Abstract
Background: In this investigation the effects of tricyclic drugs on cellular respiration were studied using the anaplastic astrocytoma cell line IPSB-18 by use of a Clark-type oxygen electrode which measured changes in cellular respiration rate (oxygen consumption), in a dose–response assay. Materials and Methods: The drugs investigated were clomipramine, norclomipramine, amitriptyline and doxepin. In addition, the combined effects of dexamethasone and clomipramine on cellular respiration were investigated. Results: It was established that at lower concentrations (0.14 mM-0.5 mM) amitriptyline was the most potent inhibitor of cellular respiration. Previous studies have indicated that inhibition of cellular respiration is considered an indicator of apoptosis. Overall, it appeared that clomipramine and its metabolite norclomipramine were the most potent inhibitors of cellular respiration in glioma cells over the concentration range 0.5-0.9 mM. Dexamethasone was able to induce inhibition of cellular respiration both alone in glioma cells, and in combination with clomipramine, where it had an additive or synergistic effect, thereby increasing cell death. Conclusion: The extensive research currently ongoing and previously reported regarding the use of clomipramine as a potential antineoplastic agent aimed at targeting the mitochondria of gliomas is promising.
It is established that impairment of mitochondrial function may lead to adenosine triphosphate (ATP) depletion and necrotic cell death (1). However, more recently, mitochondria have been heavily implicated in the regulation of apoptotic cell death and in cancer formation (2). Tumour cell invasion is highly ATP dependent, but mitochondria within tumour cells often display abnormalities in their number, structure and function. Mitochondrial respiration is decreased in neoplastic tissue, along with a lowering of the number of mitochondria. These findings indicate that tumour cells rely upon anaerobic glycolysis as an energy source and this enables them to survive under hypoxic conditions (3). There are at least three known mechanisms by which mitochondria can trigger apoptosis, although these effects may be inter-related. Apoptosis may be triggered by disruption of electron transport, oxidative phosphorylation and ATP transport, release of proteins that trigger activation of caspases and alteration of cellular redox potential (4). A number of agents appear to target the mitochondria and promote the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation, resulting in cell death (1). Caspases are cysteine proteases and exist in a latent state in ‘healthy’ cells. In response to damage or a malfunction to vital metabolic processes, cells generate signals that lead to activation of caspases, which result in apoptotic cell death (5).
However, defects in apoptosis signaling pathways are common in cancer cells. Moreover, tumour development, progression and resistance to radiotherapy and chemotherapy are all the direct result of defects in the regulation of apoptosis (6), due to raised apoptotic thresholds. It is the identification of the specific proteins responsible for the regulation of apoptosis that will lead to the development of clinical therapies directed at altering the levels of expression of pro-apoptotic proteins and enhancing the effects of current radiotherapy and chemotherapy. The Bcl-2 proto-oncogene represses a number of cellular apoptotic pathways and is known to be expressed in increasing amounts in glial tumours of higher malignancy (7). Application of antisense oligonucleotides to the Bcl-2 gene resulted in an increase in apoptotic cell death. This result indicates that Bcl-2 plays a role in tumour progression of gliomas by acting as an oncogene and inhibition of the Bcl-2 gene could have a therapeutic effect (7).
It has been determined that chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than 3 and 8 (8). Caspases 1, 2, 3, 7, 8 and 9 are constitutively expressed in most human malignant glioma cell lines and drug-induced apoptosis involves delayed activation of caspases 2, 7 and 9, and is blocked by broad-spectrum caspase inhibitors (8). It has been established that the cytotoxic effects of many chemotherapeutic agents are mediated via apoptotic pathways; therefore developing drugs that target the mitochondria may provide a new strategy to induce apoptosis in tumour cells (9).
Tricyclic antidepressants (TCAs) are an important group of antidepressants in clinical use (10). Their name is derived from their characteristic three-ring nucleus and they have been in clinical use for over 40 years. TCAs were first thought to be useful as antihistamines with sedative properties and later as antipsychotics. Clinical observations led them to be reclassified as antidepressant agents. Generally, all members of this drug family act as mixed norepinephrine and serotonin uptake inhibitors (11).
