The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

Breast cancer cell culture

Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). According to the ATCC guidelines for growth medium, cell cultures were maintained under standard conditions (37 °C and 5% CO₂ humidified environment). MDA-MB-231 and MCF-7 cells were cultured and allowed to grow into a monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, Dublin, Ireland) and 1% antibiotics (100U/mL penicillin and 100 μg/mL streptomycin)²⁷. Cells were checked every day using the ZOE Fluorescent Cell Imager and split when reaching a 70–80% confluency using trypsin–EDTA (Sigma-Aldrich, St. Louis, MO, USA). The trypan exclusion method and a hemocytometer were used to assess and determine the viability of the cells before plating.

Plant extract preparation

The Drug Enforcement Office in Zahle, Beqaa Governorate, provided the dried samples of a Lebanese cannabis strain in October 2019 from the Beqaa valley in Yammoune (Yammoune: 34◦07′46.4′′North, 36◦01′40.8′′East; altitude, 1375 ± 10 m). The plant material was securely stored at the Lebanese American University and a plant specimen was deposited at the University Department of Natural Sciences’ herbarium (ID, 2019–0011). Lebanese Cannabis flower sample (10 g) was air-dried and extraction using ethanol was performed as previously described by Shebaby et al. yielding 1.17 g of crude cannabis oil extract (COE)²⁶. The phytochemical compounds of the Lebanese C. sativa plant were separated via liquid column chromatography (LCC). The column ran for four days at different rates using a normal-phase silica gel column and a 4:3:3 ratio of chloroform, hexane, and diethyl ether, respectively, as the eluent mobile phase. The samples were collected in test tubes and the solvent was evaporated using the rotary evaporator. Following that, the collected samples were analyzed via thin layer chromatography (TLC) that was carried out for every five tubes. The Retention Factor (Rf) was calculated by measuring the traveled distances using the ImageJ software and tubes with similar TLC behavior were combined. The tubes were later evaluated by Gas chromatography coupled to Mass spectrometry using a Shimadzu GCMS-QP2020NX as previously described²⁶.

The extract preparation of CBD and THC compounds to be used on cancer cells was achieved by dissolving 12.5 mg of THC and 37.5 mg of CBD in ethanol. Following ethanol evaporation using rotary evaporator, the CBD and THC (3:1) were then dissolved in 1 mL DMSO reaching a final concentration of 50 mg/mL stock solution, stored at − 20 °C for later use. The composition and percentages of CBD and THC in this purified preparation were further confirmed (Supplementary Table 1 and Figure S1) by GC–MS analysis as previously described²⁶.

Cell viability assay

The viability of MDA-MB-231 and MCF-7 was assessed using the MTS (Promega, Wisconsin, USA) cell viability reagent. Cells were harvested, counted and prepared for seeding in triplicates at a density of 1 × 10⁵ cells/mL overnight. Cells were then treated with various concentrations of CBD/THC mixture (10, 12.5, 15, 17.5, 20 and 25 μg/mL) for 24 and 48 h. After the desired incubation period, MTS reagent was used to quantify cell viability and determine cell proliferation. Following removal of cell culture medium, 100uL of the MTS working solution prepared according to the manufacturer’s instructions was added to each well²⁸. This assay is based on using the MTS tetrazolium salt, which when combined with metabolically active cells results in the formation of a formazan dye which could be detected at 492 nm using the Varioskan™ LUX multimode microplate reader. The findings of three separate trials are included and the relative cell proliferation was calculated as follows:

Cell cycle progression analysis using flow cytometer

Both, MDA-MB-231 and MCF-7 cells were seeded at a density of 1 × 10⁵ cells/mL in 6 well-plates overnight followed by treatment for 24 and 48 h with 12.5, 15 and 17.5 μg/mL of the cannabinoid mixture and 30uM cisplatin (Abcam, Cambridge, United Kindgom), used as positive control. After the desired incubation period, culture medium was collected, and cells were washed with PBS and detached using trypsin–EDTA reagent. Cells were centrifuged, washed, fixed with ice-cold PBS and 70% ethanol and stored at − 80 °C, overnight²⁹. Fixed cells were centrifuged the following day, counted using hemocytometer, and stained with 50 μg/mL Propidium Iodide (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 μg/mL RNase (Roche, Basel, Switzerland) for 45 min in the dark. Using the Incyte mode on the Guava® easyCyteTM flow cytometer (Luminex Corporation, Austin, TX, USA), the DNA content was assessed and distributed among the various cell cycle phases according to the degree of PI binding relative to the control. Cells with DNA contents less than 2n were classified into the pre-G0/G1 phase, those with DNA content between 2 and 4n into the synthesis phase (S phase) while cells with 2n and 4n DNA contents were grouped into the G0/G1 and G2/M phases, respectively.

