Cannabidiol and fluorinated derivative anti-cancer properties against glioblastoma multiforme cell lines, and synergy with imidazotetrazine agents

doi:10.21203/rs.3.rs-3639079/v1

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Cannabidiol and fluorinated derivative anti-cancer properties against glioblastoma multiforme cell lines, and synergy with imidazotetrazine agents

https://doi.org/10.21203/rs.3.rs-3639079/v1

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Background

Glioblastoma multiforme (GBM) is an aggressive cancer with poor prognosis, partly due to resistance to the standard chemotherapy treatment, temozolomide (TMZ). Phytocannabinoid cannabidiol (CBD) has exhibited anti-cancer effects against GBM, however, the ability of CBD to overcome common resistance mechanisms to TMZ have not yet been investigated. 4’-Fluoro-cannabidiol (4’-F-CBD, or HUF-101/PECS-101) is a derivative of CBD, that exhibits increased activity compared to CBD during in vivo behavioural studies.

Methods

This work investigated the anti-cancer activity of cannabinoids against GBM cells sensitive to and representing major resistance mechanisms to TMZ. The cannabinoids were also studied in combination with imidazotetrazine agents, and the OrbiSIMS technique was used to investigate the mechanism of action of CBD.

Results

CBD and 4’-F-CBD were found to overcome two major resistance mechanisms (methylguanine DNA-methyltransferase (MGMT) activity and DNA mismatch repair (MMR)-deficiency). Synergistic responses were observed when cells were exposed to cannabinoids and imidazotetrazine agents. Synergy was increased with T25 and 4’-F-CBD. OrbiSIMS analysis highlighted the presence of methylated-DNA, a previously unknown anti-cancer mechanism of action of CBD.

Conclusions

This work demonstrates the anti-cancer activity of 4’-F-CBD and the synergy of cannabinoids with imidazotetrazine agents for the first time and expands understanding of CBD mechanism of action.

It has been reported that cannabinoids exhibit anti-cancer properties^1–3. Most activity of cannabinoids is considered to be a result of interaction with cannabinoid receptors 1 and 2 (CB1 and CB2) of the endocannabinoid system. It has been demonstrated that CB1 and CB2 receptor expression can be altered in cancers, often upregulated (for example in hepatocellular carcinoma) and can be correlated with cancer cell invasion, proliferation and apoptosis^3–4. However, the roles of cannabinoids and cannabinoid receptor regulation in cancers is not yet fully understood. In particular, cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC) are often studied together^1,2,5. These cannabinoids are usually assessed in combination at a ratio of 1:1 CBD:THC (such as in Sativex®), and sometimes in combination with other anti-cancer agents, such as temozolomide (TMZ). Indeed, phase I/II clinical trials in glioblastoma multiforme (GBM) patients have found that Sativex® was safe to administer with TMZ^6–8, and further studies are underway to study the efficacy of this drug combination with radiotherapy^9–10. Cannabinoids are reported to exhibit effects against several cancers. CBD itself has demonstrated activity against colorectal, breast, glioma, cervical and lung cancers^3,11.

There are varied reports on the anti-cancer mechanisms of action of CBD^5,11–13. Whilst CBD is understood to have multiple targets, with a rich and diverse pharmacology, most of the pathways involved are only hypothesised. The suspected pathways involved are via transient receptor potential cation channel subfamily V member 2 (TRPV-2), increased reactive oxygen species generation and increased endoplasmic reticulum stress, with some effects thought to be a result of interaction with the endocannabinoid system^{1,3,5,11–13}. Additionally, the behavioural effects of CBD have been reported to involve deoxyribonucleic acid (DNA)-methylation, predominantly at the C⁵-cytosine in cytosine-phosphate-guanine (CpG) islands^14–15. DNA-methylation has not been reported as a mechanism of anti-cancer activity of CBD, as far as we are aware. However, the methylation of cytosine in CpG islands indicates that nucleotide base methylation does occur as a result of exposure to CBD, and therefore, DNA-methylation may be a possible mechanism of CBD anti-cancer activity^14–15. Inhibition of CB1, CB2 and TRPV-2 receptors has also been shown to reverse some of the anti-cancer effects of CBD, however the pathways involved are not yet fully understood^3,11.

4’-Fluoro-cannabidiol (4’-F-CBD), also referred to as HUF-101 and PECS-101 in the literature, is a recently synthesised CBD derivative^16–17. 4’-F-CBD is reported to exhibit increased potency over CBD in in vivo behavioural assays^16,18–19. Additionally, there is a recent report that 4’-F-CBD can prevent chemotherapy-induced pain¹⁷. However, to the best of our knowledge, the anti-cancer properties of 4’-F-CBD have not yet been studied.

Glioblastoma multiforme (GBM) is an aggressive grade IV brain cancer with a dismal prognosis of 5% 5-year survival²⁰. Contributing to the poor prognosis is the common resistance of GBM to the standard of care chemotherapy, TMZ. TMZ is a DNA-alkylating agent, predominantly methylating DNA purines at N³-adenine, N⁷- and O⁶-guanine positions. N-methylation is generally repaired quickly by base excision repair, but O-methylation is not^21–22. O-methylation leads to a mis-pair of guanine with thymine (rather than cytosine) during DNA replication, triggering DNA mismatch repair (MMR), leading to cell death via apoptosis or autophagy²³. There are two major resistance mechanisms to TMZ demonstrated in GBM. Firstly, an over-expression of O⁶-methylguanine-DNA methyltransferase (MGMT) allows the cells to repair DNA-methylation at the O⁶-guanine position, restoring guanine. Secondly, MMR deficiency allows O⁶-methylguanine to be tolerated^22,24. One method to try to overcome these common resistance mechanisms to TMZ is to synthesise analogues of the molecule. T25 is a N3-propargyl, C8-thiazole analogue of TMZ, created to overcome resistance by MGMT over-expression. DNA-alkylation with the propargyl group (rather than methyl of TMZ), means that MGMT is not able to recognise and remove the DNA-alkylation, and the cells are therefore still sensitive to treatment^23,25–26. C8-thiazole, replacing carboxamide, enhances in vitro drug metabolism and pharmacokinetic (DMPK) properties, including stability; crucially, T25 is not a substrate for P-glycoprotein, an important efflux pump expressed by blood brain barrier (BBB) epithelia²⁷.

