stained, and examined under a stereo microscope. Only colonies with more than 50 cells were counted to calculate the surviving fractions. The radiation survival curves were generated by plotting the surviving fractions against the radiation dose. The plating efficiency and surviving fraction were calculated using the following equations:
2.6Calculation of the Combination Index
The cells were seeded at a density of 5 × 104 cells per well in a 24‐well plate. Before irradiation, they were pre‐treated with various BCP concentrations for 4 h. The BCP‐containing medium was replaced with a regular medium 24 h after treatment. Cell viability was assessed using the MTT assay after an additional 48‐h incubation. The combination index was calculated from cell viability data to evaluate the interactions between RT and BCP (Hsu et al. 2014).
2.7 γH2AX Immunofluorescent Staining
The cells were first seeded on the coverslips and placed in a six‐well plate before treatment. Following a 4‐h treatment with BCP, the cells were subjected to X‐ray irradiation. U87MG cells were treated with 70 μM BCP and an 8 Gy X‐ray dose, whereas GL261 cells were treated with 60 μM BCP and a 5 Gy X‐ray dose. At 24 and 48 h post‐treatment, the cells were washed with PBS, fixed with ice‐cold acetone for 5 min, and then blocked with 0.5% Tween 20 solution containing 1% BSA for 45 min. The cells were then incubated with an anti‐γH2AX antibody (#9718, Cell Signaling) overnight at 4°C. After incubation, the coverslips were stained with Alexa Fluor 488‐conjugated secondary antibody (#ab150077) and DAPI solution, followed by mounting. Cell images were captured using an Olympus BX61 fluorescence microscope equipped with a CCD camera. Image analysis was performed using ImageJ software to quantify fluorescent signals.
2.8Western Blotting
The cells were treated as described above. The cells were collected and lysed with RIPA buffer containing 1 mM PMSF, and the protein concentrations were determined using the Bradford assay. A total of 30 μg of cell lysates were separated on a 12% SDS‐PAGE gel, followed by protein transfer onto a PVDF membrane. After blocking with 5% nonfat milk or 5% BSA at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against the target proteins. Next, the membranes were incubated with HRP‐conjugated secondary antibodies (Genetex, Irvine, CA, USA) for 1 h at room temperature. Protein expression was detected using enhanced chemiluminescence (ECL) reagent (#LF08‐500, Visual Protein, Taipei, Taiwan), and the signals were captured using the Luminescence/Fluorescence Image System LAS‐4000. Band intensities were quantified using ImageJ software.
The primary antibodies used include the following: CB2 (ARG40813, Arigo, Taiwan), PPARγ (ARG55241, Arigo), phosphor‐AKT (4058S, Cell Signaling), phosphor‐ERK (9101S, Cell Signaling), Cyclin D1 (2978S, Cell Signaling), Cyclin E1 (A22360, ABclonal, Germany), CDK2 (A0094, ABclonal), phosphor‐RB (AP0444, ABclonal), Bax (GTX109683, Genetex), Bcl‐xL (2764 T, Cell Signaling), cleaved caspase 3 (ARG57512), NF𝛋‐B p65 (8242S, Cell Signaling), and β‐actin (GTX109639, Genetex). β‐actin served as an internal control.
2.9 NF‐κB p65 Transcription Factor Assay
The human nuclear factor kappa‐B p65 subunit (NF‐κB/p65) concentration in cellular samples following the different treatments was determined using an ELISA kit (E‐EL‐H1388, Elabscience Biotechnology Inc., Houston, TX, USA). The ELISA assay was performed following manufacturer‐provided protocols. The optical density was measured at 450 nm using a Multimode microplate reader (TECAN Infinite 200 PRO, Switzerland).
2.10Therapeutic Evaluation of the GL261 Tumor‐Bearing Mice Model
First, 2 × 106 GL261 cells were subcutaneously inoculated into the right flank of 6‐week‐old male C57BL/6 mice purchased from the National Laboratory Animal Center, Taiwan. Treatments were initiated when the tumor volume reached 80–100 mm3. The GL261‐bearing mice were randomly divided into four groups: control (CTRL), RT, BCP, and combination (COMB). Olive oil was used as the vehicle for diluting BCP. In the CTRL and RT groups, the mice were intraperitoneally administered 50 μL of olive oil daily for 14 days. Mice in the BCP and COMB groups were intraperitoneally injected with 200 mg/kg BCP for 14 days. On day 4 after the first vehicle or BCP dose, mice in the RT and COMB groups received 4 Gy of X‐ray radiation. Figure S1 shows the treatment protocol. Tumor volumes and body weight were monitored every 3 days until the tumor sizes reached 1500 mm3, which was set as the experimental endpoint. The Institutional Animal Care and Use Committee of National Yang Ming Chiao Tung University approved all animal procedures (IACUC No. 1101012). The tumor volume was calculated using Equation (4), and the tumor inhibition rate for each treatment was calculated based on the tumor volume on day 19 using Equation (5).
