Cell culture

CEM (acute lymphoblastic), HEL-92 (erythroblastic), HL60 (acute promyelocytic), and MOLT-4 (acute lymphoblastic) leukemic cell lines were obtained from the Cancer Research UK Laboratories (London, United Kingdom) and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. No antibiotics were used in our experiments, and all cell lines were incubated in a humidified atmosphere with 5% CO2 in air at 37°C.

To study the effect of THC on cell growth, cells (2 × 105/mL) growing exponentially were reset in fresh culture medium supplemented with THC (0-100 μM; Sigma, Dorset, United Kingdom). Aliquots were removed at 6, 24, and 48 hours for assessment of cell number, cell viability, and cell cycle distribution.

The concentration required to reduce cell viability by 50% (IC50) was determined using the sigmoid Emax model as follows: EP = EC – [(Emax×Cn)/(IC50n + Cn)], where EP is predicted effect, EC is control effect, Emax is maximum effect, C is concentration of drug, and n is the sigmoid-fit factor.13

As THC binds to both the CB1 and CB2 receptors, we assessed the effects that THC had through individual receptors by culturing THC-treated cells with the respective antagonist. Specifically, cells were cultured with THC at the Bmax concentration (concentration just achieving maximum binding) with the CB1-R antagonist AM28114 (Tocris Cookson, Bristol, United Kingdom) and/or the CB2-R antagonist AM63015 (Tocris) (both 0-100 μM). Cell parameters were then assessed on day 2. The effect on cell viability of culturing cells with the specific CB2-R agonist palmitoylethanolamide (PEA)16(0-100 μM; Tocris) was also studied in a similar fashion. Additionally, the effect of PEA on THC-induced cytotoxicity was investigated by culturing CEM, HEL-92, and HL60 cells with THC in the presence or absence of PEA (both at IC50 concentrations) prior to assessment of cell viability on day 2.

DNA analysis

The distinct phases of the cell cycle were distinguished by flow cytometry, according to methods described previously.17 Acquisition of data was performed within 1 hour using a FACSCalibur (BD Biosciences, Oxford, United Kingdom). Five thousand cells were analyzed for each data point, and the percentages of cells in sub-G1 (apoptotic fraction, cells with a reduced DNA content but similar morphology), G1, S, and G2/M phases were determined using the cell cycle analysis program WinMDI v2.4 (http://facs.scripps.edu/software.html).

Biotinylation of THC and determination of CB1 and CB2 cannabinoid receptor levels

THC was irreversibly biotinylated with biotin-XX-NHS (CN Biosciences, Nottingham, United Kingdom) using methods reported previously.18 Briefly, a stock concentration of THC (10 mM) was incubated with biotin-XX-NHS (2 mg/mL) at a ratio of 1:5 for 6 hours at room temperature. Phosphate-buffered saline containing 0.1% bovine serum albumin was added and incubated for 1 hour at 4°C. The mixture was then centrifuged in a Microcon YM-100 centrifugal filter unit (Millipore, Watford, United Kingdom). The residue (biotinylated-THC) was then collected from the filter head by reverse centrifugation and made up to a volume of 100 μL with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (Solution A).

To assess total receptor binding, cells (1 × 106) were resuspended in 100 μL PBS and incubated with 10 μL biotinylated THC for 1 hour. Following 3 washing steps in Solution A, cells were incubated with streptavidin–fluorescein isothiocyanate (FITC) conjugate (1:100; CN Biosciences, Nottingham, United Kingdom) for 45 minutes at 4°C. Cells were washed thrice and resuspended in 500 μL PBS for flow cytometric analysis using the FACSCalibur. Five thousand events were recorded, and the mean FITC-fluorescence in each sample was measured and assessed using CellQuest v2.8 (BD Biosciences). As there were no good selective CB1-R and CB2-R agonists that could be biotinylated like THC and used to detect the individual cannabinoid receptors, we assessed the amounts of individual cannabinoid receptors by culturing cells with biotinylated THC, in the presence or absence of selective CB1-R and CB2-R antagonists (10 μM AM281 and AM630). The extent of THC binding was then established like before.