GBM is difficult to treat due to the location, as the physical BBB protects the brain, restricting the movement of most therapeutic agents into the brain²⁴. CBD is known to cross the BBB, and many of the observed effects of CBD are a result of interaction with the endocannabinoid system in the brain^28–32. There are reports of CBD anti-cancer activity against GBM, although these are usually in combination studies with THC or TMZ^{3,4,11,33–34}. The combination of CBD and TMZ has been reported to cause both an additive and synergistic response in vitro^35–36. However, the few reports of CBD activity alone against GBM demonstrate a good response, with the concentration required to inhibit cell growth by 50% (GI₅₀) ranging from 10.67 ± 0.58 µM against GL216³⁷ and 12.75 ± 9.7 µM against U87MG^{34,36,38–40} to 21.6 ± 3.5 µM against U373MG³⁸.

Using an in vivo U87MG GBM mouse model, when CBD, THC and TMZ were administered in combination, tumour growth was reduced by a larger extent than after administration of TMZ alone³³. CBD has also been shown to be effective in in vivo GBM models U87, U251, GSC3832 and GSC387 at 15–20 mg/Kg, in combination treatments with THC and TMZ^{3,33,38,41–43}. This has been demonstrated after intravenous, intraperitoneal, subcutaneous and oral administration^2,38. CBD has also been investigated in combination with radiotherapy in a mouse GL216 model, resulting in almost 90% apoptosis^2,37.

To the best of our knowledge, there are no reports investigating the activity of CBD alone against TMZ-resistant GBM. However, there is a report of CBD activity against the colorectal cancer cell line, HCT116¹². HCT116 cells exhibit a deficiency of MMR and are therefore commonly used as a model to represent this resistance mechanism (or tolerance) to TMZ. In the study, CBD was administered alone and found to inhibit cell growth with a GI₅₀ of 10.8 µM after 24 h exposure¹². The common resistance mechanisms to GBM treatment with TMZ prevent the conversion of DNA-methylation to cell death^22,24. As discussed, CBD is thought to act via multiple pathways^{1,3,5,11–13}, and therefore may be able to overcome the two major resistance mechanisms to GBM treatment, MGMT over-expression and MMR deficiency.

The aims of this work were to assess the anti-cancer activity of CBD and 4’-F-CBD against GBM. Cells sensitive to TMZ treatment and those representing the two major resistance mechanisms (over-expression of MGMT and MMR deficiency) have been studied to understand whether the cannabinoids` activity is impacted by these resistance mechanisms. As a synergistic response of CBD treatment with TMZ has been reported previously, and clinical evaluation of TMZ in combination with Sativex is underway, herein, combination treatments of cannabinoids (CBD and 4’-F-CBD) and TMZ or derivative, T25, were studied. Finally, 3D Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) analysis was used as a novel approach to study the mechanisms of anti-cancer action of CBD. The 3D OrbiSIMS allows label-free imaging at the subcellular level by combining time of flight and Orbitrap detectors for analysis with high spatial resolution and mass resolving power (240,000 at m/z 200)⁴⁴.

Materials

Plant-derived and synthetic CBD were purchased from THC Pharm (Frankfurt, Germany). 1-Fluoropyridinium triflate was purchased from Fluorochem (Derbyshire, UK). Isolute HM-N was purchased from Biogate (Hengoed, UK). Cell lines U373-V and U373-M were supplied by Schering Plough (NJ, USA). Cell lines HCT116 and MRC-5 were supplied from ATCC (VA, USA). RPMI-1640, minimum essential medium, foetal bovine serum (FBS), non-essential amino acids, geneticin G418, gentamicin, L-glutamine, penicillin/streptomycin, sterile Hepes buffer, sterile cell culture sodium bicarbonate, ethylenediaminetetraacetic acid, 10× trypsin- ethylenediaminetetraacetic acid solution, TMZ, ammonium formate, indium tin oxide-coated glass slides, dry dichloromethane, deuterated chloroform (CDCl₃) and sterile dimethyl sulfoxide were purchased from Sigma Aldrich (Dorset, UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar (Heysham, UK). T25 was synthesised within the University of Nottingham by Helen Summers. All other solvents and reagents used were of high performance liquid chromatography grade or higher, purchased from ThermoFisher Scientific (Leicestershire, UK).

General Chemistry

A Buchi Rotavapor consisting of a V-850 vacuum controller, R-210 rotavapor and B-491 heating bath was used for drying. A Biotage SP4 flash chromatography system was used for separation with a normal phase puriFlash (PF-15SIHP-F0004, Interchim, Montluçon, France) column cartridge. A flow rate of 5 mL/min was used with line A (hexane) and line B (20% ether in hexane). The column cartridge was equilibrated with 5% line B for 3 column volumes (CV) first. After equilibration, the product was loaded onto the column. The gradient used was 0–2 CV 5% line B, 2–12 CV 5–10% line B, 12–22 CV10% line B, 22–32 CV 10–20% line B, 32–35 CV 20% line B. Separation was confirmed with thin layer chromatography on silica precoated aluminium backed 60 F₂₅₄ plates (Merck, Darmstadt, Germany) using 6% ether in hexane. Compounds were visualised by a UV lamp at 254 nm.