2.11Tumor Sectioning and Immunohistochemistry
After the experiment ended, the tumors were embedded in paraffin and sectioned into slices 10‐μm thick using a microtome. Antigen retrieval was performed after deparaffinization and sample rehydration. Antibodies targeting cleaved caspase‐3 (GTX86952, Genetex) and NF‐κB p65 (8242S, Cell Signaling) were used for immunohistochemistry. Images were captured using a light microscope equipped with a digital camera.
2.12Statistical Analysis
Statistical analysis was conducted using GraphPad Prism 9 software version 9.5.0 (GraphPad Software Inc.; San Diego, CA, USA). Student's t‐test was used to compare differences between two groups, and one‐way ANOVA was used when analyzing more than two groups. Two‐way ANOVA was used to determine statistical differences between groups in vivo. A p‐value < 0.05 was considered statistically significant. Data are presented as mean ± standard error mean (SEM). For in vitro experiments, the results were obtained from at least three independent experiments. For in vivo therapeutic evaluations, experiments were conducted twice with n = 4 per group.
3Results
3.1 BCP Synergistically Enhanced the RT‐Induced Cell Death Effect
We first investigated the effects of CB2 expression on GBM prognosis by retrieving data from the Xena database and comparing overall survival time between GBM patients with high and low CB2 expression. Patients with higher CB2 expression levels had significantly poorer overall survival compared with those with lower CB2 expression (p < 0.05) (Figure S2). This suggests that CB2 is a potential target for GBM treatment. Western blotting was conducted to validate the CB2 expression in U87MG and GL261 cells (Figure 1A). BCP as a CB2 agonist was confirmed by the MTT assay results, which caused a dose‐dependent reduction in cell viability in both cell lines (Figure 1B,C), with IC50 values of approximately 140 and 120 μM for U87MG and GL261 cells, respectively.
BCP‐mediated radiosensitization was evaluated using the MTT assay results obtained 72 h post‐BCP treatment. As shown in Figure 2A,B, cell viability decreased with increasing BCP or radiation dose. Calculating the combination index (CI) based on the MTT results provided insights into the interaction between BCP and RT. A CI value < 1 indicates a synergistic interaction between treatments. Figure 2C,D illustrate the CI values obtained under various conditions. BCP synergistically enhanced RT efficacy in U87MG cells in a dose‐dependent manner at irradiation doses exceeding 8 Gy. Notably, BCP‐mediated synergistic radiosensitization was more robust in GL261 cells, even at lower irradiation doses. These findings indicate that BCP can act as a radiosensitizer and enhance RT‐induced cytotoxicity in GBM cells, especially when combined with higher irradiation doses.
Combination treatments using half of the IC50 dose of BCP at 8 and 5 Gy irradiation doses were used in U87MG and GL261 cells, respectively. These chosen irradiation doses yielded similar surviving fractions, as indicated by the colony formation assay (Figure S3).
3.2 BCP Inhibits RT‐Induced DNA Damage Repair
Ionizing radiation (IR) causes various forms of DNA damage, potentially resulting in cell death if the damage is irreparable. Upon DNA damage, cells activate several proteins, including 53BP1 and γH2AX, to initiate DNA repair mechanisms. To investigate the effects of BCP on IR‐induced DNA damage repair, we conducted γH2AX immunofluorescence staining. The RT and COMB groups exhibited significantly increased γH2AX levels compared with the CTRL group in both cell lines at 24 h (Figure 3). However, BCP treatment did not increase γH2AX levels in either cell line. At 48 h, the COMB group maintained the highest γH2AX level among all groups, whereas the γH2AX signals in the RT group were reversed to baseline levels, similar to those of the CTRL group in both cell lines. These results suggest that BCP sensitizes GBM cells to RT by inhibiting RT‐induced DNA damage repair, as evidenced by the higher γH2AX signal level observed in the COMB group compared with the RT group at 48 h.
3.3 BCP Enhances RT‐Induced G2/M Arrest and Halts Cell Cycle Progression in GBM Cells
The combination of BCP and RT augmented RT‐induced G2/M arrest in both cell lines (Figure 4), with the more pronounced effects found in GL261 cells (Figure 4B). Interestingly, RT did not change the frequency of the S phase in either cell line. Alternatively, the RT‐induced G2/M arrest was correlated with the G0/G1 reduction, especially in GL261 cells. Although the G0/G1 reduction was modest in the U87MG cells, the GL261 cells showed a nearly 20% and 30% reduction after RT and combination treatment, respectively.