Assessment of gene expression

Samples for analyses of gene expression were prepared from CEM cells cultured with an IC50 THC, in a manner previously described.19 Briefly, aliquots (10 × 106 cells) were removed at 3 hours and homogenized in TRIzol reagent (Invitrogen, Paisley, United Kingdom). Total RNA was then chloroform extracted and precipitated using iso-propanol and ethanol. RNA quality was examined using the Agilent 2100 Bioanalyzer (Agilent Technologies UK, Cheshire, United Kingdom). Biotinylated copy RNA transcripts were produced from total RNA and hybridized to the human genome U133A oligonucleotide array comprising around 23 000 genes (Affymetrix UK, High Wycombe, United Kingdom). Samples were run in triplicate, and data analyses were performed using GeneSpring v6.1 (Silicon Genetics, Redwood City, CA). Only consistent changes in gene expression, relative to the untreated control samples that were detected in all 3 replicates, were reported.

Immunoblotting analysis for p53, cleaved PARP, total extracellular signal-related kinase protein (pERK), and phosphorylated pERK

Total cellular protein was solubilized and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide with a 5% stacking gel as described previously.17 Primary antibody probing was performed with anti-p53 (clone DO-7; 1:500; Dako, Cambridge, United Kingdom), anti-cleaved poly (adenosine diphosphateribose) polymerase (PARP; 1:1000; Cell Signaling Technology, Hitchin, United Kingdom), anti–phospho-p44/42 MAPK (1:1000; Cell Signaling), or anti–p44/42 MAPK (1:100; Cell Signaling). Anti–β-actin was used as a loading control (1:200; Oncogene Research Products, Boston, MA). Following a washing step in 0.1% Tween in tris-buffered saline (100 mM Tris (tris(hydroxymethyl)aminomethane), 150 mM NaCl, pH 7.6) horseradish peroxidase–conjugated anti-species immunoglobulin G1 (IgG1) was used as the secondary antibody (Dako). Bands were visualized by the electrochemiluminescence (ECL) detection system (Amersham Biosciences, Little Chalfont, United Kingdom).

Statistical analysis

All statistical analyses were performed using Minitab version 10 (State College, PA). Data were normally distributed as established by Shapiro-Wilk testing, and parametric analyses were used throughout. Differences between variables and control cultures, as determined by analysis of variance, were further characterized by paired Student t tests.

THC is cytotoxic to leukemic cell lines

Concentration-dependent decreases in cell viability were seen in all cell lines cultured with THC for 2 days (Figure 1A). The concentrations required to reduce cell viability by 50% (IC50) were calculated and were similar in CEM and HL60 cells (26.5 μM and 21.1 μM, respectively), but higher in HEL-92 cells (61.4 μM). There were concomitant increases in apoptotic cells and reductions in the number of cells in the other phases of the cell cycle (Figure 1B-D). Apoptosis was confirmed in CEM cells by the presence of the cleaved PARP in cells cultured with THC as early as 3 hours (Figure 5C). When the subdiploid (sub-G1) portions of the histograms were excluded from the analyses, there was still no evidence of arrests at any phases of the cell cycles (Table 1).

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Figure 1.

Effect of THC on cell growth parameters. CEM, HEL-92, and HL60 cells were cultured continuously with THC (0-100 μM) for 2 days. (A) Cell viability was assessed by trypan blue dye analyses after 6 hours and 2 days, and the concentration required to cause a 50% reduction in cell viability (IC50) was calculated and is shown in the inset box. The effect of 2-day cultures with THC on cell cycle distribution was also assessed in the cell lines (B-D), and representative cell cycle histograms of cells cultured with 60 μM THC are shown (E). Each data point represents the means of at least 3 separate experiments, and SDs have been omitted for clarity.