Liquid chromatography mass spectrometry (LC-MS) was used to verify the product. A Shimadzu UFLCXR system was used with an Applied Biosystems API3000 to visualise spectra. Separation was achieved using a Phenomenex Gemini-NX C18 110A column (50 mm × 2 mm × 3 µm) at 40°C. A flow rate of 0.5 mL/min was used with 0.1% formic acid in water in line A and 0.1% formic acid in acetonitrile in line B. The gradient used was 0.0–1.0 min 5% line B, 1.0–3.0 mins 5–98% line B, 3.0–5.0 mins 98% line B, 5.0–5.5 mins 98–5% line B, 5.5–6.5 mins 5% line B.

Bruker 400 Ultrashield nuclear magnetic resonance (NMR) was used to assess the product by hydrogen (¹H) NMR at 400 MHz using CDCl₃ (δ = 7.26). MestReNova software version 14.2.2 (Mestrelab Research, Santiago de Compostela, Spain) was used to process the data. Chemical shifts (δ) are reported in parts per million (ppm). Coupling constants (J) are recorded in Hz, and the multiplicities are described as singlet (s), doublet (d), triplet (t), multiplet (m) or broad (br).

4’-Fluoro-cannabidiol

The synthesis of 4’-F-CBD is shown in Fig. 1 and was first reported by Breuer et al¹⁶, this method was followed, with modifications to improve the separation of the product from any unreacted CBD.

Synthetic CBD was used as an initial starting point for the synthesis. 1-Fluoropyridinium triflate (79 mg, 0.3 mmol), CBD (100 mg, 0.3 mmol) and 4.5 mL dry dichloromethane were stirred overnight in a nitrogen environment at room temperature. The yellow product was washed with (3 × 5 mL) aqueous sodium bicarbonate (NaHCO₃). The organic layer was then dried over sodium sulphate (Na₂SO₄) anhydrous, filtered and dried onto isolute (1–2 spatulas). Separation of 4’-F-CBD from any unreacted CBD was performed by Biotage SP4 flash chromatography and confirmed by thin layer chromatography.

Characterisation reported by Breuer et al¹⁶: total yield (27%), ¹H NMR (300 MHz, CDCl₃) δ = 6.17 (s, 1H, Ar), 5.52 (s, 1H), 4.56 (s, 1H), 4.44 (s, 1H), 3.92 (s, 1H), 2.50 (br, 2H), 2.19–2.05 (br, 2H), 1.77 (s, 3H), 0.86 (t, 3H), LC-MS [M + H]⁺ m/z = 332.

Characterisation found: total yield (42%), this is higher than reported due to improved separation by flash chromatography. ¹H NMR (400 MHz, CDCl₃) δ = 6.20 (d, J = 6.3, 1H, Ar), 5.72 (br, s, 1H, OH), 5.56 (d, J = 2.6, 1H, CH = C), 5.03 (br, s, 1H, OH), 4.60 (s, 1H, CH = C), 4.47 (s, 1H, CH = C), 3.94 (d, J = 10.1, 1H, Ar-CH), 2.69–2.40 (m, 3H, CH₃-C = C), 2.28–2.20 (br, m, 1H, CH-C = C), 2.17–2.07 (m, 1H, CH-C = C), 1.88–1.75 (m, 2H, CH₂), 1.71 (d, J = 1.3, 3H, CH₂-CH), 1.63–1.54 (m, 5H, CH₃, CH₂), 1.35 (dd, J = 7.3, 2.0, 2H, CH₂), 1.35–1.23 (m, 2H, CH₂), 0.91 (t, J = 6.8, 3H, CH₃). Whilst Breuer et al¹⁶ did not report all ¹H NMR peaks, those they did report match those found, and the additional peaks could all be assigned to the structure as described. LC-MS: [M + H]⁺ calculated m/z = 332.5, found m/z = 332.9, retention time: 3.26 mins, purity 95%. LC-MS characterisation matches that reported by Breuer et al¹⁶.

Cell Culture

Human GBM cell lines U373-V (MGMT-low, +MMR) and U373-M (+ MGMT, +MMR) and human colorectal cancer cell line HCT116 (MGMT-low, -MMR) were used in this work. Cell lines U373-V and U373-M were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% non-essential amino acids, 50 µg/mL gentamycin and 400 µg/mL G418. Cell line HCT116 was cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Non-tumourigenic foetal lung fibroblasts (MRC-5) were cultured in minimum essential medium supplemented with 10% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM Hepes buffer and 0.075% sodium bicarbonate. All cell lines were cultured in an incubator with 5% CO₂ at 37°C.

MTT Assay

The MTT assay was used to evaluate the growth and viability of all cell lines used upon treatment with CBD and 4’-F-CBD alone and combinations of CBD and TMZ, CBD and T25, 4’-F-CBD and TMZ, and 4’-F-CBD and T25. Briefly, cells were seeded into 96-well plates at the following densities: 3 days` exposure: all cell lines: 3 ×10³ cells/well; 6 days` exposure: U373-V and U373-M cells: 650 cells/well, HCT116 and MRC-5 cells: 400 cells/well. After the cells were allowed to attach overnight, they were exposed to test agents for either 3 or 6 days. MTT assays were performed at the time of treatment (T0) and following the exposure time for cells treated and non-treated controls. MTT was added, and following 2 h incubation, the formazan crystals were dissolved in 150 µL sterile dimethyl sulfoxide and absorbance was read at λ = 570 nm on a PerkinElmer EnVision plate reader. GI₅₀ and combination index (CI) values were calculated using Equations 1–3.