In U87MG cells, RT upregulated Cyclin D1, Cyclin E1, CDK2, and pRB, which were suppressed by the combination treatment (Figure 4C). Likewise, GL261 cells had similar changes in Cyclin E1 and pRB after RT and combination treatment (Figure 4D). Unlike in the U87MG cells, RT inhibited Cyclin D1 expression in the GL261 cells and was further enhanced when combined with BCP.
3.4 BCP Enhances PPARγ and Pro‐Apoptotic Protein Expression While Reducing Survival‐Associated Protein and NF‐κB Levels in RT‐Treated GBM Cells
Changes in protein expression resulting from different treatments were evaluated via western blotting to elucidate the possible mechanisms underlying BCP‐mediated radiosensitization. Figure 5A,B demonstrates that combining BCP with RT elevated PPARγ, Bax, and cleaved caspase‐3 levels in both cell lines, indicating that the combination treatment may promote cell apoptosis. Given that the MTT assay only reflects a relative cell survival without differentiating between growth inhibition and apoptosis, we then analyzed the changes in pAKT and pERK in cells subjected to different treatments. AKT and ERK activation plays a critical role in sustaining cell survival by inhibiting apoptosis and promoting proliferation when encountering genotoxic stress like RT (Kim et al. 2006; Lu et al. 2020; Qiao et al. 2017). Figure 5C,D shows that BCP repressed RT‐mediated pAKT and pERK upregulation in both cell lines. These findings suggest that BCP enhances the radiosensitivity of GBM cells by activating the PPARγ signaling pathway while suppressing the AKT and ERK signaling pathways to promote apoptosis.
AKT and ERK activation can lead to the nuclear translocation of NF‐𝛋B, a transcription factor that regulates multiple cellular responses, potentially resulting in treatment resistance and tumor recurrence. Given that BCP represses RT‐induced AKT and ERK activation, we further examined the NF‐𝛋B levels in each group. Figure 6 shows that the COMB group exhibited the lowest NF‐𝛋B (p65 subunit) expression among all groups in both U87MG (Figure 6A) and GL261 cells (Figure 6B), indicating that BCP may prevent RT‐induced radioresistance by reversing the NF‐𝛋B activation. In addition, the fluorescent images of NF‐𝛋B and NF‐𝛋B activity assay show a similar trend, suggesting that BCP combined with RT downregulates NF‐𝛋B signal transduction (Figure S4).
3.5 BCP Augments the Therapeutic Efficacy of RT in the Subcutaneous GL261 Tumor‐Bearing Mice Model
We established a subcutaneous GL261 tumor‐bearing mice model to evaluate the therapeutic efficacy of the combined treatment in vivo. Figure S1 shows the experimental design in detail. Mice in the BCP and CTRL groups were administered 200 mg/kg BCP or an equal volume of vehicle for 14 days, whereas those in the COMB and RT groups received 4 Gy RT on day 4 after the first BCP or vehicle dose. Consistent with the in vitro results, the combination treatment exhibited the most significant tumor inhibition among all groups, followed by the RT and BCP groups (Figure 7A). RT‐induced tumor suppression was observed from days 7–10 in the RT and COMB groups. However, RT‐mediated tumor suppression was transient, as a substantial increase in tumor volume was observed from days 10–19 in the RT group. Conversely, the combination treatment effectively repressed tumor growth. On day 19, the tumor volume differed significantly between the COMB and RT groups, indicating that BCP enhances the therapeutic efficacy of RT.
The tumor inhibition rates on day 19 for the BCP, RT, and COMB groups were 15.3% ± 28.4%, 61.2% ± 21.7%, and 83.9% ± 12.7%, respectively (Figure 7B). The tumor inhibition rate in the COMB group suggested that BCP synergistically enhanced the RT‐mediated tumor suppression. Notably, 200 mg/kg BCP did not inhibit tumor growth in vivo as the tumor growth curve of the BCP group was similar to that of the CTRL group. Furthermore, the overall survival time of the different treatments was recorded (Figure 7C). The COMB group had the longest survival time compared with the other groups. The therapeutic evaluation confirmed that BCP is an ideal radiosensitizer for treating GL261 tumors in vivo. Regarding general toxicity, all treatments had negligible effects on body weight, as shown in the body weight tracking results (Figure 7D). Figure S6 shows the individual tumor growth curves in long‐term survival studies. Additionally, BCP combined with RT increased cleaved caspase‐3 and decreased NF‐κB levels in tumors (Figure 8A,B), consistent with the in vitro findings. The combination treatment resulted in the most profound tumor suppression and extended survival time without causing general toxicity in tumor‐bearing mice.