Apoptosis investigation using flow cytometer

Both cell lines were seeded (1 × 10⁵ cells/mL) in 6 well-plates and treated with 12.5, 15 and 17.5 μg/mL CBD/THC and 30uM cisplatin for 24 and 48 h. After desired incubation period, cells were washed using PBS and detached using trypsin. After centrifugation (1400 g, 5 min and 4 °C), cells were counted, and stained for 5 min with the staining solution (1X Binding buffer, 1X PI and 1X Annexin-V) in the dark. Flow cytometry (Luminex Corporation, Austin, TX, USA) was used to obtain the data (5,000 events/sample) which was analyzed using the Incyte mode on the Guava® easyCyteTM software. The cell population in the four quadrants was examined and defined as follows: the lower left quadrant (Ann^–/PI^–) denotes viable cells, the lower right (Ann⁺/PI) and upper right (Ann⁺/PI⁺) quadrants represent cells at early and late stages of apoptosis, respectively, while the upper left quadrant (Ann^–/PI⁺) represents necrotic cells.

Cell death detection enzyme-linked immunosorbent assay (ELISA)

Breast cancer cells were plated overnight in a 6 well-plate at a density of 1 × 10⁵ cells/mL. The following day, cells were treated with 12.5, 15 and 17.5 μg/mL CBD/THC mixture and 30 μM cisplatin, in fresh medium, for a duration of 24 h. After the desired incubation period, and using the Cell Death detection ELISA kit (Roche, Basel, Switzerland), a sandwich enzyme-immunoassay procedure was followed as previously described by El Khoury et al.³⁰.This assay is used to measure cytoplasmic histone-associated DNA fragments (mono- and oligo-nucleosomes) released into the cytoplasm of cells undergoing apoptosis. Following the manufacturer’s instructions, cells were collected, lysed and transferred into a pre-coated microplate with anti-histone antibody with high specificity to H1, H2A, H2B, H3 and H4 histone subunits. After sufficient time, the wells were washed followed by the addition of anti-DNA antibody conjugated to a peroxidase enzyme (anti-DNA-POD) that binds to single- and double-stranded DNA. Upon the addition of the ABTS (2,2’-azino-di-[3-ehtylbenzthiazoline sulfonate (6)]) peroxidase substrate, the absorbance A₄₀₅ was measured using the Varioskan™ LUX multimode microplate reader, allowing for the determination of the retained peroxidase enzyme in the immunocomplex. The results were used to calculate the specific enrichment of mono- and oligo-nucleosomes released into the cytoplasm of apoptotic cells using the following formula:

Inhibition of caspases using Z-VAD-FMK

Breast cancer cells were seeded in 96 well-plates at a density of 1 × 10⁵ cells/mL and then treated with caspases inhibitor, Z-VAD-FMK (50 μM) for 30 min prior to the addition of the CBD/THC (12.5, 15 and 17.5 μg/mL) mixture for 24 and 48 h. To assess the caspase-dependent cytotoxic effect of the treatment, MTS viability reagent was used as described previously to quantify metabolically active cells by spectrophotometry using the Varioskan™ LUX multimode microplate reader at 492 nm. The effect of Z-VAD-FMK inhibition on the proliferation of both cell lines was assessed and compared to cells treated only with CBD/THC mixture.

Inhibition of autophagy using chloroquine and wortmannin

Breast cancer cells were seeded in 96 well-plates at a density of 1 × 10⁵ cells/mL. To evaluate the autophagy-dependent cell death, breast cancer cells were pre-exposed to 50 nM wortmannin (Wort) or 10 μM chloroquine (CQ) for 30 min, inhibitors of autophagosome and autophagolysosome formation, respectively. Cells were then treated with increasing concentrations of CBD/THC mixture for 24 h followed by the addition of MTS viability reagent, as detailed above. Cell viability was determined by quantifying formazan formation relative to control, untreated cells, which was further compared to cells treated with CBD/THC only.

