Abstract
Aim: Chlorpromazine has activity against a large variety of cancer types. However, this phenothiazine produces a plethora of serious side-effects. We have studied thioridazine (TZ), a phenothiazine neuroleptic that is much milder, for activity against multidrug-resistant (MDR) cancer cells, as well as against the overexpressed ABCB1 transporter (P-glycoprotein) that is the cause for the MDR phenotype of these cancer cells. Materials and Methods: MDR mouse T-lymphoma cells, transfected with the human gene ABCB1 that codes for the transporter ABCB1, were incubated with TZ for various periods of time and examined for evidence of apoptosis. Concentrations of TZ were evaluated for activity against the ABCB1 transporter with the aid of an automated ethidium bromide (EB) method. Results: TZ induces apoptosis of MDR cancer cell line, as well as inhibits the activity of the overexpressed ABCB1 transporter of these cells. Conclusion: Because thioridazine has been in moderately safe use for over 40 years for the therapy of psychosis, it has the potential to serve as an adjuvant with anticancer agents, rendering the a priori MDR cancer cells susceptible to the anticancer agent.
- Multidrug-resistant cancer cells
- MDR mouse T-lymphoma cells
- ABCB1 transporter
- P-glycoprotein
- phenothiazines
- thioridazine
- apoptosis
In the developing world, survival rates from cancer are far lower due to poor diagnostic facilities, hence diagnoses are made during late stages of cancer often precluding any form of therapeutic success, poor competence in diagnostic and therapeutic aspects, and low availability of effective but also costly forms of therapy (chemotherapy, radiotherapy). Regardless of economic support, chemotherapy of cancer is highly problematic, especially when therapy promotes the development of multidrug resistance (MDR), hence cancer becomes refractory not only to the initial chemotherapeutic agent, but to many other anticancer drugs as well (1, 2). The development of an MDR phenotype of cancer cell has been known for many decades to be due to the overexpression of transporters that extrude the anticancer agent before it reaches its intended target (3). Consequently, there has been an intensive and extensive search for agents that inhibit the transporter, thereby rendering the cancer susceptible to any anticancer agent. As an example, over 100,000 compounds have been screened by the National Cancer Institute (NCI) for activity against MDR transporters of 60 cancer cell lines (4). However, because these transporters are also present and vital to normal cells, as of the time of this writing, no inhibitor of any transporter responsible for the MDR phenotype of cancer has successfully passed clinical evaluation.
Transporters that render cancer cells multidrug-resistant. When tumor resistance develops against a single particular chemotherapeutic agent, in many cases the resulting phenotype shows a wide range of resistance to that chemotherapeutic agent as well as to many structurally unrelated noxious compounds (MDR pattern) (5). The major mechanism of MDR is the expression of ATP-dependent transporters known as the ATP-binding cassette (ABC) family (6). In humans, the three major types of MDR proteins include members of the ABCB (ABCB1/MDR1/p-glycoprotein), the ABCC (ABCC1/MRP1, ABCC2/MRP2, probably also ABCC3–6, and ABCC10–11), and the ABCG (ABCG2/MXR/BCRP) subfamily. On the basis of a great deal of clinical and experimental work, it has been established that these pumps recognize a very wide range of unrelated drug substrates. ABCB1 preferentially extrudes large hydrophobic molecules, while ABCC1 and ABCG2 can transport both hydrophobic drugs and large anionic compounds, e.g. drug conjugates (7). The investigation of the relationship between ABC expression levels and sensitivity to drugs or possible drug candidates is of great importance because an agent that is eventually shown to inhibit an ABC transporter of a cancer cell line can contribute to further overexpression of other ABC transporters responsible for the MDR phenotype (8).
Apoptosis-inducing activity of thioridazine on MDR mouse T-lymphoma cells after 90 min.
