Introduction
The endocannabinoid system consists of cannabinoid (CB) receptors, endogenous lipid signalling molecules generated in the cell membranes from phospholipid precursors termed endocannabinoids and the enzymes that produce and degrade these natural ligands. Two major types of cannabinoid receptors, CB1 and CB2, are G protein-coupled membrane metabotropic receptors expressed predominantly but not exclusively in the central nervous system and peripheral immune system, respectively. The endocannabinoid system is implicated in a variety of physiological and pathological processes such as control of behaviour, control of energy expenditure, inflammation, analgesia, cancer, etc. Two best studied endocannabinoids are anandamide (AEA) and 2-arachidonoylglycerol (2-AG) [21].
Anandamide is the first identified endocannabinoid [10] which acts on both CB1 and CB2, receptors [14]. Anandamide also binds transient receptor potential vanilloid type 1 receptor (capsaicin receptor, TRPV1), a non-selective cation channel activated by a variety of physical and chemical stimuli [33]. This prototypic substance has been reported to exert substantial antiproliferative and proapoptotic activity toward various cancers [see, for example: 1, 9, 12, 17, 18, 27]. For in vitro studies investigators used different cancer cell cultures, employed various methods and protocols to estimate AEA antiproliferative and/or cytotoxic properties, and identified involvement of different receptors in anticancer activity of this compound. For example, Mimeault et al. [30] have shown that micromolar concentrations of AEA exert strong inhibitory effects on EGF-stimulated proliferation of prostate carcinoma cells by acting through CB1 receptors, whereas according to Contassot et al. [5], micromolar concentrations of AEA induced apoptosis in the long term and recently established glioma cultures acting on TRPV1, but not CB1 and/or CB2 receptors. Diversity of results is explained by complexity of mechanisms through which cannabinoids and their receptors impact proliferation and apoptosis of cancer cells, and by different reactions of various types of cancer cells [16].
One of the more frequently used experimental model systems for malignancy is rat C6 glioma. This rapidly proliferating cell line, originally obtained by exposing the rat to N,N’-nitrosomethylurea, presents several features similar to human glioblastomas and enables bothin vitro and in vivo experiments [15]. The effects of AEA on rat C6 glioma cell line have been repeatedly investigated [3,7,23-26] and in several of these studies the MTT cell viability assay was used. Although numerous alternatives have been proposed, the MTT test (introduced by Mosmann in 1983) is still much in use for cellular chemosensitivity assays [34]. Considering its extreme simplicity, it might have been expected that the MTT test should be a robust and dependable tool for assessing viability of cell cultures exposed to AEA. However, even these studies resulted in discordant results.
In the present study we report on our attempts to standardize the MTT cell viability assay and use it to characterise the in vitro response of the C6 rat glioma cell line to short-term, 24h exposure to AEA. Results of our experiments suggest some general remarks concerning methodology and interpretation of the in vitro cytotoxicity tests.
Material and methods
Cell culture
Rat C6 glioma cells were obtained from the American Type Culture Collection (Manassas, VA) and used over a passage range of 2 to 15. Cells were cultured in Petri’s dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ ml streptomycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 in air and the medium was changed every 3 to 4 days.
MTT assay
To determine the effects of anandamide upon cell viability, we measured the redox-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich) – experimental scheme in Fig. 1. C6 glioma cells were seeded in 96-well flat bottom microplates at a density of 2 × 104 cells per well in DMEM supplemented with 10% FBS and penicillin-streptomycin. After 24 h incubation at 37°C, the medium was removed and a new culture medium containing anandamide (Tocris Bioscience, UK) was added (assay volume 200 µl). The cells were then further incubated at 37°C for 24 h, after which 20 µl (5 mg/mL) solution of MTT dissolved in PBS and in medium with or without FBS was added to each well following Table I and Fig. 1. After 3 h of incubation media were removed and MTT metabolic product – formazan was dissolved in 200 µl DMSO and plate was placed on a shaking table for 15 minutes. Absorbance at 570 nm was measured by using Epoch microplate reader (BioTek). Experiments were performed according to five protocols shown in Table I and experimental scheme in Fig. 1.