Immunofluorescence

Breast cancer cells were seeded at 1 × 10⁵ cells/mL in 6 well plates overnight, and then treated with CQ (10uM), Wort (50 nM), CQ + Wort, and CBD/THC (10 and 12. 5 μg/mL) with and without Wort (50 nM) for 24 h. The following day, cells were prepared for immunofluorescence. As such, cells were washed with PBS (1X), fixed using 4% paraformaldehyde (PFA) and permeabilized using 0.1% Triton-X. Blocking using 1% bovine serum albumin (BSA) was performed for 1 h at room temperature followed by primary antibody incubation (anti-LC3B) overnight. The following day, washing and secondary goat anti-rabbit IgG antibody carrying Alexa fluor 488, was performed along with DAPI staining prior to visualizing the samples using the ZOE Fluorescent Cell Imager³¹.

Wound healing assay

To evaluate the migration properties of aggressive breast cancer cell lines post-exposure to cannabinoids (CBD and THC), a wound healing assay was performed. Cells were cultured overnight, in 24 well-plates, at a density of 2 × 10⁵ cells/mL and were allowed to reach a confluent monolayer. The following day, a scratch using a sterile micropipette tip was performed across the middle of the well, creating a wound region. Each well was gently washed with PBS to remove non-adherent cells before adding fresh medium with the various concentrations of treatment (IC₅₀/4 = 3.125 μg/mL and IC₅₀/2 = 6.25 μg/mL). These sub-IC₅₀ concentrations were used to confirm that any alterations in wound healing reflect alterations in migration rather than cytotoxic effects. Using the ZOE fluorescent cell imager microscope, images were taken at 0 and 24 h after wounding and the wound widths were analyzed using the ImageJ software. The data reported represents the average of 3 separate trials, each including measurements at 16 different points for each experimental condition.

Trans membrane migration and invasion assays

To further evaluate the effectiveness of the CBD/THC mixture on the motility of aggressive breast cancer cells, migration and invasion assays were performed using trans-well chambers (24-well chamber; 8 μm pore size). On the first day, TNBC cells were cultured and starved for 24 h in incomplete media supplemented with 0.5% v/v FBS. The following day, cells were collected and seeded in the upper side of the trans-well chamber at a density of 5 × 10⁵ cells/mL in serum-free medium for the migration assay or in pre-coated inserts with Matrigel for the invasion assay. Matrigel (Corning, NY, United States) was thawed on ice at 4 °C, diluted in serum-free medium (1:4), and finally 60uL were added to each insert, 1 h prior to the addition of cells with treatment (IC₅₀/4 and IC₅₀/2). In the migration assay, the cells were incubated for 24 h while the assessment of cell invasion was performed after 48 h. Following the desired incubation period, inserts were washed twice with PBS and a cotton swab was used to remove the remaining cells from the upper side of the insert. Cells on the lower side of the insert were fixed with PFA (4%) for 2 min, then permeabilized using methanol (100%) for 20 min and stained for 15 min with DAPI (1 μg/mL). Membranes were then washed and observed on the inverted fluorescent microscope and several images from different fields were used to quantify the number of migratory and invasive cells using the ImageJ software.

Protein extraction, quantification and preparation

To evaluate the effect of the CBD/THC mixture on the expression of regulatory proteins, MDA-MB-231 and MCF-7 cells were cultured in petri dishes and allowed to grow overnight. The following day, cells were treated with 12.5, 15 and 17.5 μg/mL cannabinoid mixture. After 12 or 24 h, cells were scraped and lysed using a lysis buffer prepared with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and EDTA solution to prevent the proteolytic degradation during protein extraction. Lysate was centrifuged at 18,000 g for 10 min at 4 °C and the lysate was aliquoted and stored at − 80 °C for later use³². Protein quantification was performed using the DC Protein Assay reagents (BioRad, Hercules, CA, USA) following the manufacturer’s instructions. Laemmli buffer solution containing β-mercaptoethanol was used for the preparation of protein samples (25–50 μg), subjected to 100 °C for 10 min before loading into the polyacrylamide gels.