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Figure 5.

Effect of THC on p53 protein expression. CEM and HL60 cells were cultured with an IC50concentration (day 2), and p53 expression was assessed by immunoblot analysis at 6, 24, and 48 hours (A). Similarly, culturing MOLT-4 cells with THC for 2 days also showed no increase in p53 levels, while culturing with cisplatin (cddp) did (B). The culture of CEM cells with THC at 1 × and 5 × IC50 resulted in duration-independent increases in cleaved PARP and decreases in phosphorylated-pERK (C).

THC was also cytotoxic in the cell lines after only 6 hours of culture (percentage of viable cells [%V] IC50, 48.2, 65.8, 73.2 μM in CEM, HL60, and HEL-92, respectively), and this was associated with varying degrees of apoptosis in the cell lines (percentage of apoptotic cells [%A] at 6 hours, 1.2% ± 0.6% in CEM cells to 38.0% ± 1.1% in HL60 cells; Figure 1E). To establish whether THC cytotoxicity was exclusive to cancer cells, the efficacy of THC was investigated in peripheral blood mononuclear cells. Results showed THC was as potent in these normal cells (day 2 IC50, 48.1 μM).

Leukemic cell lines express varying levels of CB1-R and CB2-R

The levels of CB1-R and CB2-R were assessed by a novel nonradioactive method, and results indicated clear differences in their levels in the cell lines tested. Initially, we measured total THC binding to each of the cell lines, without distinguishing the 2 subsets of cannabinoid receptors (total binding) (Figure 2). We next blocked each receptor subset in turn by using specific antagonists, as a way of assessing the level of individual receptor type (Figure 3A-B). As an example, total THC binding to CEM cells was 4.82 ± 0.09 units. Blocking CB1-R with a specific CB1-R antagonist reduced the degree of THC binding to 0.87 ± 0.03 units, while blocking the CB2-R reduced THC binding to 3.29 ± 0.08 units. Blockade of both receptor subtypes, resulted in THC binding of just 0.41 ± 0.21 units (Figure 3B).

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Figure 2.

Binding profiles for THC. CEM (•), HEL-92 (HEL; ▪), and HL60 (▴) cell lines were cultured with biotinylated THC. Cells were then incubated with FITC-conjugated streptavidin, and the extent of fluorescence was assessed by flow cytometry. The maximum specific binding (Bmax) and the dissociation constant (Kd) values in the cell lines are shown in the inset box. The IgG1 isotype control (control) is also shown, and each data point represents the means and SDs of at least 3 separate experiments.

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Figure 3.

Assessing the levels of CB1-R and CB2-R. (A) CEM, HEL-92 (HEL), and HL60 cell lines were cocultured with a biotinylated THC (at Bmax concentration) and increasing concentrations of either the CB1-R or CB2-R antagonist (CB1-A or CB2-A). Mean fluorescence, as a measure of biotinylated THC binding, was then assessed by flow cytometry. (B) Similarly, the total cannabinoid-receptor level in each cell line was assessed by coculturing cells with biotinylated THC (Δ9-THC) and each antagonist (CB1-A and CB2-A); all were used at a maximum-blocking concentration. Each data point represents the mean of at least 3 separate experiments, and SDs have only been shown in panel B.