4Discussion
Our study elucidated the potential of combining BCP with RT to enhance the therapeutic efficacy against GBM. Initially, we evaluated the radiosensitization effect of BCP by calculating the CI based on the MTT assay results. Figure 2 demonstrates that BCP significantly and synergistically increased RT‐mediated cell killing, as evidenced by CI values < 1, particularly at higher radiation doses in both cell lines. RT induces DNA double‐strand breaks (DSBs), activating DNA damage response (DDR) pathways for repair. However, cancer cells may succumb to DNA damage if repair mechanisms fail (Lord, Garrett, and Ashworth 2006). Phosphorylation of histone H2AX is a crucial step in DDR, marking sites of DSBs. Therefore, increased γH2AX signal postirradiation reflects DNA damage. As shown in Figure 3, elevated γH2AX signal persisted at 48 h postirradiation in the COMB group, suggesting that BCP inhibits RT‐induced DSB repair. Similar inhibition of DNA damage repair by cannabinoids has been reported in brain tumors treated with RT and temozolomide (TMZ) (Scott, Dalgleish, and Liu 2014; Soroceanu et al. 2022).
RT‐induced DNA damage triggers G2/M arrest as a protective mechanism against cell division in the presence of damaged DNA (Pawlik and Keyomarsi 2004). Our findings demonstrate that BCP enhances RT‐induced G2/M arrest in both cell lines (Figure 4A,B). Additionally, markers critical for the G1‐S transition, including Cyclin E1, CDK2, and pRB, were downregulated in the COMB group compared with the RT and BCP groups, suggesting inhibition of cell cycle progression. Interestingly, unlike previous reports on A549 and NCI‐H358 cells (Chung et al. 2019), BCP‐induced G1 arrest and CDK2, CDK4, CDK6, cyclin D1, and cyclin E1 downregulation were not observed in this study. These results may be related to the lower BCP concentration used in this study (half the IC50 value determined using the MTT assay). Echoing the western blotting results, the γH2AX staining (Figure 3) demonstrated that the combination treatment enhanced the RT‐induced DNA damage by inhibiting DNA repair and halting cell cycle progression.
RT can activate pro‐survival mechanisms, including the AKT and ERK signaling pathways, leading to NF‐𝛋B activation and expression of downstream effectors, thereby promoting treatment resistance (Bai, Ueno, and Vogt 2009; Chen et al. 2016). Correlations between increased AKT phosphorylation and reduced RT efficacy have been established in various cancers, including head and neck squamous cell carcinoma (Gupta et al. 2002), lung carcinoma (Blackhall et al. 2003), breast cancer (Schmitz et al. 2004), prostate cancer (Liao et al. 2003), and glioblastoma (Choe et al. 2003). Impaired treatment efficacy is often associated with AKT‐mediated inhibition of apoptosis and cell death (Kim et al. 2006; Vivanco and Sawyers 2002). Our study revealed that BCP suppressed RT‐induced pAKT and pERK activation and increased apoptosis‐related protein expression in the COMB group (Figure 5). Several studies have shown that radiosensitivity can be elevated by inhibiting the PI3K/AKT cascade, thereby impairing DNA damage repair (Bussink, van der Kogel, and Kaanders 2008; Mohapatra et al. 2022). In this study, the combination treatment exhibited sustained enhancement of DNA damage signals and decreased pAKT levels compared with the RT group, suggesting that BCP sensitizes GBM cells to RT by inhibiting pro‐survival pathways and DNA damage repair.
Furthermore, several cannabinoids, including BCP, have been shown to activate the PPARγ signaling pathway in a CB2‐dependent manner (Youssef, El‐Fayoumi, and Mahmoud 2019) rather than directly binding to PPARγ (Mueller and Jungbauer 2009; O'Sullivan and Kendall 2010). PPARγ and its ligands regulate apoptosis, and combining PPARγ ligands with other anticancer therapeutics can further enhance apoptosis (Elrod and Sun 2008). In this study, BCP and combination treatment significantly elevated PPARγ and cleaved caspase‐3 levels (Figure 5A–B), potentially contributing to enhanced apoptosis and radiosensitivity. Elevated PPARγ levels enhance the radiosensitivity of A549 cells (Kaur et al. 2019), consistent with our findings. Our results show that BCP enhances the efficacy of RT by activating PPARγ and potentially upregulating apoptosis, as evidenced by increased cleaved caspase‐3 levels after treatment (Smaili et al. 2001).