Cytoplasmic cellular fractionation

Subcellular fractionation was performed to separate nuclear, membrane and cytoplasmic cellular fractions. A protocol adapted from Abcam was followed to perform this protein extraction. Both breast cancer cell lines were seeded at a density of 1 × 10⁵ cells/mL overnight followed by the treatment with 12.5, 15 and 17.5 μg/mL of CBD/THC for 24 h. After the desired incubation period, cell lysis was performed on ice using a fractionation buffer prepared with protease inhibitors. Cells were then scraped and centrifuged at 2800 g for 5 min. The supernatant containing the cytoplasmic, mitochondrial and membrane factions was centrifuged again at 13,600 g (5 min) allowing the separation of the mitochondrial fraction in the pellet from the cytoplasmic fraction in the supernatant^33,34. Each fraction was collected and quantified using the DC Protein Assay reagents. Proteins were prepared in Laemmli buffer solution containing β-mercaptoethanol, then denatured at 100 °C for 10 min before loading into the polyacrylamide gels.

Western blot analysis

The effect of CBD and THC mixture on the protein expression was evaluated in both cell lines, via Western Blotting. Protein samples were loaded onto the gel to be separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes which were subsequently blocked in 5% BSA solution for 1 h at room temperature and then incubated with primary antibody, overnight at 4°C³⁵. All primary antibodies were diluted according to the manufacturer’s recommendation in blocking buffer solution as follows: 1:3000 anti-β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) used as loading control, and 1:1000 anti-Bax, anti-Bcl2, anti-cleaved PAPRP, anti-cytochrome c, anti-cleaved caspase 3, anti-beclin1, anti-LC3B, anti-MMP2 and anti-MMP9. Membranes were then washed and incubated with HRP-conjugated secondary antibody (BioRad, Hercules, CA, USA) for 1 h at room temperature³⁶. The chemiluminescence Clarity Western ECL Substrates (BioRad, Hercules, CA, USA) were used to detect proteins expression through the revelation of blot images using the ChemiDoc imaging instrument. ImageJ software was used for quantitative analysis of the target protein bands as normalized to the endogenous β-actin control.

Detection of reactive oxygen species

Breast cancer cell lines were seeded in 96 well-plates at a density of 2 × 10⁵ cells/mL and allowed to adhere overnight. The following day, MDA-MB-231 and MCF-7 cells were treated for 45 min with 25 μM of the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) according to the manufacturer’s recommendation (Abcam, Cambridge, United Kingdom). After the desired incubation time, cells were washed and treated with 12.5, 15 and 17.5 μg/mL of CBD/THC mixture diluted in culture media without phenol red for 2 h and 24 h. Hydrogen peroxide (H₂O₂) was used as a potent ROS inducer and NAC, a potent ROS inhibitor was used. The oxidative conversion of H₂DCFDA to its highly fluorescent 2′,7′-dichlorofluorescein (DCF) upon ROS presence, was quantified using the fluorometric Varioskan™ LUX multimode microplate reader³⁷.

Statistical analysis

All the reported data represents the mean value ± standard deviation of three independent trials for each experiment. Statistical analyses were performed using GraphPad Prism8 by calculating p-value using t-tests, one- or two-way ANOVA depending on the experiment. GraphPad Prism8 was used for the preparation of the figures. All significant differences were reported as follows: * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: 0.0001 < p < 0.001.

Cannabinoids mixture inhibits the proliferation of breast cancer cell lines by promoting cellular fragmentation