In the 1970's, it was found that TCAs showed selective inhibition of mitochondrial activity in yeast cells. It was surmised that the wide range of actions shown by the TCAs in vivo was due to interactions with membranes and membrane-bound enzymes, in particular with the mitochondrial membrane (12), resulting in inhibition of cellular respiration and limitation of ATP production. Further experiments showed that cancer cells were much more susceptible to the inhibitory effects of TCAs than were non-transformed cells. After treatment with TCAs, it was observed that the respiration rate of transformed cells was significantly less than their normal counterparts in oxygen electrode studies. It was concluded that antimitochondrial drugs (e.g. TCAs) depress mitochondrial activity in cancer cells and ‘kill’ them, whereas non-transformed cells were able to recover after treatment (13). This mode of action of the TCAs was found to be a common feature amongst members of the group but there appears to be no clear relationship between chemical structure and pharmacological action. However, it appears that the chlorine-containing drugs are more toxic to the functions of the mitochondrial membrane than others (14). Imipramine, clomipramine (CIMP) and citalopram induce apoptosis in cancer cells and this process is associated with an early increase in the production of reactive oxygen species (ROS) and subsequent loss of mitochondrial membrane potential (15). The literature suggests that TCAs can induce apoptosis in acute myeloid leukaemic cells (15) and gliomas (16-18). Recent studies have shown that the tricyclic CIMP, produced dose-dependent toxicity in gliomas, but not in foetal astrocytes (17, 18). The drug inhibited complex III of the respiratory chain, resulting in elevated levels of ROS, cytochrome c release and caspase-activated apoptosis (17). The data presented by these studies indicates that CIMP may be useful in the treatment of patients with primary brain tumours; it is estimated that there are over 350 anecdotal cases of patients with a range of different primary brain tumours that are taking, or who have taken, CIMP in the UK. With respect to these cases, there have been numerous reports of survival benefit and increased quality of life (2, 17). More recently, the combination treatment of CIMP and imatinib (Gleevec) in C6 rat glioma demonstrated inhibition of cell growth, and enhanced apoptotic cell death. These findings verified the synergistic antiproliferative and cytotoxic effects of this combination in vitro and suggest the potential for clinical application (19).
The cornerstone of conventional therapy for malignant, primary brain tumour has been a combination of surgery, radio- and chemotherapy. Due to the limited success of these therapeutic strategies, alternative molecular targets and approaches such as immunotherapy merit consideration (20). Additionally, combinatorial and custom-built strategies should be developed to target the biological properties of gliomas (cellular heterogeneity and local diffuse invasion) as well as to circumvent the obstacle of the blood-brain barrier and their inherent chemoresistance.
It has been proposed from ongoing research that clomipramine causes glioma cells to undergo apoptosis (17, 18, 21). It has also been suggested that clomipramine is selective for neoplastic glial cells, as it had no effect on non-neoplastic, foetal astrocytes (17). This present investigation continued with this same theme of previous research in that glioma cell viability was determined using the anaplastic astrocytoma cell line IPSB-18 (22) by use of a Clark-type oxygen electrode which measured changes in respiration rate (oxygen consumption), in a dose–response assay. TCAs were evaluated to determine the order of potency of the compounds in the same manner. The drugs investigated were clomipramine hydrochloride (CIMP); norclomipramine hydrochloride (a metabolite of CIMP) (NORCIMP); amitriptyline hydrochloride (AMIT); and doxepine hydrochloride (DOX).
The next stage of research was to investigate the potential interaction dexamethasone (DEX) had with CIMP on cellular respiration. In these experiments, cells were pre-treated with DEX and then treated with a single dose of CIMP during oxygen electrode studies. This was an important aspect to investigate as glioma patients being treated with CIMP frequently receive dexamethasone to reduce peritumoural oedema and consequent raised intracranial pressure.
Materials and Methods
Cell culture. The anaplastic astrocytoma-derived cell line IPSB-18 (22) (at passages 46-50) was maintained as a monolayer culture in Dulbecco's modified Eagle's medium (DMEM, without pyruvate, with glucose, glutamax and phenol red) (61965; Gibco, UK) and supplemented 10% heat inactivated foetal bovine serum (FBS) (F-7524; Sigma Aldrich, UK) and referred to as complete media, washed with Hank's Balanced Salt Solution (HBSS) (14170; Gibco) and harvested using TripLE™ Express (12605; Gibco, UK).
Drugs and solutions. The following drugs were used: CIMP, NORCIMP, AMIT, DOX and DEX all obtained from Sigma-Aldrich. All tricyclics were dissolved in 70% ethanol to give a stock solution of 100 mM.