Cytotoxic effect of THC is not mediated via the CB1-R and CB2-R

To investigate the role of the cannabinoid receptors in the cytotoxic response, cells were cultured with an IC50 concentration of THC in the presence of the receptor antagonists. Initially, we assessed the cytotoxic effect of the antagonists alone on the cell lines, and results showed the CB1-R antagonist to have no significant effect on cell viability. However, the CB2-R antagonist caused concentration-dependent decreases in cell viability (Figure 4A). Coculturing cells with THC (IC50) and increasing concentrations of the CB1-R antagonist resulted in no loss of cytotoxicity in the cells, indicating that the CB1-R was not responsible for this effect (Figure 4B). The effect of coculturing cells with THC and the CB2-R antagonist was more difficult to interpret as the CB2-R antagonist had a cytotoxic effect in its own right (Figure 4C). However, there was no reversal of THC-induced cytotoxicity at any concentration. The cytotoxic effects of THC were not alleviated by the addition of both antagonists concurrently (%V on day 2 in CEM, HEL-92, and HL60 66.7% ± 6.1%, 72.7% ± 6.4%, and 75.3% ± 3.1%, respectively, in THC-treated cells versus 69.0% ± 8.5%, 68.0% ± 2.0%, and 74.7% ± 3.1%, respectively, in cells cultured with THC and both antagonists). We addressed the possibility that the CB2-R antagonist may have been acting as a putative reverse CB1-R agonist by culturing cells with both the CB2-R and increasing concentrations of CB1-R antagonists. Results showed that the CB1-R antagonist did not alleviate the cytotoxic effect of the CB2-R antagonist (Figure 4D). As the CB2-R antagonist caused cell death on its own, we further explored the role of the CB2-R in mediating cell kill with the use of a specific CB2-R agonist (PEA). Results showed that PEA had no significant cytotoxic effect in the cell lines (Figure 4E). Similarly, PEA did not significantly antagonize/interfere with THC-induced cytotoxicity (Figure 4F).

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Figure 4.

Effect CB-R antagonists of cell viability. CEM, HEL-92, and HL60 cells were cultured continuously with either the CB1-R or the CB2-R antagonist (0-50 μM) for 2 days. As the trend in the effects of the individual antagonists were similar in that the CB1-R alone had no effect on viability while the CB2-R was cytotoxic, only the results of the effects on CEM are shown graphically, with the IC50s seen in the cell lines shown in the inset box (A). Cells were also cultured for 2 days with a combination of THC (IC50) and increasing concentrations of either the CB1-R antagonist (B) or the CB2-R antagonist (C). The effect of the CB1-R antagonist on the cytotoxic effect of the CB2-R antagonist (IC50) (D), and the cytotoxic effect of the putative CB2-R agonist PEA (E), were also studied. The effect of PEA in CEM, HEL-92, and HL60 cells at IC50 concentrations on THC-induced cytotoxicity were assessed on day 2 (F). White bars show untreated samples; light gray bars, THC at IC50; dark gray bars, PEA alone; and black bars, the effect of both agents used concomitantly. Each data point represents the means and SDs of at least 3 separate experiments.

THC does not increase p53 expression

To investigate whether p53 protein levels were affected by THC treatment, whole-cell lysates from CEM and HL60 cells were analyzed by immunoblotting. Results showed no significant changes to p53 (Figure 5). As these cell lines possess mutated p53 responses, similar immunoblotting experiments were performed using the acute myeloid leukemic cell line MOLT-4, which expressed wild-type p53. These cells were cultured with IC50concentrations of either THC or cisplatin, and results recapitulated the previous data, showing no increases in p53 levels following culture with THC. In contrast, significant increases in p53 levels were seen following cisplatin treatment (Figure 5).

THC does not interact with common cytotoxic agents

To investigate interactions between THC and established chemotherapeutic agents (cisplatin), cells were cultured with a combination of the 2 at IC25concentrations. Results from these simple pilot experiments showed additivity in the magnitude of cell kill when using the 2 agents simultaneously. Specifically, the estimated cytotoxicity (numerically the sum of the effects of the drugs used individually) was comparable to the measured cytotoxicity (the effect when the 2 drugs were used concurrently)17(Figure 6A). Due to the absence of clear hyperadditivity in these early experiments involving IC25 concentrations of both drugs, we next investigated whether or not lower concentration of THC (IC5 and IC10) could enhance cisplatin-induced cytotoxicity. Results showed that there were no significant increases in cell kill when culturing both drugs together at lower doses (Figure 6B).