Previous research has shown that PPARγ overexpression combined with RT can impair DNA repair, increase γH2AX foci, and potentially induce cell death in non‐small cell lung carcinoma (Kaur et al. 2019). Similarly, our study revealed that combining BCP and RT in GBM cells triggered PPARγ activation, thereby intensifying apoptosis. ERK activation promotes cell proliferation and survival and protects cells against RT‐induced cell death (Lu et al. 2020). Among all groups, the RT group exhibited the highest pERK expression (Figure 5C,D), consistent with observations from studies using other cannabinoids, including Anandamide, CBD, and THC (Pagano et al. 2021; Solinas et al. 2013). Changes in CB2 expression were also evaluated (Figure S5), revealing that RT increases CB2 expression in GBM cells, which could be inhibited by the combination with BCP. However, further investigation is required to elucidate the role of CB2 in this context. Nonetheless, our in vitro results suggest that BCP holds promise as a potential radiosensitizer.
Furthermore, our in vivo study demonstrated that the combination treatment significantly inhibited tumor growth and extended survival time in GBM‐bearing mice (Figure 7). Consistent with findings from other studies (Nishiga et al. 2022; Scott, Dalgleish, and Liu 2014), we observed transient tumor inhibition following treatment with RT, with tumor regrowth observed from day 13 onward in our study. This phenomenon aligns with clinical observations in patients with GBM, who often experience temporary tumor disappearance followed by rapid regrowth after RT. Unfortunately, tumor recurrence frequently occurs a few months after treatment, leading to relatively shorter progression‐free and overall survival in patients with GBM compared with those with other cancer types (Lautenschlaeger et al. 2022; Tesileanu et al. 2022). GBM recurrence is associated with various factors, including the presence of cancer stem cells and treatment‐mediated reshaping of the tumor microenvironment (Ali et al. 2020).
Our study found that combining BCP with RT led to sustained tumor suppression, with significantly smaller tumors observed in the COMB group compared with the RT or BCP alone. A synergistic enhancement of treatment efficacy was observed when BCP was combined with RT. The combination treatment also extended the lifespan of mice with GBM by 34% compared with RT alone, suggesting its potential to improve survival time in patients with GBM.
Cannabinoids have garnered significant attention for their anticancer properties, either when used alone or in conjunction with other therapies (Lopez‐Valero, Saiz‐Ladera, et al. 2018; Scott, Dalgleish, and Liu 2014; Soroceanu et al. 2022). For instance, combining CBD with TMZ has proven beneficial in enhancing outcomes for patients with GBM compared with those receiving TMZ alone (Twelves et al. 2021). However, the radiosensitization potential of cannabinoids, particularly BCP, remains unclear. To the best of our knowledge, this study fills this gap by being the first to show that BCP can act as a radiosensitizer for GBM, as demonstrated in both cell culture and tumor‐bearing mice.
Although our study provides valuable insights into the potential radiosensitization effects of BCP against GBM, several limitations warrant consideration for future research. First, employing an orthotopic GBM model would better mimic the tumor microenvironment observed in clinical settings, including factors like the blood–brain barrier and the unique immune milieu in the brain. Second, validating the BCP‐mediated radiosensitization using fractionated RT administered to patients could enhance the clinical relevance of our findings. Lastly, exploring the effects of BCP treatment on immune cells is crucial because several immune cells express CB2 receptors. Therefore, future studies should assess the effects of this combination therapy on the antitumor immune response to provide a comprehensive understanding of its efficacy.
5Conclusions
This study provides evidence supporting the potential for BCP to enhance RT efficacy in GBM. BCP was shown to improve RT outcomes by inhibiting DNA damage repair, activating PPARγ‐dependent cell apoptosis, and suppressing cell proliferation by inhibiting pAKT, pERK, and NF‐𝛋B activation. Furthermore, the combination of BCP and RT significantly inhibited tumor growth in GBM‐bearing mice. These findings underscore the therapeutic value of BCP as an adjunctive therapy for GBM radiotherapy. Importantly, BCP targets and activates CB2 receptors, which are overexpressed in GBM cells but minimally expressed in normal brain tissues. In conclusion, BCP has emerged as a promising novel radiosensitizer that can enhance the efficacy of RT in GBM without causing severe side effects.
Ethics Statement
All animal studies were approved by the Institutional Animal Care and Use Committee of the National Yang Ming Chiao Tung University (IACUC No. 1101012).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Acknowledgments
We thank the animal support from the National Laboratory Animal Center (Taiwan), and the technical support from the National Yang Ming Chiao Tung University Instrumentation Resource Center.
Data Availability Statement
The data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.