Phytocannabinoids possess anticancer activity when used alone, and a number have also been shown to combine favourably with each other
The phytocannabinoids (CANN) are a group of related compounds extracted from the cannabis plant (
A number of cells express the cannabinoid receptor (CBR), of which there are a number of sub-types (e.g. CBR1, CBR2), and it is believed that signalling through this G-protein coupled receptor is required for CANN anticancer action. Most of the
Results from numerous
The necessity for receptors to be present in order to elicit these cell killing mechanisms may not, however, be absolute; anticancer activity has been seen in leukaemia cells that is independent of the receptors (
As part of our ongoing research efforts investigating the potential benefits of CANNs in a leukaemia setting, we have examined further the effects of these drugs combined with others on cell growth and survival. We paired CANNs together and specifically examined the activity of these mixes in leukaemia cells, both alone and in combination with a number of common anti-leukaemia drugs. We have adopted a number of practical models to assess drug-drug interactions, and also assessed the importance of drug sequence in determining the overall efficacy of the differing treatments.
The human cancer cell lines CEM (acute lymphocytic leukaemia) and HL60 (promyelocytic leukaemia) were purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK), and grown in RPMI-1640 medium (Sigma-Aldrich Co., Ltd., Dorset, UK) supplemented with 10% foetal bovine serum (FBS) and 2 mM L-glutamine. All cell lines were incubated in a humidified atmosphere with 5% CO2 in air at 37°C, and discarded after ~12 passages. Authentication of the cell lines was performed by the service provider using the AmpFISTR Identifier Plus PCR amplification kit looking for the presence of <10 known loci for each of the cell lines.
Cytarabine (CYT: Sigma) and vincristine (VIN: Sigma) were reconstituted in PBS at a stock concentration of 10 mM, and kept at −20°C for no more than four weeks. Cannabidiol (CBD), cannabigerol (CBG) and THC (all provided by GW Research Ltd., Cambridge, UK) were dissolved in ethanol to appropriate concentrations that ensured a final ethanol concentration in cell cultures <0.1%. For experiments with treatments, the amount of FBS in the cell culture medium was reduced to 5%. One aim of the current study was to investigate the benefit of using two different CANNs together in a pair. The combinations used here mimic a number of current and recent clinical trials where a proprietary product containing CBD and THC was used (
To study the effect of the CANNs on cell growth, leukaemia cells that were growing exponentially were seeded into 96-well plates at a density of 1.5×104/well. CANNs were then added to the wells at various concentrations, ensuring an equal volume of 200
Cells (1.5×104/well) growing exponentially were reset in fresh culture medium and aliquoted into 96-well plates. A CANN-pair (either CBD+THC or CBD+CBG) was combined with CYT or VIN at concentrations that were equal ratios of their respective IC50 according to methodologies described previously (
The ability of CANNs to modify the efficacy CYT and VIN was studied by assessing and comparing the IC50 of the anti-leukaemia drugs in the absence and presence of the CANNs. The CANNs tested were CBD+CBG and CBD+THC (the modulating drug in this setting), and these were used at a single total sub-optimal concentration of 1
CEM and HL60 cells were seeded into 6-well plates at a density of 1×105/well and then treated according to a culture schedule that lasted a total of 96 h. The treatment would involve two separate phases; each lasting 48 h. One set of drugs would be administered in the first 48 h phase and a second set of drugs in the following 48 h phase. The culture medium would be removed by centrifugation after the first treatment to be replaced with fresh medium in an attempt to remove the drugs used in the first phase of treatment. The drugs studied were either CBD+CBG (4
Western blot analyses were performed as previously described (
All statistical analyses were performed using GraphPad Prism or Microsoft Excel, and differences between treatments and control groups were determined by one-way ANOVA and subsequently by paired tests. Data values were presented as the means and SDs of at least three separate experiments.