Determining cellular oxygen consumption – tricyclic treatment. To determine inhibition of cellular oxygen consumption of anaplastic astrocytoma cells on addition of the tricyclic drugs, a Clark-type oxygen electrode (Oxygraph, Hansatech Instruments, Norfolk, UK) with a water jacket maintained at 37°C was used. Cells were harvested from culture, resuspended in 0.5 ml of complete medium, and stored with centrifuge tube caps loosened in the water bath. Cells were centrifuged and resuspended in 0.5 ml of clear DMEM when required for experimentation. The change in the medium was undertaken for two reasons; firstly, it has been found that mammalian cells are more sensitive to antimitochondrial agents when glucose is replaced with glutamine as the carbon energy source and secondly, the cell sample remaining at the end of the experiment was used in a subsequent colorimetric protein assay. Therefore, the use of the clear medium was advantageous to avoid interference with results. A cell suspension of 0.5 ml was added to the electrode chamber and the rate of oxygen consumption was measured using ‘Oxygraph’ software. After 3 minutes, 10 μl of drug at different concentrations (dose-response assay) were added to the cell suspension via the capillary using a Hamilton syringe. For 20 min following this addition, the cells were left to incubate with the drugs. The rate of oxygen consumption was measured at five different time points (T1 2-3 min; T2 7-8 min; T3 12-13 min; T4 17-18 min and T5 22-23 min) to determine change over time. T1 was considered to be the ‘control’ value of oxygen consumption per experiment (before the addition of any drug) and the change in oxygen consumption in response to the drugs was expressed as percentage change in response from that of the control.
The drug treatment regimes were taken from previous experiments with CIMP and the range used was 0-0.9 mM (17) (final concentration in electrode chamber) for all drugs. All experiments were performed in triplicate and an average of the results taken. All oxygen consumption rates were corrected for cellular protein content as measured by the Bio-Rad protein assay.
Determining cellular oxygen consumption – tricyclic treatment with DEX pre-treatment. Cells were initially treated with a range (0-5 μM final concentration) of DEX and left to incubate with the drug overnight (24 hours). Cells were harvested the subsequent day by scraping the bottom of the flask and not by using trypsin. The cells were resuspended and stored as before. A cell suspension of 0.5 ml was added to the electrode chamber and the rate of oxygen consumption was measured using ‘Oxygraph’ software. This time point was considered as the initial response to pre-treatment with DEX. After 3 min, 10 μl of 0.5 mM CLOM were added to the cell suspension via the capillary using a Hamilton syringe. This concentration was used because data suggested from the previous experiment that at this concentration a sub-maximal response from the cells was achieved, in this way it would be easier to determine the effect of DEX, if any, on the cells. For 20 min following this addition, the cells were left to incubate with the drug. The rate of oxygen consumption was measured at the same time points as previously (T1 2-3 min; T2 7-8 min; T3 12-13 min; T4 17-18 min and T5 22-23 min) to determine change over time. DEX at 0 μM was considered as the control response; subsequent concentrations were compared to this, and the change in oxygen consumption in response to the drugs was expressed as percentage change in response from that of the control. All experiments were performed in triplicate and an average of the results taken. All oxygen consumption rates were corrected for cellular protein content as measured by the Bio-Rad protein assay.
Results
Effects on IPSB-18 cell line oxygen consumption. Effects of 0.25 mM CIMP, NORCIMP, AMIT and DOX: At this concentration, it was noted that NORCIMP did not induce a significant change in oxygen consumption of the cells and remained the least potent tricyclic. However, CIMP began to induce a significant change in oxygen consumption (p<0.05-0.01). DOX appeared to be less effective at this concentration, while AMIT was the most potent suppressor of oxygen consumption (p<0.01 at all time points, 29% inhibition of oxygen consumption (IOOC, was measured at T5 between the control value at this time point and the value achieved by the TCA in question.). This resulted in an order of potency of: AMIT>CIMP>DOX>NORCIMP (Figure 1). Effects of 0.5 mM CIMP, NORCIMP, AMIT and DOX: Once again, at this concentration, it was noted that NORCIMP did not induce a significant change in oxygen consumption of the cells. DOX induced a lower but still significant effect on oxygen consumption with p<0.05 across all time points, than was seen at the previous concentration. CIMP again appeared at the middle of the range potency with p<0.05-0.01 being seen, as well as no significance. AMIT consistently showed significant inhibition of oxygen consumption with p<0.01 (26%) across all time points. At this concentration, it was observed that AMIT and DOX had remarkably similar potency for the inhibition of oxygen consumption by IPSB-18 cells. This resulted in an order of potency of: AMIT=DOX>CIMP=NORCIMP (Figure 2).