We investigated the efficacy of two major compounds isolated from the Lebanese C. sativa plant, CBD and THC, on the viability and proliferation of breast cancer cell lines. Cytotoxicity assay was performed using the MTS reagent. As reported in Fig. 1, CBD/THC mixture (3:1) showed a significant inhibition of MDA-MB-231 and MCF-7 proliferation in a dose- and time- dependent manner. After 24 and 48 h, both cell lines showed a gradual decrease in absorption value (A₄₉₂) upon treatment as compared to the untreated cells. A significant decrease in MDA-MB-231 proliferation was determined reaching 86.4%, 63.57% and 31.37% at 12.5, 15 and 17.5 μg/mL 24 h post-treatment, respectively, while reporting a significantly higher effect after 48 h of treatment reaching 43.2%, 17.79% and 8.4% at 12.5, 15 and 17.5 μg/mL, respectively (Fig. 1A). The half-maximal inhibitory concentrations (IC₅₀) on MDA-MB-231 cells are 16.18 μg/mL at 24 h and 12.12 μg/mL at 48 h treatment. However, a milder yet significant effect was observed on MCF-7 cell line, showing a decrease in cell proliferation reaching 77.11%, 71.99% and 62.6% after 24 h and 56.51%, 49.80% and 30.57% after 48 h of treatment with 12.5, 15 and 17.5 μg/mL, respectively (Fig. 1B). The mixture of CBD and THC was found to exhibit a noticeably higher inhibitory effect on the proliferation of cells as compared to the individual compounds in concentrations that are comparable to the mixture (Supplementary Fig. 2A & B). The IC₅₀ concentrations are 19.43 μg/mL after 24 h and 13.62 μg/mL after 48 h of treatment. Thus, our results indicate a dose- and time-dependent anti-proliferative effect on both breast cancer cell lines, MDA-MB-231 and MCF-7 cells. Based on these results, subsequent experiments were performed using CBD/THC mixture concentrations (12.5, 15 and 17.5 μg/mL) the closest to IC₅₀ at 24 and 48 h on both cell lines.

Cell cycle analysis of MDA-MB-231 and MCF-7 cells in response to CBD/THC treatment was then performed by evaluating the DNA content using the flow cytometer in accordance with PI binding affinity. Cannabinoid mixture significantly decreased the G0-G1 (DNA = 2n), S (2n < DNA < 4n) and G2/M (DNA = 4n) content while increasing the pre-G (DNA < 2n) content in both cell lines in a dose- and time-dependent manner (Fig. 2). This mixture significantly decreased the G0-G1 content in MDA-MB-231 cells from 38.7% in control cells to 16.26% at 24 h and from 38.76% to 18.86% at 48 h of 17.5 μg/mL treatment. Along with this, a significant increase in the pre-G subpopulation was noticed from 8.32% and 9.17% in control cells to 59.86% and 67.96% at 17.5 μg/mL treatment at 24 and 48 h, respectively (Fig. 2C and D). The effect of treatment on MCF-7 cell cycle progression was milder when compared to MDA-MB-231 cell line. The data showed a significant decrease from 57.10% (24 h) and 51.46% (48 h) in control cells to 44.09% (24 h) and 40.63% (48 h) at 17.5 μg/mL in G0-G1 content while showing a significant increase in the pre-G content from 8.05% (24 h) and 14.98% (48 h) in control cells reaching 29.22% (24 h) and 33.78% (48 h) at the highest concentration of treatment (17.5 μg/mL) (Fig. 2G and H). Our data suggests that CBD/THC mixture inhibits the proliferation of breast cancer cells by promoting cellular fragmentation rather than inducing a cell cycle arrest.

Cannabinoid mixture promotes the activation of the apoptotic cell death mechanism in breast cancer cells

To determine whether the mixture of CBD and THC promotes the activation of the programmed apoptotic cell death mechanism, MDA-MB-231 and MCF-7 cells were treated with 12.5, 15 and 17.5 μg/mL of CBD and THC for 24 and 48 h and analyzed using flow cytometry following Annexin V and PI dual staining. As shown in Fig. 3, a dose- and time-dependent increase in apoptotic cells was observed in both cell lines. The TNBC cells showed a significant decrease in the percentage of viable cells (Ann^–/PI^–) from 87.97% in control cells to 39.37% upon exposure to 17.5 μg/mL along with a significant increase in the total percentage of apoptotic cells (Ann⁺/PI^– and Ann⁺/PI⁺) from 9.59% to 52.02% at the highest concentration of treatment (Fig. 3A). The effect was noticeably higher after 48 h of treatment, where the results showed a significant decrease from 89.63% to 39.81% in viable cells (Ann^–/PI^–) and a significant increase from 7.70% to 47.16% in apoptotic cells (Ann⁺/PI^– and Ann⁺/PI⁺) at 17.5 μg/mL of CBD/THC treatment (Fig. 3B). On the other hand, the results on MCF-7 cells showed a decrease in the percentage of viable cells (Ann^–/PI^–) from 85.75% to 68.63% coupled to an increase in the total apoptotic cells (Ann⁺/PI^– and Ann⁺/PI⁺) from 10.12% to 20.63% at 17.5 μg/mL as compared to control cells (Fig. 3C). A higher effect was observed after 48 h of treatment as shown by the significant decrease in viable cells (Ann^–/PI^–) from 82.82% to 51.69% along with a significant increase in the total apoptotic cell population (Ann⁺/PI^– and Ann⁺/PI⁺) from 13.16% to 39.17% upon exposure to 17.5 μg/mL of CBD/THC (Fig. 3D). Our results demonstrate the promising effect of this cannabinoid mixture on promoting apoptosis in MDA-MB-231 and MCF-7 cell lines in a dose- and time-dependent manner.