The activity of phenothiazines towards cancer cells. Phenothiazines have been shown to have neuroleptic, antimycobacterial and anticancer properties (19-17). It has been demonstrated that chlorpromazine (CPZ) inhibits the growth of cancer cells whenever studied (17-21). Moreover, other phenothiazines such as promethazine (22) and thioridazine (23-25) also inhibit a variety of cancer types. In fact, the properties of phenothiazines associated with inhibitory effects on cancer cell growth can be reproduced with inhibitors of calmodulin (26-29). These studies suggest that calcium signaling pathways and calcium-dependent enzymes are targets of phenothiazines (30). Because some of these pathways are involved in the generation of ATP, and ATP is required for the activity of ABC transporters which, when overexpressed, result in the MDR phenotype of cancer cells, it is obvious that studies that focus on the activity of phenothiazines towards the ABC transporters of cancer cells are needed. Since unlike CPZ, thioridazine (TZ) is much milder and produces fewer negative side-effects than CPZ (30), we have studied the activity of this phenothiazine on the viability of MDR mouse lymphoma cells transfected with the human ABCB1 gene that codes for ABCB1, the transporter that when overexpressed, renders the cell immune to anticancer agents (31-33), as well as on the activity of the ABCB1 transporter itself.
Materials and Methods
Cell lines. L5178 mouse T-cell lymphoma cells (ECACC cat. no. 87111908, U.S. FDA, Silver Spring, MD, USA) were transfected with pHa ABCB1/A retrovirus (31-33), as described elsewhere (34, 35). The ABCB1-expressing cell lines were selected by culturing the infected cells with 60 ng/ml of colchicine (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) to maintain the MDR phenotype. L5178 mouse T-cell lymphoma cells (parental) and the human ABCB1-gene transfected sub-line were cultured in McCoy's 5A medium (Sigma-Aldrich) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich), L-glutamine (Sigma-Aldrich) and antibiotics (penicillin, streptomycin) (Sigma-Aldrich) at 37°C and in an atmosphere with 5% CO2.
Compounds. Ethidium bromide (EB), verapamil, reserpine and TZ were purchased from Sigma-Aldrich Quimica SA, Madrid, Spain.
Viability assay. The cytotoxicity of EB and the efflux pump inhibitors (EPIs), verapamil, reserpine and TZ was determined by the trypan blue exclusion method, previously described in detail (36). The effect of the aforementioned agents on apoptosis was determined with the aid of the Annexin V-FITC Apoptosis Detection Kit (Calbiochem EMD Biosciences, Inc. La Jolla, CA, USA) strictly in accordance with manufacturer's instructions (31-33).
EB accumulation assay. The cells were adjusted to a density of 2×106 cells/ml, centrifuged at 2000 ×g for 2 min and re-suspended in phosphate-buffered saline (PBS) solutions of pH 7.4 supplemented with glucose. The cell suspension was distributed in 90 μl aliquots into 0.2 ml tubes. The tested compounds were individually added at different concentrations in 5 μl volumes of their stock solutions and the samples were incubated for 10 min at 25°C. After this incubation, 5 μl (1 μg/ml final concentration) of EB (20 μg/ml stock solution) were added to the samples and the tubes were placed into a Rotor-Gene™ 3000 thermocycler (Corbett Research, Sydney, Australia) and the fluorescence monitored on a real-time basis. Prior to the assay, the instrument was programmed for temperature (37°C), the appropriate excitation and emission wavelengths of EB (530 nm bandpass and 585 nm highpass, respectively), and the time and number of cycles for the recording of the fluorescence. The results were evaluated by Rotor-Gene Analysis Software 6.1 (Build 93) provided by Corbett Research (Sydney, Australia). A complete description of the method has been previously presented in detail (31-33). The essence of the method is that a progressive increase of fluorescence, induced by the agent in question, provides an estimate of the inhibition of efflux of EB promoted by that agent.
Ethidium bromide accumulation by MDR mouse T-lymphoma cells overexpressing the ABCB1 transporter in the presence of verapamil (VP), thioridazine (TZ) and reserpine (RES). Cells (2×106 cells/ml) were suspended in 100 μl of phosphate-buffered saline solutions (pH 7.4) supplemented with 1 mg/l of EB with and without agent at given concentrations, then the fluorescence was continuously monitored.