Statistical analysis
The concentration-dependent effects of AEA upon C6 cell proliferation were analysed for statistical significant differences between each concentration and the corresponding control data using one-way analysis of variance (ANOVA) with post hoc Duncan’s test. The influence of the presence of serum or albumin in medium on the cytotoxic effect of AEA was analysed using two-way analysis of variance (ANOVA) with post hoc Duncan’s test. All data are presented as the means ± SEM.
Results
Protocol I
The activity of anandamide in C6 glioma cells while the drug was administered in DMEM without serum and MTT was added in DMEM without serum has been found cytotoxic in all concentrations in a dose-dependent manner (Fig. 2A).
Protocol II
In the case of anandamide administered in DMEM without serum and MTT added in DMEM with serum, the drug was cytotoxic in all concentrations in a dose-dependent manner similar to the result obtained in Protocol I (Fig. 2B).
Protocol III
Anandamide administration in DMEM with serum and MTT addition in DMEM without serum did not induce any cytotoxic effect (Fig. 3A).
Protocol IV
Anandamide given in DMEM with serum while MTT was administered in DMEM with serum, did evoke no cytotoxic effect, whereas the opposite effect was observed – the greater number of viable cells after AEA treatment at a dose of 3.75 µM (Fig. 3B).
Protocol V
There was no cytotoxic effect following AEA administration in DMEM with albumin (Fig. 4).
Influence of serum or albumin presence in the medium on cytotoxicity of anandamide in C6 glioma cells
The present study showed a significant dose-dependent cytotoxic effect of AEA on C6 glioma cells
(p < 0.001) in experiments when medium without serum was used. The experiments demonstrated a significant influence of the presence of serum in the medium on the occurrence of cytotoxicity of AEA in C6 glioma cells (p < 0.001). Our study indicated a significant interaction between the cytotoxic effect of AEA on C6 glioma cells and the presence or absence of serum (p < 0.001) or albumin (p < 0.001) in the medium.
Discussion
Tetrazolium salts are widely used to assess in vitro cytotoxicity of candidate anticancer drugs and other bioactive substances. The popularity of this assay lays in its unsurpassed simplicity and low cost. In our studies we have chosen the MTT test assuming that it would enable us to obtain reproducible results. We have made a choice of a relatively short (24 h) incubation time of the cell culture with AEA, because there is an indication that cytotoxicity of AEA toward the C6 rat glioma is evoked by TRPV1 receptors. Capsaicin, the agonist of TRPV1 receptor, was markedly cytotoxic toward C6 glioma cells [2]. In the other malignant cell model system (Jurkat leukemia line) it has been shown that capsaicin TRPV1-mediated cytotoxicity is the consequence of an increase in intracellular production of free radicals, which is full-blown already 6 h after capsaicin challenge [28].
Our first and a bit surprising finding was that when AEA was added to the culture medium, after 24 h no decrease in the MTT-related optical density was detectable. There was no dose-response relationship, and in fact the readings were in some cases suggestive of an increase in MTT dye due to the addition of AEA to the cell culture. These observations prompted us to critically review the MTT method which we were using.
Several potential sources of errors in the MTT cell viability assay have been identified. For example, Marshall et al. [29] pointed out that changes in cell culture environment, such as glucose depletion or change in pH of the medium, may influence the reduction of tetrazolium salts independently of changes in cell viability. However, in our experiment such effect should be minimized by a relatively short incubation time.
Es-Sady et al. [11] and Pagliacci et al. [32] reported that alterations in cell cycle phase redistribution by compounds under study (ursolic acid and genistein, respectively) significantly influenced the mitochondrial number and/or function, and in this way influenced MTT reduction to formazan independently of the effect on cell viability. A similar mechanism should not be a priori excluded in the case of AEA, as it has been shown that nanomolar concentrations of AEA (and some other cannabinoids as well) appeared to promote mitogenic kinase signalling and induce proliferation of model cancer cells, including a glioblastoma and a lung cancer cell line [20].