To further explore the potent pro-apoptotic activity of CBD/THC mixture on breast cancer cell lines, DNA fragmentation, a major hallmark of apoptosis was quantified using the ELISA method. Both cell lines showed a significant increase in the enrichment of DNA fragments while increasing the concentration of cannabinoid treatment. MDA-MB-231 cells reported a significant increase reaching 3.76, 9.48 and 11.64 upon exposure to 12.5, 15 and 17.5 μg/mL of treatment, respectively. The effect was noticed to be higher than the positive control used, Cisplatin (30uM) in this case (Fig. 4A). CBD and THC mixture showed similar effects on MCF-7 cells, with an increase in enrichment factor reaching 2.1, 3.93 and 4.77 at 12.5, 15 and 17.5 μg/mL treatment, respectively. Cisplatin, used as a positive control, showed a similar effect to CBD and THC treatment on DNA fragmentation in this cell line (Fig. 4B). At a molecular level, DNA fragmentation was further evaluated through PARP cleavage analysis via western blotting (Fig. 4C). Cannabinoid treatment induced a significant increase in the cleaved-PARP expression in both breast cancer cell lines reaching 2- and 3.5-fold increase at the highest concentration of treatment (17.5 μg/mL) in MDA-MB-231 and MCF-7 cells, respectively (Fig. 4D–E). These results confirmed the promising effect of the cannabinoid mixture on promoting DNA fragmentation in MDA-MB-231 and MCF-7 cells, a major hallmark of apoptosis.

To decipher the molecular pathways through which the cannabinoid mixture inhibits the proliferation of breast cancer cell lines, western blot analysis of regulatory apoptotic markers was performed. The expression of major proteins involved in the activation or repression of the apoptotic pathway was assessed. The expression of the pro-apoptotic protein Bax was not altered and remained constant while the expression of the anti-apoptotic Bcl-2 protein was significantly reduced as compared to the control in both cell lines treated with CBD/THC (Fig. 5). The ratio of Bax to Bcl2 was calculated and found to be significantly increasing dose-dependently in both cell lines. The data showed a 1.7- fold increase in MCF-7 cells and 5- fold increase in MDA-MB-231 cells upon treatment with the highest concentration of CBD and THC (17.5 μg/mL) (Fig. 5B–C). These results reveal the promising pro-apoptotic activity of cannabis compounds on breast cancer cells via the activation of the mitochondrial-dependent pathway. To further confirm the activation of the intrinsic apoptotic pathway, cytochrome c release into the cytosol was assessed. Thus, we aimed at evaluating the expression of cytochrome-c isolated from total and cytosolic protein fractions via western immunoblotting. No significant change in the total cytochrome c expression was observed in both cell lines. However, subcellular fractionation revealed the increase in the cytosolic cytochrome c expression in MCF-7 (15-folds increase) and MDA-MB-231 (3.5-folds increase) (Fig. 5B–C). The release of cytochrome c into the cytoplasm further supports the activation of the mitochondrial-dependent apoptotic pathway.