\(GI50A= \left(\frac{\left(C-T0\right)}{2}\right)\) + T0 Eq. (1)

GI₅₀ \(= \left(\left(\frac{(E-D)}{(X-Y)}\right)*\left(X-GI50A\right)\right)+D\) Eq. (2)

Where:

GI50A = Absorbance at GI₅₀

C = Mean absorbance of control (cells only)

T0 = Mean absorbance of control (T0)

X = High absorbance (absorbance at data point just above GI50A)

Y = Low absorbance (absorbance at data point just below GI50A)

D = High concentration (concentration at absorbance data point just above GI50A)

E = Low concentration (concentration at absorbance data point just below GI50A)

\(CI= \left(\frac{D1}{DX1}\right)+ \left(\frac{D2}{DX2}\right)\) Eq. (3)

Where:

CI = Combination index

D1 = GI₅₀ of test agent A when in combination with test agent B

DX1 = GI₅₀ of test agent A alone

D2 = GI₅₀ of test agent B when in combination with test agent A

DX2 = GI₅₀ of test agent B alone

Cell Viability

Results of the MTT assays were confirmed by viable cell count assays. Cells were seeded into 6-well plates at the following densities: U373-V and U373-M cells: 4 ×10⁴ cells/well, HCT116 and MRC-5 cells: 2 ×10⁴ cells/well. After the cells were allowed to attach overnight, they were exposed to test agents for either 3 or 6 days. Following the exposure time, cells washed with PBS and harvested with trypsin-ethylenediaminetetraacetic acid solution. The viable cells were then counted using a haemocytometer under a Nikon Eclipse TS100 microscope.

Preparation of cells for 3D OrbiSIMS analysis

Cell samples were prepared for analysis by 3D OrbiSIMS following a method based on Newman et al (2017)⁴⁵. U373-V cells treated with CBD, CBD and TMZ, and CBD and T25 were assessed by 3D OrbiSIMS.

Indium tin oxide-coated glass slides were placed into a petri dish and seeding U373-V cells at a density of 1.6 ×10⁵ cells/well into the dish. Petri dishes were placed in the incubator at 5% CO₂, 37°C. Cells were exposed to the GI₅₀ value of test agents for 3, 6, 24 and 72 h. For cells treated with a combination of test agents, the concentrations were based on combination MTT assays to represent ~ 75% growth inhibition, shown in Table 1.

Table 1

Concentration of test agents for preparation of cell exposure, dosed in combination for 3D OrbiSIMS analysis.
Test agent A	Test agent A concentration (µM)	Test agent B	Test agent B concentration (µM)
CBD	11	TMZ	2
CBD	7	T25	14

Following the exposure time, the slides were harvested. The cells were washed (3 × 1 mL) with 150 mM ammonium formate solution at pH 7.4. The glass slides were then dipped into liquid nitrogen and freeze-dried in a benchtop freeze dryer (VirTis SP Scientific Sentry 2.0) at -50°C for 1 h. Once removed from the freeze drier, the slides were sealed in petri dishes with parafilm and stored at -80°C until analysis.

3D OrbiSIMS Analysis

The 3D OrbiSIMS technique uses a HybridSIMS instrument (IONTOF GmbH), which incorporates both time of flight and Q Exactive HF Orbitrap analysers. Samples were analysed using the single ion beam Orbitrap depth profiling mode, utilising a 20 keV Ar₃₀₀₀⁺ gas cluster ion beam of 20 µm diameter (duty cycle of 4%) and a target current of 0.2 nA. Both positive and negative mode ion polarity spectra were acquired with a mass range of m/z = 75–1125. The profile was performed over an area of 200 × 200 µm using random raster mode. The injection time was set to 500 ms and 80 scans were taken for each analysis over an average of 120 s. A low energy electron floodgun was used for charge compensation, additionally, the pressure in the main chamber was regulated using Ar gas to 9 ×10^− 7 mbar to enhance the charge compensation. The mass resolution was 240,000 at m/z 200.

3D OrbiSIMS data were acquired and analysed using SurfaceLab 7 software (IONTOF GmbH, Münster, Germany). Peak lists were automatically generated for all of the spectra with a minimum count value applied of 10,000 and subsequently combined using the ‘union’ function with a catch mass radius of 2 ppm. All data were normalised to the total ion count (TIC) of that analysis. All assignments are based on accurate mass to within 2 ppm, and those reported throughout are putative. Data were chemically filtered using molecular formula prediction software, SIMS-MFP version 1.1 (University of Nottingham, Nottingham, UK)⁴⁴, into groups containing fatty acids (C_nH_nO₂), sulfatides (C_nH_nN₁O₁₁₋₁₂S₁) and glycerophospholipids (C_nH_nO_8/13P or C_nH_nNO₇₋₁₀P)⁴⁶. Data groups were then analysed using multivariate analysis software, simsMVA⁴⁷. The data groups were mean-centred, and the principal component analysis (PCA) function was used in algorithm mode, retaining all components. The scores and variance were used to find principal components exhibiting differences between the groups, and loadings allowed visualisation of the principal components.

Statistical Analysis

Chemical structures and schemes were prepared using ChemDraw version 21.0.0 (PerkinElmer Informatics, MA, U.S.A.). One-way ANOVA with Dunnett’s multiple comparisons, or multiple t-tests where appropriate were performed in Prism version 9.3.1 (GraphPad, CA, U.S.A.) to assess the significant differences between sample groups. Differences were considered statistically significant when the p-value was < 0.05 (α = 0.05). All data (n ≥ 3) are represented as mean ± standard deviation (SD).