Assay for apoptosis induction. The assay was carried out using Annexin V-FITC Apoptosis Detection Kit from Calbiochem (EMD Biosciences, Inc. La Jolla, CA, USA) according to the manufacturer's instructions. The cell suspension was adjusted to approximately 1×106 cells/ml. The cell suspension was distributed into 0.5 ml aliquots (5×105 cells) and transferred to Eppendorf tubes. The cells were incubated in the presence of the compounds for 90 min at 37°C, with 12H-benzo[α]phenothiazine (M627) being used as positive control at a final concentration of 50 μg/ml (37), then the samples were washed in PBS. The cells were centrifuged at 2000×g for 3 minutes. The harvested cells were re-suspended in culture medium and distributed to 24-well tissue culture plate in 0.5 ml aliquots, followed by the incubation of the plate for 24 h at 37°C in 5% CO2. After the incubation, the apoptosis assay was carried out according to the rapid protocol of the kit. The fluorescence was analyzed immediately using a Becton Dickinson FACStar™ flow cytometer (Becton Dickinson and Company, USA).
Results and Discussion
The induction of apoptosis of the MDR mouse lymphoma cells by concentrations of TZ is presented in Table I. Briefly, concentrations below the ones that, produce toxicity (20 mg/l) induced apoptosis in a concentration-dependent manner.
To assess the effect of TZ on apoptosis of MDR mouse T-lymphoma cells, after determination of the cytotoxic activity of thioridazine (IC50 9.56 μg/ml after 24 h), three concentrations of TZ (5, 10 and 20 μg/ml) were selected for apoptosis studies. During apoptosis, the plasma membrane undergoes multiple changes: the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane can be detected by annexin V. To discriminate between apoptotic and necrotic cells, the annexin V-FITC binding assay was combined with propidium iodide staining, but due to increased membrane permeability, late apoptotic cells cannot be discriminated from necrotic cells. Apoptosis was measured by quantifying the proportion of annexin V-FITC and propidium iodide positive cells using flow cytometry. As shown in Table I, the treatment with 5 μg/ml TZ for 90 min induced early apoptosis in 15.49% of the cell population and 6.6% of the cells underwent late apoptosis. The apoptosis-inducing potential of TZ was more potent at the concentration of 10 μg/ml, because 49.54% of the cells were early-apoptotic and 27.26% showed late-apoptotic and necrotic features, although the proportion of dead cells was 3.4%. The induction with 20 μg/ml of TZ was toxic for the population.
The evaluation of agents and TZ for activity against the overexpressed ABCB1 transporter of the MDR mouse lymphoma cells is depicted in Figure I. Briefly, verapamil and reserpine that serve as positive controls inhibit ABCB1 activity in a concentration-dependent manner. In comparison to these effects, TZ inhibits the activity of ABCB1 in a similar concentration-dependent manner and on a μg/ml basis, the inhibitory activity is far greater than that of verapamil and reserpine.
The results obtained in the current study suggest that TZ induces apoptosis of the MDR lymphoma cells and inhibits the activity of their overexpressed ABCB1 transporter. However, the concentrations that produce these results are extremely high and greater than the ones used for the therapy of psychosis – namely, ca. 0.5 μg/ml. Nevertheless, TZ can serve as a lead compound for the development of derivatives which may be more active at concentrations that are non-toxic and achievable in the patient. At the time of this writing, these derivatives have been produced and will be the subject of further studies.
Acknowledgements
G. Spengler was supported by TÁMOP-4.2.1/B-09/1/KONV-2010-0005 – Creating the Center of Excellence at the University of Szeged supported by the European Union and co-financed by the European Regional Fund. L. Amaral was supported by BCC grant SFRH/BCC/51099/2010 provided by the Fundação para a Ciência e a Tecnologia (FCT) of Portugal and PTDC/SAU-FCF/102807/2008 provided by the UPMM. This work was supported by EU-FSE/FEDER-PTDC/BIA-MIC/105509/2008 and EU-FSE/FEDERPTDC/ SAU-FCF/102807/2008 from the Fundação para a Ciência e a Tecnologia (FCT) of Portugal.
- Received September 30, 2011.
- Revision received November 7, 2011.
- Accepted November 8, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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