The activation of the execution apoptotic pathway through caspase-3 cleavage was evaluated to further confirm the prominent effect of cannabinoids on promoting apoptosis in breast cancer cells. Western blot analysis revealed a consistent upregulated expression of cleaved caspase-3 after 24 h of treatment. A significant increase in the cleaved-caspase 3 expression in both breast cancer cell lines was noted, reaching 2.5- and 15.52-fold increase at the highest concentration of treatment (17.5 μg/mL) in MDA-MB-231 and MCF-7 cells, respectively (Fig. 5B–C). These results demonstrate the pro-apoptotic activity of CBD/THC mixture as revealed by the activation of apoptosis via the execution pathway. Moreover, the caspase inhibitor, Z-VAD-FMK, was used to further determine the role of caspases in promoting cell death. Therefore, MTS assay was performed on both cell lines pre-treated with Z-VAD for 30 min followed by the addition of CBD/THC mixture for 24 and 48 h. The viability of MDA-MB-231 cells significantly decreased, reaching 28% post treatment with 17.5 μg/mL of CBD/THC. However, the cell viability was found to be significantly higher, reaching approximately 80%, when the TNBC cells were treated for 24 h with Z-VAD in combination with 17.5 μg/mL of CBD/THC (Fig. 5D). On the other hand, Z-VAD treatment did not affect the response of MCF-7 cells to the cannabinoid treatment for 24 h. Cell viability assay revealed a decrease in percent viability of MCF-7 cells reaching 60% when treated with cannabinoids alone or in combination with Z-VAD (Fig. 5E). However, a significantly higher effect of Z-VAD on reversing the anti-proliferative effect of the cannabinoid mixture in MDA-MB-231 and MCF-7 cells was observed reaching 88% and 57% viability, respectively, as compared to 14% and 33% viability when treated with 17.5 μg/mL cannabinoids alone. These results further support our previous findings demonstrating the activation of apoptosis in a caspase-dependent manner, via the onset of the execution apoptotic pathway.

To determine if the activation of the intrinsic apoptotic pathway in this case was due to oxidative stress, DCFDA assay was performed to quantify the production of ROS in MDA-MB-231 and MCF-7 cells exposed to the cannabinoid treatment. The TNBC cells exhibited a weak yet significant decrease in ROS levels upon exposure for 2 h to the cannabinoid mixture reaching 0.6-fold upon treatment with the highest concentration of CBD/THC mixture (17.5 μg/mL). However, the instant ROS production levels were found to be moderately decreased in MCF-7 cells after 2 h of treatment reaching approximately 0.75-fold. Therefore, we aimed at assessing the time-dependent effect of CBD/THC mixture on inhibiting ROS production after 24 h of treatment. Our results showed an enhanced inhibition of ROS production in MDA-MB-231 and MCF-7 cell lines in a dose-dependent manner. A significant decrease in ROS production was observed reaching 0.17 in MDA-MB-231 and 0.2 in MCF-7 cells upon exposure to 17.5 μg/mL for 24 h (Fig. 5F). Our results were compared to the potent ROS inhibitor, NAC, and the ROS inducer H₂O₂ used on both cell lines. The dose- and time-dependent inhibition of ROS formation demonstrates the antioxidant properties of this treatment suggesting that the activation of the intrinsic apoptotic pathway is not dependent on oxidative stress, but rather on internal stimuli responsible for its activation.

Cannabinoids mixture promotes alteration to the regulatory role of autophagy in breast cancer cells