Cancer cell growth inhibition by cannabinoids

The anti-cancer activity of cannabinoids CBD and 4’-F-CBD was assessed against a vector control GBM cell line (U373-V) and two cell lines representing common resistance mechanisms to GBM treatment with TMZ (U373-M, MGMT-transfected U373-V isogenic partner, and MMR-deficient HCT116 colorectal cancer). Exposure periods of 6-days as well as 3-days were studied because TMZ is understood to require at least one cell cycle in order to exhibit its cytotoxic effect²². This is observed in Fig. 2, where the GI₅₀ of TMZ against the U373-V cell line falls significantly (p < 0.001) from 147 ± 55 µM after 3-days exposure to 10 ± 2 µM following 6-days exposure. After 3-days exposure, T25, CBD and 4’-F-CBD exhibited significantly lower GI₅₀ values compared to TMZ against all cell lines studied. For U373-M and HCT116 cell lines (representing resistance to TMZ treatment), both cannabinoids and T25 also showed significantly lower GI₅₀ values than TMZ following 6-days exposure.

Test agents were also assessed against non-tumourigenic MRC-5 fibroblasts, as shown in Fig. 2. TMZ was shown to be the least active, with a GI₅₀ of 323 µM after 3-days exposure, or 724 µM after 6-days exposure. CBD appears to be the most active, with a GI₅₀ of 5 µM and 7 µM (3- and 6-days exposure). 4’-F-CBD and T25 both demonstrated GI₅₀ values between 37–58 µM.

Synergy of cannabidinoids with imidazotetrazine anti-cancer agents

Combination treatments of CBD with TMZ or T25 against the three cell lines were studied by MTT assay and confirmed by cell count assays. The CI indicating the cell response to the combined treatments are shown in Table 2. Briefly, CI = 1 indicates an additive response, CI > 1 is antagonistic and CI < 1 shows a synergistic response. This is demonstrated in the representative isobolograms of combinations against the U373-V cell line shown in Fig. 3.

Table 2

CIs of cannabinoids (CBD or 4’-F-CBD) administered in combination with TMZ or T25 after 3- or 6-days exposure against U373-V (GBM control, -MGMT, +MMR), U373-M (GBM, +MGMT, +MMR) and HCT116 (-MGMT, -MMR) cell lines. Data represented as a range for the different combination concentrations tested. The full CI data for each combination are shown in supplementary information 1.
Combination	Treatment time (days)	Combination Index (CI)
Combination	Treatment time (days)	U373-V	U373-M	HCT116
CBD/TMZ	3	0.10–0.89	0.23–0.74	0.11–1.06
CBD/TMZ	6	0.12–0.76	0.21–0.74	0.05–0.95
CBD/T25	3	0.33–0.65	0.22–0.57	0.53–0.93
4’-F-CBD/TMZ	3	0.16–1.01	0.13–0.59	0.37–0.74
4’-F-CBD/TMZ	6	0.09–0.53	0.05–0.50	0.48–0.79
4’-F-CBD/T25	3	0.21–0.52	0.19–0.75	0.37–0.79

Combining exposure of all three cell lines to CBD and an imidazotetrazine agent (TMZ or T25) resulted in a consistently synergistic response. Table 2 shows that only against the HCT116 cell line was there a combination that did not provide a synergistic response, CI = 1, when TMZ (304.5 µM) was used with CBD (7.5 µM) after only 3-days exposure (when TMZ is less effective, as shown in Fig. 2). However, when the cells were exposed to the test agents for 6-days (required to observe the full effects of TMZ), the lowest CI (greatest synergy) was observed following exposure for 6-days of HCT116 cells to CBD (1.3 µM) and TMZ (0.5 µM). Table 2 and Fig. 3 also show that as well as the more synergistic response, the combination of CBD and T25 also provided the most consistent response, with a smaller range in CI values.

Combinations treatments of 4’-F-CBD with TMZ or T25 were also assessed against the three cell lines, showing that the combination of 4’-F-CBD with TMZ or T25 resulted in a synergistic response in all three cell lines. The only exception was following 3-days exposure of 4’-F-CBD and TMZ to the U373-V cell line. Similarly to the only additive response observed in the CBD combination studies, this was at low concentrations of the test agents, and at 3-days where TMZ does not exhibit its full effect. Table 2 shows that the lowest CI of 0.09 was observed following exposure of U373-V to 4’-F-CBD and TMZ for 6-days.

Indications of anti-cancer mechanism of cannabidiol activity

The anti-cancer mechanisms of action of CBD against U373-V cells were investigated by the 3D OrbiSIMS technique with cells analysed following exposure to CBD either alone, with TMZ or with T25 for up to 3-days. Using the spectra acquired, a targeted search for secondary ions indicative of the suspected mechanisms of action was conducted including glutathione (C₁₀H₁₆N₃O₆S⁻) as an indicator of oxidative stress⁴⁸, ceramide (C₆₃H₁₂₄NO₆S⁻) as an indicator of CB1 activity⁴⁹, and anandamide (C₂₂H₃₆NO₂⁻) as an indicator of interaction with the endocannabinoid system⁵⁰. These were not observed with 3D OrbiSIMS analysis; however, DNA and methylated-DNA ions were observed. From the secondary ion intensity values shown in Fig. 4, it can be seen that cells exposed to CBD for 24 h exhibited significantly higher methylated-DNA content compared to the control samples of non-dosed cells. Significant differences were not observed following exposure to CBD alone for 3, 6 or 72 h. Supplementary information 2 shows more details of the detection of methylated-DNA shown in Fig. 4. Further analysis of the 3D OrbiSIMS data using PCA revealed that cells exposed to CBD alone exhibited an increase in fatty acid content. Following exposure for 3 and 6 h, an increase in palmitic, stearic and octatriacontanoic acids was observed, as well as a decrease in oleic acid. After 72 h exposure, only an increase in palmitic acid was observed. A detailed illustration of the PCA conducted using the 3D OrbiSIMS data demonstrating the difference in fatty acid composition of samples is shown in supplementary information 3.