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Figure 6.

Effect of combining THC with cisplatin. The viability in the cell lines treated concurrently with IC25 concentrations of THC and cisplatin (▦) were compared with the calculated viability (□) (A). This estimated value was determined as the sum of the viabilities in cells treated with either THC or cisplatin alone. Cells were also cultured with small concentrations of THC (IC5, ⋄; and IC10, ▴) or left untreated (▪) to investigate whether these suboptimal doses could enhance the cytotoxic effects of cisplatin. Each data point represents the means and SDs of at least 4 separate experiments.

THC induces changes in the gene expression profile

Microarray analyses were performed on CEM cells cultured with THC. We used a low concentration (1 × IC50 at day 2) and a short exposure time, which resulted in a data set with few gene expression changes. However, because of the nature of the treatment, the changes observed were likely to be drug specific and as a direct result of drug exposure, rather than nonspecific secondary effects like cell death or cell cycle arrest. First of all, we looked specifically at the expression of genes that had previously been identified as having a role in THC-induced cell death, such as genes in the ras/MAPK pathway, and those involved in ceramide metabolism. Genes in the MAPK signaling cascade that showed altered expression were DUSP6 (encoding dual specificity phosphatase 6/MAPK phosphatase 3 [MKP3]) and MAP2K2(mitogen-activated protein kinase kinase 2/MEK2). Interestingly, MKP3 and MAP2K2 have the same intracellular target, the extracellular signal-regulated kinase 2 protein (ERK2/mitogen-activated protein kinase 1). They have, however, opposing functions: MEK2 phosphorylates, and thereby activates ERK2, whereas MKP3 dephosphorylates ERK2, taking it to an inactive state. Accordingly, the expression changes in response to THC observed were an increase in MKP3 expression in all 3 samples and a decrease in MAP2K2 expression (Table 2). Both changes are consistent with decreased MAPK signaling. There were no significant changes seen in genes involved in ceramide metabolism or in any other mechanism associated with THC-induced cell death. Additionally, there were no changes in the cytokine genes associated with THC-induced immune suppression. Many of these genes were in fact absent. Second, we examined the genes exhibiting the greatest degree of change in expression. The top 10 up- and down-regulated genes are listed in Table 2. Of particular note, both MKP3 and MAP2K2 were among the most significantly changed genes. Although 2 genes encoding histone H1 isoforms were the 2 most significantly down-regulated genes, there were no other gene patterns that were immediately apparent. We used a t test and fold-change filtering program (www.cgal.icnet.uk) as a plug-in for the GeneSpring v6.1 software to find the most significantly changed genes. Using a 2-fold cut-off and a delta-value of less than 0.1, 3 genes were found to be significantly changed in the THC-treated samples; these were the DUSP6gene, encoding MKP3, and the histone H1 isoforms H2BK and H4C. The scatter plot of 1 of the treated samples normalized against the controls (Figure 7) revealed additional genes to these 3 that were outside the 2-fold cut-off lines; however, these outliers were either closer to the 2-fold cut-off or were lower in intensity, which decreased the confidence of the measurements. Although these changes may have been biologically relevant, they were not deemed significant.

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Figure 7.

Scatterplot of CEM cells treated with THC. Data are normalized against the controls, showing 2-fold cut-off lines. Only the genes for DUSP6 (increased), H1H2BK, and H1H4C(both decreased) were significantly altered in the THC-treated cells (P < .1).

THC decreases phosphorylated pERK protein expression

To investigate whether the changes in the gene expression of MAPK-associated species, whole cell lysates from CEM cells cultured with 1 × and 5 × IC50 THC were analyzed by immunoblotting. Results indicated clear decreases in the expression of pERK as early as 3 hours of incubation with 1 × IC50 THC (Figure 5C).