Effects of 0.75 mM CIMP, NORCIMP, AMIT and DOX: Interestingly, at this concentration it was observed that AMIT no longer had a significant inhibitory effect on oxygen consumption of the IPSB-18 cell line. CIMP maintained its significant inhibitory effect on oxygen consumption with p<0.05-0.01 across all time points and DOX with p<0.01 across all time points. It was also noted at this concentration that NORCIMP began to exert a significant inhibitory effect on oxygen consumption and remarkably was the most potent tricyclic at this concentration (24% IOOC). The order of potency was: NORCIMP=DOX>AMIT=CIMP (Figure 3).
Effects of 0.9 mM CIMP, NORCIMP, AMIT and DOX: Once again, at this concentration, it was noted that AMIT did not induce a significant change in the oxygen consumption of the cells. DOX potency began to decline again with wavering significance over time (p<0.05-0.01). NORCIMP again appeared to be the most potent of the tricyclics at this concentration with the greatest inhibition of oxygen consumption (27%, p<0.01), however the most significant results occurred with CIMP, which achieved significance of p<0.001 across all time points (20% IOOC). The order of potency was: NORCIMP>CIMP>DOX>AMIT (Figure 4).
The tricyclics used in this investigation did not exert their effects in a dose-dependent manner. This can be clearly demonstrated using AMIT as an example. At lower concentrations, this drug is more potent than it is at higher concentrations and vice versa with NORCIMP.
Effects of pre-treatment with DEX on initial rate of oxygen consumption of the cell line IPSB-18. An interesting finding of this investigation was the observation that pre-treatment with DEX (even at the lowest concentration) significantly reduced the initial rate of respiration of the cells by ~3 nmol/ml (p<0.01-0.001) (Figure 5). The initial rates were obtained from the readings from the oxygen electrode before the addition of 0.5 mM CIMP.
Effects of pre-treatment of IPSB-18 cell line with DEX and chronic treatment with 0.5 mM CIMP with regard to change in oxygen consumption over time. Another interesting observation regarding the results generated with DEX pre-treatment and chronic CIMP treatment was the finding that DEX appeared to exert an additive inhibitory effect with CIMP on oxygen consumption in a dose-dependent manner. DEX at 0.25 μM and 0.5 μM significantly changed the rate of oxygen consumption across all time points (26% IOOC at 0.25 μM, p<0.05-0.001). The higher DEX concentrations of 1 μM and 5 μM achieved a significant inhibition of oxygen consumption above that of the control values across all time points (p<0.05) (Figure 6).
Discussion
Determination of potency of TCAs on cellular respiration on the IPSB-18 cell line. The TCAs as a group did not display dose-dependent kinetics. It was established that at the lower concentrations of 0.14-0.5 mM AMIT were the most potent inhibitors of cellular respiration as measured by oxygen consumption using the oxygen electrode. Previous studies have indicated that inhibition of cellular respiration is an indicator of apoptosis (9). However, at the higher doses 0.75 and 0.9 mM, AMIT was found to be the least potent TCA. This observation may be due to resistance, desensitization or tachyphylaxis. This is where the effect of the drug gradually diminishes when it is given continuously, repeatedly or at large doses, and this can occur over the space of a few minutes (10). Many different mechanisms can give rise to this type of phenomenon, including change or loss of the site of action for the drug (in this case the mitochondria), increased metabolic degradation of the drug and the possible active extrusion of the drug from the cell (10). This extrusion can occur by an increase in expression of the cell surface, energy-dependent transport protein, P-glycoprotein. This provides protection of the cells against environmental toxins (drugs) by ‘picking up’ the drug as it enters the cell membrane and expelling it through a core in the transporter. If this is the case, then this indicates that the cells are using glycolysis as their energy source, as energy is required for this process, and as cellular respiration is depleted, the production of ATP via conventional pathways is limited. Other reasons may include a decrease of the drug being taken up into the mitochondria; insufficient activation or increased deactivation of the drug may also occur (11). It has also been established that when using in vitro tests to assess the potential of antidepressants (especially the TCAs), many drugs are metabolized to pharmacologically active substances in vivo, and it is often unclear whether the parent drug or the metabolite is actually responsible for the antidepressant effect observed (11). A recent study concerned with regional distribution of CIMP and its metabolite in regions of the rat brain stated that CIMP concentration was found to be highest in the cerebral cortex (2.9 μg/ml), and in decreasing concentrations in the hypothalamus, striatum, cerebellum, hippocampus and brain stem (23), with the metabolite concentration being highest also in the cerebral cortex but at a significantly lower concentration (0.67 μg/ml), and in decreasing concentrations in the hypothalamus, striatum, hippocampus, cerebellum and brain stem (23). In the clinical setting, it has been determined that the relationship between plasma concentration and therapeutic effect is not simple. For example, when nortriptyline (a metabolite of AMIT) reaches an excessively high plasma concentration, the antidepressant activity is reduced (11); whether this is the same for the effect on inhibition on cellular respiration is unclear. This particular drug has a narrow therapeutic window; however, this activity has not been determined in the other TCAs. Therefore, it may be concluded that high doses of AMIT in vitro may be outside the therapeutic window and hence less effect is seen. In addition, at higher doses the drug may bind to receptors on the cell surface rather than directly interact with the mitochondria due to a saturated solution of the drug.