Figure 4 demonstrates the significant increase in secondary ions with respect to methylated-guanine, cytosine, adenine and thymine after 24 h exposure of the U373-V cells to CBD. It is also observed in Fig. 4 that following 3 h exposure of the cells to CBD with T25, methylated-guanine, cytosine and thymine were also observed at significantly higher levels than in the control sample. T25 is thought to create propargyl-adducts on DNA, not methyl. Table 3 demonstrates this for the first time, showing secondary ions related to propargylated-DNA were found following exposure of cells to CBD and T25. The cells exposed to CBD and T25 also exhibited a change in the fatty acid composition, showing an increase in palmitic and octatriacontanoic acids. Exposure of cells to CBD and TMZ also resulted in an increase in secondary ions putatively assigned to arachidonic, cinnamic and palmitic acids.

Table 3

3D OrbiSIMS analysis of U373-V cells exposed to CBD for 3 h, CBD and T25 for 3 h and a non-treated control. Peak intensity (secondary ion counts) normalised to the TIC, with deviation below in ppm, of propargylated-DNA. Data presented as an average of n = 3 technical repeats. One-way ANOVA was performed, α = 0.05, to compare treated samples to the control, no significant differences were found.
Sample		Propargyl-Guanine	Propargyl-Cytosine	Propargyl-Adenine	Propargyl-Thymine
	Formula	C₈H₆N₅O⁻	C₇H₆N₃O⁻	C₈H₆N₅⁻	C₇H₅N₂O₂⁻
	m/z	188.1675	148.1431	172.1685	149.1276
Control		0	1.09 ×10^− 5 ± 2.70 ×10^− 6	0	7.56 ×10^− 6 ± 2.65 ×10^− 6
Control		-	0.4	-	-0.3
CBD 3 h		0	0	0	0
CBD 3 h		-	-	-	-
CBD/T25 3 h		4.16 ×10^− 5 ± 0	1.77 ×10^− 4 ± 1.47 ×10^− 4	3.48 ×10^− 5 ± 0	1.16 ×10^− 4 ± 9.82 ×10^− 5
CBD/T25 3 h		-0.1	0.1	0.1	-0.4

The exploration of the anti-cancer effects of cannabinoids is a growing area of research. CBD has been shown in the literature to exhibit anti-cancer properties against cancers including breast, colorectal, lung and GBM^5,12. The ability of CBD to inhibit GBM cell growth in vitro is usually studied in combination with either THC or TMZ^33–34. This has led to phase I/II clinical trials in GBM patients^6–8. Further clinical trials are underway to study the efficacy of combinations of radiotherapy and chemotherapy with TMZ and a mixture of CBD and THC, against GBM⁹, as well as daily administration of CBD with TMZ¹⁰. As discussed above, the anti-cancer activity of CBD alone against GBM has been studied in cell lines including U87MG (GI₅₀ = 12.75 ± 9.7 µM)^{34,36,38–40}, GL216 (GI₅₀ = 10.67 ± 0.58 µM)³⁷ and U373MG (GI₅₀ = 21.6 ± 3.5 µM)³⁸. These cell lines do not possess the over-expression of MGMT or deficiency of MMR that result in the major GBM resistance mechanisms to TMZ, represented in this work by human GBM cell line U373-M and colorectal cancer cell line HCT116, respectively. CBD anti-cancer activity has been studied against the HCT116 cell line previously for its effects against colorectal cancer¹². In this work, HCT116 cells were utilised to represent the second major resistance mechanism to TMZ, a deficiency of MMR. To the best of our knowledge, the anti-cancer properties of 4’-F-CBD have not been studied before. The potential advantages of treating GBM with 4’-F-CBD, compared to CBD, are, briefly, that 4’-F-CBD is reported to have increased potency in in vivo behavioural assays compared to CBD, suggesting potentially increased binding at the molecular level, or increased delivery to the brain^16,18–19. There is also the opportunity that the fluorine atom on 4’-F-CBD provides for imaging and potentially theranostic purposes^51–52.

The activity of all agents was assessed against the non-tumourigenic MRC-5 cell line to indicate any cancer-selectivity. Figure 2 demonstrates that TMZ showed the most cancer-selectivity (GI₅₀ = 323 µM following 3-days exposure) and CBD the least (GI₅₀ = 5 µM following 3-days exposure). Once the effect of the test agents on the non-tumourigenic cell line was established, their anti-cancer activity could be investigated against GBM cell lines. Studying the U373-V cell line, the TMZ GI₅₀ falls from 147 ± 55 µM after 3-days to 10 ± 2 µM after 6-days exposure (Fig. 2). This is consistent with reported literature⁵³ as TMZ must undergo ring opening to MTIC, before it is able to methylate the DNA, most impactfully at the O⁶-guanine position^21–22. O-methylation leads to a mis-pair of guanine with thymine (rather than cytosine) during DNA replication, damaging the cells and triggering the pathway to cell death via apoptosis or autophagy²³. This process comprises multiple rounds of futile DNA incision and thymine re-insertion before DNA-replication fork collapse, thus 6-days exposure is required to see the full impact of TMZ treatment. Interestingly, this is not true for imidazotetrazine analogue T25, indeed, all other test agents studied showed inhibitory effects on the cell growth after 3-days exposure. The TMZ GI₅₀ is high for the two cell lines representing common (clinical) resistance mechanisms, as expected and demonstrated in the literature²².