Our previous studies showed a small number of CANNs could be used together to induce a cytotoxic response that was hyperadditive in nature. We therefore expanded this initial work by pairing CBD, CBG and THC in different permutations, and assessing their effects on cell numbers after 72 h treatment. IC50 values for the individual CANNs were determined, and these were compared with IC50 achieved when the matching CANN-pair was used. Results showed the virtual IC50 of the mixtures were generally lower than those for the CANNs when used individually (
In this basic paired-model, CEM cells were more responsive to treatments, as the combination of two CANNs generally resulted in an improvement in activity. Moreover, combinations including CBD as one of the partners in a pair usually resulted in a greater reduction in cell number (IC50 values in CEM for CBD/THC, CBD/CBG and CBG/THC were 3.6±0.19, 2.8±0.24 and 11±0.55
Median-effect analyses were employed to assess the interactions between each CANN-pair and common anti-leukaemia drugs. Guided by our initial results showing CBD-containing pairs to be most efficacious, we selected the CBD/CBG and CBD/THC pairs and combined them with either CYT or VIN. CI-values were then calculated by using these results and used as a way of understanding the drug-interactions (
Results showed that outcome of the interactions were dependent upon both drug and cell line. They also hinted that combinations involving VIN would more likely result in enhanced activity, whilst those with CYT may cause additivity/mild antagonism (
A second model of drug interaction was employed in our studies. This experiment was designed to test the ability of a CANN-pair to sensitise cells to the effects of CYT or VIN. Specifically, the ability of a sub-effective concentration of CANN to alter the efficacy of CYT or VIN was determined by comparing the IC50 values of the chemotherapy agents in the absence or presence of the modulating CANN drug. Results showed adding CANNs to cells cultured with CYT or VIN only changed the cell number IC50 values for each chemotherapy drug to a small extent (
The reduction in viability was associated with an increase in apoptosis, as shown by flow cytometry, which was generally higher in combinations involving CBD/THC (
Having seen synergistic interactions between CANNs and anti-leukaemia drugs when they were used simultaneously, we next assessed the impact of using the drugs sequentially. Consequently, cells were cultured according to schedules that consisted of two rounds of treatment, each lasting 48 h. Each round of treatment was separated by a washing step to remove drug from the medium. The order of the drugs were swapped in equivalent experiments to assess the counter-order of drugs. In some cases, a treatment schedule could involve the use of a CANN-pair in the first round of treatment followed by no treatment in the second. This mimicked a 'recovery' schedule. The duration of each treatment phase was 48 h to ensure that cells were not overgrown by the end of the full treatment regimen, which lasted 96 h.
Results showed that, generally, the percentages of cells within the sub-G1 population of the cell cycle were low in CEM cells following any treatments (
In accordance with our earlier published data, the greatest number of cells present in a sub-G1 population was seen following the schedule where HL60 cells were cultured with CBD/THC in the first phase of treatment followed by no treatment in the last phase (92%), in imitation of a recovery phase. This was considerably higher than the percentage seen when the cells were cultured with CBD/THC in both rounds of treatment (66%) (
This work was a continuation study performed to investigate further the effects that CANNs may have on leukaemia cells. Our earlier studies showed that a number of CANNs were capable of eliciting death in cancer cells when used alone or in combination with each other; however, the benefits of using these with pre-existing chemotherapy drugs had not been investigated. In the current study, we showed that combining CANNs with the anti-leukaemia agents CYT and VIN resulted in enhanced overall activity. Furthermore, cooperation between CANN and chemotherapy was sequence-dependent, with a greater level of cell killing seen when the CANNs were used after the chemotherapy.
There is an increasing body of evidence showing that CANNs derived from the cannabis plant possess anticancer activity (
Although anticancer studies involving CANNs have rightly concentrated on cancers of the brain (
These studies fully support the possibility that mixing CANNs could result in a product that is optimised for anticancer effect. Crucially, it is important to note that not all the individual components of a combination need to elicit a direct cytotoxic effect, but instead can merely support the effect of its corresponding pair/partner. This cooperative phenomenon has been described using a number of terms such as an entourage-effect, a bystander-effect and a compensatory-effect; however, the overall effect for a combination is simply to induce a measurable response that is greater than the sum of component's individual ones (
In our current studies, initial experiments were performed to assess the activity of various CANN pairs and to identify the most active mix. Our results suggested that pairs comprising CBD were most active. In agreement with our earlier results, pairing CBD with CBG was as active as CBD with THC. The mechanism of this cooperative interaction between CANNs is unknown, but may simply be a reflection of the sum of the anticancer properties of the individual agents used (
After confirming that these CANNs could be paired without a loss of anticancer action, we next mimicked the current clinical path by assessing the effect of combining CBD/THC with common anti-leukaemia drugs. We first determined the value of using the CANN pair and chemotherapy at the same time, and results showed clear improvements in the cytotoxic response. This was indicated by significant improvements in the IC50 of CYT and VIN if CBD/THC was included in the treatment. For example, in HL60 cells, the IC50 for VIN was 20 nM; however, this was reduced to 3.2 nM if a sub-toxic dose of CBD/THC was used with it. Furthermore, improvements in the IC50 were associated with increases in apoptosis. Generally, substituting the CBD/THC pair with CBD/CBG had little effect on the IC50 for CYT and VIN; however, the IC50 values were reduced and chemotherapy efficacy improved in some instances.