To further elucidate whether the mixture of CBD and THC can promote the activation of another cell death mechanism, western immunoblotting of major autophagic markers, namely LC3B and Beclin-1, was performed. Our results showed a significant increase in the expression level of the autophagosome membrane-bound protein, LC3B, in breast cancer cells upon treatment with CBD/THC for 24 h (Fig. 6). The quantification of LC3B revealed a sevenfold and fourfold increased expression in MDA-MB-231 and MCF-7 cells, respectively. However, our results showed a significant dose-dependent decrease in the expression level of Beclin-1 in both breast cancer cell lines. The quantification of the blots showed a decreased expression reaching 0.5 in MDA-MB-231 and 0.7 in MCF-7 cells upon treatment with cannabinoid mixture. Additionally, the expression of LC3B, Beclin-1 and cleaved caspase 3 proteins was also evaluated after 12 h of treatment to assess the time-dependent alterations in autophagy. Our western blot analysis revealed a significant 1.5-fold increase in the expression of LC3B in MDA-MB-231 and MCF-7 cells while also promoting a downregulated expression of Beclin-1 reaching 0.8 and 0.5 in MDA-MB-231 and MCF-7, respectively. The expression of cleaved caspase 3 was also evaluated 12 h post-treatment and was found to be significantly upregulated in both cell lines further confirming an earlier activation of apoptosis. Given the consistent increase in LC3B observed, we aimed to investigate whether autophagy is responsible for breast cancer cell death; hence, cytotoxicity assay was performed following the treatment of breast cancer cells with the cannabinoid mixture alone or in combination with autophagy inhibitors, CQ or Wort. MTS assay was conducted, and no significant effect was observed between the cells treated with CBD/THC alone or in combination with CQ for 24 h. The MDA-MB-231 cell viability reached 27% following treatment with 17.5 μg/mL CBD/THC mixture and 30% upon treatment with CBD/THC and CQ for 24 h. Similarly, MCF-7 cell viability decreased reaching 61% and 65% upon treatment with CBD/THC mixture alone and in combination with CQ, respectively (Fig. 6D). Since CQ inhibits the last stages of autophagy, namely autophagic flux, we then aimed at assessing the effect of Wort, an inhibitor of earlier stages of autophagy. Our results revealed no reversal effect upon exposure of breast cancer cells to cannabinoids alone or in combination with Wort reaching 44% and 46% viability, respectively, supporting therefore our previous findings using CQ (Fig. 6E). To further confirm the ability of Wort to reduce the formation of complexes needed for phagophore formation, visual assessment was performed via immunofluorescence using LC3B antibody. Microscopic images revealed the ability of Wort to reverse the effect of CQ as observed by the reduced accumulation of autophagosomes in the cytosol. Immunostaining also showed an even more pronounced accumulation of autophagosomes in the cytosol of cells treated with CBD/THC as compared to cells treated with CQ alone. Taking these results together, we can suggest that the mixture of CBD/THC is promoting cellular death via apoptosis activation rather than autophagy while also acting as an autophagic flux inhibitor in breast cancer cells.

Cannabinoids mixture exhibits anti-metastatic properties by inhibiting the migratory and invasive capacities of TNBC cells

From a different perspective, we aimed at assessing the anti-metastatic properties of CBD and THC on the most aggressive type of breast cancer, namely TNBC cells. Wound healing and trans-well migration and invasion assays were performed. In the wound healing assay, the TNBC cells were co-cultured with low doses of CBD/THC mixture (IC₅₀/4 and IC₅₀/2) knowing to exhibit a mild effect on cell viability, following the scratch formation. In Fig. 7, images from time points 0 and 24 h are shown along with the quantification results of the wound closure (Fig. 7A). Our results showed that the cannabinoid mixture significantly inhibited the migration of MDA-MB-231 cells across the wound space after 24 h, as compared to the control cells. A significant increase in the wound width was observed upon exposure of the MDA-MB-231 cells to 6.25 μg/mL (IC₅₀/2) of CBD/THC for 24 h. To further confirm the results of wound healing assay, trans-well migration assay was performed. Our results suggested that after 24 h of treatment, the motility of the TNBC cells was significantly inhibited as revealed by the prominent decrease in the number of migrated cells when compared to untreated cells (Fig. 7B) A significant decrease in migrated cells was noticed reaching 0.8 and 0.2 upon exposure to 3.12 and 6.25 μg/mL, respectively. On the other hand, we tested the inhibitory effect of CBD/THC mixture on inhibiting cell invasion through a Matrigel coat that resembles the extracellular environment. Our data revealed a significant inhibitory effect of this cannabinoid’s mixture on the invasive capacities of MDA-MB-231 cells following CBD/THC treatment (6.25 μg/mL) for 48 h reaching 0.3 folds (Fig. 7C). The anti-metastatic effect was further elucidated at a molecular level as revealed by the ability of CBD/THC to downregulate the expression of Matrix Metalloproteinases (MMP) (Fig. 7D). Our data showed a significant downregulated expression of MMP2 and MMP9, reaching 0.6 and 0.5, respectively upon exposure to 17.5 μg/mL of CBD/THC. Taking these results together, we can conclude that the CBD/THC mixture exhibits an inhibitory effect on the metastatic ability of TNBC cells.

Competing interests

The authors declare no competing interests.