The GI₅₀ values of CBD, 4’-F-CBD and T25 were consistent across cancer cell lines studied, and all values were significantly (p < 0.001) lower than that of TMZ against the two cell lines displaying TMZ resistance. This has been demonstrated previously within our group for T25²⁷, as the molecule was designed to overcome resistance mechanisms associated with TMZ treatment by creating larger DNA adducts that escape MGMT-mediated removal and tolerance following MMR-loss. The activity of CBD alone against HCT116 has also been reported in the literature, supporting the thesis that CBD activity is not impacted by resistance to TMZ conferred by MMR deficiency¹². However, this is the first time that cannabinoids have been shown to overcome the often-seen inherent resistance mechanism to TMZ treatment (although there are cases where acquired TMZ resistance has been conferred by over-expression of MGMT⁵⁴). Additionally, 4’-F-CBD demonstrated increased cancer-selectivity compared to CBD (Fig. 2) and may therefore provide a safer treatment option. These are encouraging data, as the poor outcomes for patients diagnosed with GBM demonstrate the need for new treatments.

As discussed above, synergy has previously been demonstrated between CBD and TMZ against GBM cell lines U87MG and U251^35–36,55. However, Deng et al reported that only certain concentrations resulted in a synergistic combination, whilst others resulted in an additive response³⁶. The work reported herein confirms the reports of synergy for the U373-V cell line and demonstrates synergy in the two cell lines representing clinical resistance mechanisms to TMZ for the first time. The combination of CBD with TMZ demonstrated a remarkable synergistic response with CI values as low as 0.21 and 0.05 against U373-M and HCT116, respectively (Table 2). Against the HCT116 cell line, representing a MMR deficiency, at high concentrations of TMZ, the combination resulted in an additive response, supporting the findings of Deng et al³⁶. This analysis also showed that the concentration of TMZ added does not affect the growth inhibition, and that the concentration of CBD is driving the response. This suggests that the effects of CBD are the predominant cause of the growth inhibition, potentially re-sensitising the cells to TMZ.

T25 can overcome the two major resistance mechanisms to treatment with TMZ, and a synergistic combination of CBD with T25 may lead to enhanced activity (in TMZ resistant models) (Fig. 2). The combination yielded a synergistic response at all concentrations tested for all cell lines. The CI values were lower (< 0.57 in U373-M) for this combination than the combined CBD and TMZ treatment (< 0.74 in U373-M). This demonstrates enhanced synergy of this combination, likely due to the increased activity of T25 compared to TMZ. This combination has not been studied before, and the low CI values demonstrate promise for GBM treatment.

The combined treatment of CBD with T25 was investigated further by 3D OrbiSIMS analysis. Propargylated-DNA (expected to occur following exposure to T25) was found in the samples treated with CBD and T25 (Table 3). Intensities of propargyl-cytosine and propargyl-thymine were not found to differ significantly from the non-treated control. However, propargyl-guanine and propargyl-adenine were not found to be present in the control. This provides evidence of the activity of T25. Methylated-DNA was also found to be present (Fig. 4) and methyl-guanine, methyl-cytosine and methyl-thymine were all significantly higher than in the non-treated control sample. As T25 is expected, and shown here, to deposit propargyl groups on the DNA, the methylated-DNA could be assumed to be a result of CBD activity. Of particular interest is methyl-cytosine. Methyl-cytosine at the C⁵-position of CpG islands is reported in the literature to occur after CBD exposure, however the role of CpG methylation in CBD activity is not yet clear^14–15. Additionally, CpG islands are abundant in promoter genes, including the MGMT promoter⁵⁶. Methylated MGMT promoter is an evidenced indicator of the prognosis of GBM response to therapy⁵⁷. MGMT promoter methylation silences the gene, MGMT protein is not expressed, and the tumours are more sensitive to TMZ treatment⁵⁸. The methyl-cytosine evidenced herein by exposure of GBM cells to CBD could potentially occur at CpG islands on MGMT promoters. If so, this could effectively silence MGMT, possibly contributing to the synergy observed in exposure of the cells to CBD with TMZ.

The presence of methylated-DNA at high OrbiSIMS ion intensities may represent one mechanism of anti-cancer action of CBD. DNA damage by methylation can result in mismatched pairs during replication and ultimately, lead to cell death^59–61. To the best of our knowledge, this is the first report of evidence of methylated-DNA as a potential anti-cancer mechanism of action of CBD. As discussed, a MMR deficiency (as represented by the HCT116 cell line) means that mis-matched pairs are tolerated. Therefore, this work demonstrates that by some mechanism, CBD re-sensitises MMR-deficient cells to O⁶-Me lesions. The synergy of CBD demonstrated herein with TMZ and T25 indicates that CBD also acts via a pathway other than DNA alkylation (as this is the mechanism of action of imidazotetrazine compounds). The increase in palmitic, arachidonic and cinnamic acids observed in the cells exposed to CBD are associated with oxidative stress and decreased GBM cell viability^54,61. This suggests an oxidative stress pathway could be initiated in the cells treated with a combination of CBD and imidazotetrazine agents. Cells treated with CBD were also found to contain decreased oleic acid compared to the non-treated control. Oleic acid has been shown to increase glucose utilisation and stimulate GBM cell growth⁶⁰. Oleic acid is also thought to increase the permeability of the BBB by interacting with the membranes of brain capillary endothelial cells, which form the BBB, therefore, a reduction in oleic acid would impair BBB permeability^62–63. BBB opening could potentially contribute to the increased GBM growth observed when present, due to the increased access to the blood circulation. Additionally, there are reports that oleic acid decreases P-glycoprotein-mediated drug efflux⁶⁴. This could allow enhanced TMZ levels in the brain. These findings indicate that the anti-cancer activity of CBD involves a rich and diverse pharmacology, as is suggested in the literature^5,12–13.