The sequence in which certain drugs are administered can influence the overall activity of a treatment course for a number of cancers (
In addition to their cytotoxic features (
In summary, our data showed that a number of CANNs could be used together in pairs to generate anticancer responses that are greater than would be expected if the components were used separately. These CANN pairs can then also be combined synergistically with common anti-leukaemia agents. Importantly, results also suggested that the sequence of the drugs may be crucial in determining the clinical activity of combination treatment regimens involving CANNs. Specifically, our studies recommend that if CANNs are to be combined with other anti-leukaemia drugs, that they should be used either concomitantly or after them. In conclusion, evidence of CBD activity in patients with certain forms of cancer linked with a considerable body of evidence
This work was funded by a research grant awarded to W.M.L. from GW Research Ltd. (Cambridge, UK).
Dose response curves of single agent and cannabinoid combinations. CEM and HL60 cells were grown for 48 h in the presence of increasing concentrations of the three cannabinoids, THC, CBD and CBG, either as single agents or in dual combinations prepared at a 1:1 ratio. Thus, 10
Median effect analysis of chemotherapy and cannabinoid combinations. CEM and HL60 cells were grown for 72 h in the presence of increasing concentrations of both cytarabine or vincristine and a cannabinoid-pair, combined at fractions of their respective IC50 values. CBD/THC and CBD/CBG were the two cannabinoid-pairs that were investigated, and were used at equal 1:1 ratios. Cell number was assessed at 72 h using the MTT assay and defined algorithms were then used to generate a combination index score (CI) which indicates the nature of the combination interactions (CI=1 = additivity; CI <1 = synergy; CI >1 = antagonism) (A). Representative data have also been included from experiments in CEM (B) and HL60 (C). Each data point in the column graph represents the mean and SD of at least three separate experiments.
Sensitising chemotherapy action with low doses of cannabinoids. CEM and HL60 cells were grown for 72 h in the presence of increasing concentrations of cytarabine (CYT) or vincristine (VIN). The effect of a low dose of CBD/THC or CBD/CBG on the activity of CYT and VIN was also assessed. IC50 values for cell number (A) and percentage cell viability (B) were determined by Emax models.
Sensitising chemotherapy action with low doses of cannabinoids. CEM and HL60 cells were grown for 72 h in the presence of increasing concentrations of cytarabine (CYT) or vincristine (VIN). The effect of a low dose of CBD/THC or CBD/CBG on the cell cycle distribution was assessed by flow cytometry, and the particular effects on the sub-G1 (apoptosis/cell death) fraction of cells (A) and on cyclin B expression (B) were studied more closely as read-outs for cytotoxicity and cytostasis, respectively. Each point in the column charts represent the mean and SD of three separate experiments, and SDs in (B) were omitted for clarity.
Effect of drug sequence on the cell cycle. CEM and HL60 cells were cultured according to schedules consisting of two distinct treatment stages lasting 48 h each. Treatments consisted of a cannabinoid - either CBD+THC (CT) or CBD+CBG (CG) in the first stage, followed by cytarabine (CYT) or vincristine (VIN) in the second. Parallel cultures were also performed in which the sequence of drugs was reversed. In some instances, cells were untreated (UN). Cell cycle distribution was then assessed by flow cytometry at 96 h (A). The specific effect on % sub-G1 cells using any regimen where a cannabinoid was used first was compared with those in which a cannabinoid was used second (B).