On the other hand, Zhang and Cox [35] observed a 20% higher MTT signal when viability of airway smooth muscle cells culture were assayed in 10% foetal calf serum (FCS) compared with 5% FCS. Huang et al. [22] reported that major contributions to tetrazolium salt reduction by common culture media such as albumin and fatty acids can obscure changes in the cell number during MTT and similar assays. Funk et al. [13] found that reductive activity of human serum albumin produced concentration-dependent increase in the MTT signal, although it should be mentioned that such effect did not occur when bovine serum albumin was used.
Considering that interference of blood serum and/or its major constituent albumin appeared to be the most frequently mentioned potential source of erroneous results in the MTT cell viability assay, we conducted experiments aimed at determining whether the presence of FBS influences results of the assay, and if it does, whether interference occurs during incubation of cells with AEA, or during reduction of formazan.
The results of our experiments unequivocally indicate that when serum is absent from the medium, 24 h exposure to AEA is dose-dependently cytotoxic toward C6 rat glioma cells in a low-nanomolar concentration range. However, when serum is present, no reproducible cytotoxicity of 24 h exposure to AEA in concentrations up to 10 micromoles is detected. Spectacular inhibition of AEA cytotoxicity by FBS is reproduced by substituting it with an equivalent amount of bovine serum albumin. The effect of serum (or albumin) during 24 h incubation with AEA is of such magnitude that any possible interference of serum during development of MTT signal is of secondary importance.
Blood serum is currently added to virtually every mammalian cell culture [6]. The most widely used serum supplement is foetal bovine serum (FBS), a particularly rich source of hormones, growth factors and trace elements that promote rapid cell growth. Serum has undefined and variable composition, but it is always rich in albumin. What is of importance for the MTT assay, and perhaps for other in vitro viability and/or cytotoxicity assays as well, is the fact that albumin reversibly binds numerous endogenous and exogenous compounds. This in particular concerns water-insoluble compounds such as AEA which avidly binds to albumin, similarly to fatty acids [4]. When AEA is added to culture medium containing albumin, the concentration of free AEA able to interact with TRPV1 receptors may decrease significantly, even up to 100 times. In this context particularly interesting is the case of sphingosylphosphorylcholine (SPC). Similarly to AEA, SPC is a lipid metabolite generated enzymatically from cellular membrane constituent (sphingomyelin), which acts as a ligand on some G-protein-coupled receptors. In a marked resemblance of our present results Han et al. [19] reported that exposure to SPC induced significant cytotoxicity in Jurkat cells, but both serum and albumin, by binding SPC, inhibited this cytotoxicity.
De Petrocellis et al. [8] demonstrated that the potency of anandamide at TRPV1 receptor is markedly reduced in the presence of bovine serum albumin. The authors suggested that BSA prevents the uptake of anandamide, interfering with the carrier-mediated internalisation of this compound, and the subsequent activation of receptor, which they assumed occurs intracellularly. There is, however, little support to the idea that TRPV1, membrane-spanning protein and ionic channel, is located intracellularly. It is worth mentioning that experiments, in which stimulation of cancer growth by nanomolar concentrations of AEA was observed [20], were performed on serum-starved cells, therefore effective concentration of AEA at the receptor site should be comparable to 100 times higher concentration in experiments of others, in which serum was present in culture media.
Our finding that the presence of albumin may interfere with the MTT viability assay is important not only in the context of AEA cytotoxicity towards cancers, but it also have a bearing on the general methodology and interpretation of in vitro cytotoxicity testing. Interference of serum with viability and cytotoxicity assays has been alluded to in the case of LDH assay, considered another robust and cost-effective means of assessing cytotoxicity. Its susceptibility to background signal from LDH found in supplemented growth media has been indicated as possible explanation, but no reference to drug-serum proteins interaction was mentioned [31]. On the basis of our results we would recommend that for cell viability and cytotoxicity testing with the use of MTT and other in vitro tests, serum-devoid cell cultures and short times of exposure to tested compounds should be employed.
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