The mechanism of action of 4’-F-CBD was not investigated, however, the response to exposure of cells to the cannabinoids were not significantly different. As the molecular structure of the cannabinoids is similar (Fig. 1), it would be reasonable to suggest that the activity of 4’-F-CBD could be a result of similar pathway activation to that of CBD. There was a synergistic response from all cell lines to exposure with 4’-F-CBD in combination with TMZ after both 3- and 6-days exposure (Table 2). Only the highest concentration of TMZ tested in the U373-V cell line resulted in an additive response, all other concentrations demonstrated a synergistic response (Fig. 3). Therefore, the combination of 4’-F-CBD with TMZ produced increased synergy over CBD with TMZ in all cell lines apart from after 3-days exposure to U373-V cells. The combination of 4’-F-CBD with T25 demonstrated similarly high synergistic responses, where the CI values are not significantly different to the combination of CBD with T25.

The work reported herein shows the promise cannabinoids offer for GBM treatment. The application of the 3D OrbiSIMS technique in this work demonstrates the potential of this new approach and has begun to elucidate the mechanism of anti-cancer activity of CBD. Following these findings, further work can be conducted to fully investigate the mechanisms proposed in this work. Thermus aquaticus Taq-polymerase stop assays could be conducted to interrogate the intensity of alkyl-guanine, following treatment with cannabinoids in combination with the imidazotetrazine agents (TMZ and T25). Analysis of O⁶-methylguanine adduct burden in cells would also be useful, where comparisons of cells exposed to TMZ alone or in combination with CBD. Additionally, the 3D OrbiSIMS technique has proved beneficial in this work, the technique is not chemically biased and generates a range of different ions simultaneously, so is a good starting point for complex questions which do not have a known direction for analysis. It is also relatively high throughput for in vitro studies.

This work demonstrates for the first time that CBD is able to overcome the major resistance mechanisms to TMZ standard GBM chemotherapy, MGMT over-expression and MMR-deficiency. The anti-cancer activity of 4’-F-CBD was found to be as effective as CBD against GBM, and able to overcome the two major resistance mechanisms to TMZ. To the best of our knowledge, this is the first time the anti-cancer properties of 4’-F-CBD have been studied. 4’-F-CBD was also demonstrated to be less active against non-tumourigenic cells than CBD, and therefore, a potentially safer treatment. Synergy was observed in combined treatments of the cannabinoids (both CBD and 4’-F-CBD) with TMZ and T25 for the first time. The synergistic combination of CBD and TMZ confirms reports in the literature and demonstrates that CBD is driving this synergy. Increased synergy (demonstrated by lower CI values) was observed in combinations with T25. This is encouraging as T25 itself can overcome the two major resistance mechanisms to TMZ treatment, and therefore treatment could be enhanced further by this combination. The anti-cancer pathway of activity by DNA-methylation was demonstrated after treatment with CBD for the first time. Although there is also evidence that this is not the only pathway activated, CBD activity is complex. Changes in specific fatty acids present were observed following treatment, and are indicators of the treatment effectiveness, and therefore, putative biomarkers of efficacy in vivo. The mechanism of T25 activity was also investigated by 3D OrbiSIMS and the presence of propargylated bases is evidence of DNA propargylation by the molecule. Overall, this work demonstrates the anti-cancer activity of cannabinoids CBD and 4’-F-CBD against GBM, and robust synergy in vitro with imidazotetrazine agents.

Acknowledgements

The authors would like to thank Helen Summers for synthesising the compound T25 and sharing with us.

Authors’ contributions

AB: conceptualization, methodology, investigation, analysis, writing – original draft, writing – review and editing, visualization; NK: methodology, investigation, writing – review and editing; DJS: conceptualization, methodology, resources, writing – review and editing, supervision; MRA: conceptualization, methodology, resources, writing – review and editing, supervision; PG: conceptualization, methodology, writing – review and editing, supervision; TDB: conceptualization, methodology, resources, writing – review and editing, supervision.

Ethics approval and consent to participate

N/A

Consent for publication

N/A

Data availability

The datasets generated and analysed during the current study are available in the supplementary information or available upon request.

Competing interests

The authors declare no conflict of interest.

Funding information

This work was supported by the Engineering and Physical Sciences Research Council [grant numbers EP/L01646X/1 and EP/P029868/1]. The funders had no input into the study design, collection, or analysis of data, writing or submission of the paper.

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Cannabidiol and fluorinated derivative anti-cancer properties against glioblastoma multiforme cell lines, and synergy with imidazotetrazine agents

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Materials

General Chemistry

4’-Fluoro-cannabidiol

Cell Culture

MTT Assay

Cell Viability

Preparation of cells for 3D OrbiSIMS analysis

3D OrbiSIMS Analysis

Statistical Analysis

Results

Cancer cell growth inhibition by cannabinoids

Synergy of cannabidinoids with imidazotetrazine anti-cancer agents

Indications of anti-cancer mechanism of cannabidiol activity

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1