NORCIMP behaved in a contrasting manner to AMIT, with this drug a more potent inhibitor of cellular respiration at higher concentrations and displaying no activity at lower concentrations. DOX and CIMP were in the middle of the group in terms of potency, with CIMP exerting greater potency at higher concentrations. Overall, CIMP and its metabolite NORCIMP were the most potent inhibitors of cellular respiration in tumour cells over the range 0.5-0.9 mM. The largest decrease in cellular respiration was observed with 0.9 mM CIMP, followed by NORCIMP at 0.9 mM. As stated previously, it is often unclear whether it is the parent drug or its metabolite that is more pharmacologically active; however, this investigation indicates that both CIMP and NORCIMP have similar levels of activity. The mode of action of CIMP, although not studied in this investigation has been determined previously; it is an inhibitor of complex III of the mitochondrial respiratory chain, which leads to an increase in ROS, a subsequent loss of mitochondrial membrane potential, cytochrome c release and caspase activation, which results in apoptosis (17).
Determination of the interaction between DEX and CIMP on cellular respiration of the cell line IPSB-18. It was observed that pre-treatment with DEX (before addition of CIMP) reduced the initial rate of cellular respiration of the IPSB-18 cells. DEX has previously been described as an inducer of apoptosis or necrotic cell death; indeed, from the present study this is true for glioma cells. However, the precise mode of cell death should be determined by further studies. DEX has been shown to induce a time and concentration dependent inhibition of rat C6 glioma cell proliferation (24), reduce glioma volume by 33% in mice injected with GL261 glioma cells (25), and decrease invasiveness of the human glioma cell line U87MG (26). The combination of DEX and CIMP has previously been shown to amplify cell death using the MTT assay (27). It has been suggested that DEX, via the glucocorticoid receptor located on the mitochondrial membrane, amplifies the cytotoxic effect of serum deprivation of C6 glioma cells (28). If DEX acts in this manner and in combination with CIMP, then it appears it exerts an additive or synergistic effect because it uses the same mechanism as CIMP. It was also noted in this investigation that DEX actually enhanced the ability of CIMP to inhibit cellular respiration and therefore induce apoptosis. The mode of amplification of cell death may be due to DEX initially affecting cellular respiration, and with the addition of CIMP the apoptotic threshold of the cells would be met quicker due to this additive effect. The apoptotic threshold in many neoplastic cells is often greater than that in non-transformed cells. It is of note to mention that brain tumour patients are on different doses of DEX at different stages of their treatment: patients often receive either no, low or high doses depending on raised intracranial pressure caused by surgery or radiotherapy treatments. This could potentially be tailored with CIMP therapy to maximize neoplastic cell death.
Conclusion
CIMP is the most potent of the TCAs at inducing inhibition of cellular respiration of the IPSB-18 cell line, however, in previous studies from our laboratory non-neoplastic cells were demonstrated to have the ability to recover when the drug is terminated (17) and in some cases are unaffected by treatment (18). Low-dose AMIT (0.14-0.5 mM) has emerged as a potential strategy for inducing inhibition of cellular respiration in tumour cells. Although, the mechanism of action has not been determined, it is likely to be similar to that of CIMP. This could lead to low-dose AMIT being used alongside or instead of CIMP. DEX alone is able to induce inhibition of cellular respiration in tumour cells, and in combination with CIMP, it has an additive or synergistic effect, therefore increasing cell death. The interesting results generated by this investigation indicate that further research is required into the combination of DEX and CIMP as it has been observed that as potential therapies they complement each other. The extensive research currently ongoing and previously reported regarding the use of CIMP as a potential anti-neoplastic agent aimed at malignant glioma is promising. It is hoped that this can be translated into benefits for patients suffering from this currently incurable form of neoplasm.
Acknowledgements
The authors gratefully acknowledge funding from the Samantha Dickson Brain Tumour Trust, Brainstrust, Ali's Dream, Charlie's Challenge and the Isle of Man Anticancer Association.
Footnotes
- Received November 11, 2009.
- Revision received January 20, 2010.
- Accepted January 20, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved