The MTT assay yields a relatively lower result of growth inhibition than the ATP assay depending on the chemotherapeutic drugs tested
Introduction
In the field of cancer research, there are a number of tests to assess the anti-growth effects of chemotherapeutic agents in vitro. The proper assessment of the potency of chemotherapeutic drugs requires precise and accurate in vitro laboratory tests. Thus, it is important to be aware of the limitations of the methods (Hanauske, 1993). It is also important to choose a suitable cytotoxicity assay depending on the supposed cell death mechanism (Weyermann et al., 2005). This assessment is mainly performed via the indirect measurement of the tumor cell number using some viability-related features (e.g. the dehydrogenase activity, the ATP amount) of the cells. Among these tests, two of them are of particular importance. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) and the ATP (adenosine triphosphate) assay are widely used methods to assess cell viability. The ATP and MTT assays were used for the determination of in vitro resistance and sensitivity (Wilson et al., 1990, Sevin et al., 1993, Cree et al., 1996, Kurbacher et al., 1998, Taylor et al., 2001, Furukawa, 2004, Kurbacher and Cree, 2005, Nakamura et al., 2006).
The MTT assay is comparatively cheaper (Martinez-Martos et al., 2000) as well as the more frequently used, while the ATP assay is technologically more advanced due to its luminescence-based methodology, and allows measurements of in vitro toxicity to be made with considerable precision (Cree and Andreotti, 1997). The MTT substance is reduced by mitochondrial dehydrogenases in living cells to a blue-magenta colored formazan precipitate. The absorption of dissolved formazan in the visible region correlates with the number of intact alive cells (Mosmann, 1983). Cytotoxic compounds are able to damage and destroy cells, and thus decrease the reduction of MTT to formazan.
ATP represents the most important chemical energy reservoir in cells and is used for biological synthesis, signalling, transport, and movement processes. Cellular ATP is one of the most sensitive end points in measuring cell viability (Maehara et al., 1987). When lethal cell damage occurs (e.g. via the effect of chemotherapeutic agents or mitochondrial toxins), the ATP level decreases dramatically (Andreotti et al., 1991, Garcia and Massieu, 2003). Additionally, the ATP availability in the cell determines the type of cell death (Atlante et al., 2005) although there is more intracellular substances controlling the mode of cell death (e.g. superoxide) (Wochna et al., 2005). The ATP assay is based on the reaction of luciferin to oxyluciferin catalyzed by the enzyme luciferase in the presence of Mg2+ ions and ATP yielding a luminescent signal. A linear relationship exists between the intensity of the luminescent signal and the ATP concentration (Mueller et al., 2004) or cell number (Andreotti et al., 1995). As it can be seen, although both of them basically measure the metabolic activity of cells, there is, in fact, a major difference in their principles as explained above.
We therefore investigated whether or not there is a difference and/or correlation between these two different assays. By using some anti-cancer drugs at different doses, we compared the performance of these methods in the assessment of anti-growth effects of the drugs on a lung cancer cell line (A549).
Section snippets
Cell culture
A lung cancer cell line, A549 (ECACC No. 86012804), was used in the study. The cells were cultured in DMEM supplemented with penicillin G (100 U/ml), streptomycin (100 μg/ml), l-glutamine, and 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. The ATP assay kit was obtained from DCS Innovative Diagnostika-Systeme (Hamburg, Germany), while the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) substance and all the other reagents and substances were obtained
Results
Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 show the growth inhibition curves of different drugs at six different TDCs. Taking the results of all the figures into account, the growth inhibition levels by the ATP assay were found to be higher than those by the MTT assay. This result shows that the dying cells lose relatively more ATP amount (evidenced by higher growth inhibition levels by the ATP assay), although they lose relatively less dehydrogenase activity (evidenced by lower growth inhibition
Discussion
In this study, we quantified the different outcomes of two commonly used in vitro cytotoxicity assays (the MTT and ATP assays), by employing different kinds and well-known chemotherapeutic drugs. Three outcomes of each drug were analyzed using six different TDCs in order to compare these two assays. First, the correlation between the assays was analyzed. Secondly, the differences in growth inhibition curves of the assays were studied. For this purpose, correlation analysis was performed for an
Conflict of Interest Statement
None.
Acknowledgement
We would like to thank Uludag University Research Fund for providing us with the kits, and chemicals. We are also grateful to Michaela Kendall for her great help with the English language.
References (32)
- et al.
An increase in the ATP levels occurs in cerebella granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis
Biochim. Biophys. Acta
(2005) - et al.
Measurement of cytotoxicity by ATP-based luminescence assay in primary cell cultures and cell lines
Toxicol. In Vitro
(1997) - et al.
Evaluation of different toxicity assays applied to proliferating cells and to stratified epithelium in relation to permeability enhancement with glycocholate
Toxicol. In Vitro
(2004) In vitro assays for antitumor activity: more pitfalls to come?
Eur. J. Cancer
(1993)- et al.
Beta-amyloid (1-42) affects MTT reduction in astrocytes: implications for vesicular trafficking and cell functionality
Neurochem. Int.
(2001) - et al.
The ATP assay is more sensitive than the succinate-dehydrogenase inhibition test for predicting cell viability
Eur. J. Cancer Clin. Oncol.
(1987) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Meth.
(1983)- et al.
Comparison of the usefulness of the MTT, ATP and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines
J. Biomol. Screen.
(2004) - et al.
Genistein inhibits tumor-cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts – a further pitfall in the use of the MTT assay for evaluating cell-growth and survival
Eur. J. Cancer
(1993) - et al.
Practical note on the use of cytotoxicity assays
Int. J. Pharm.
(2005)
Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells
Int. J. Toxicol.
Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian cancer
Cancer Res.
ATP tumor chemosensitivity assay
Correlation of clinical response to chemotherapy in breast cancer with ex vivo chemosensitivity
Anticancer Drugs
In vitro chemosensitivity of hepatocellular carcinoma for hepatic arterial infusion chemotherapy using the MTT assay with the combinations of antitumor drugs
Kurume Med. J.
Glutamate uptake inhibitor L-Trans-pyrrolidine 2,4-dicarboxylate becomes neurotoxic in the presence of subthreshold concentrations of mitochondrial toxin 3-nitropropionate: involvement of mitochondrial reducing activity and ATP production
J. Neurosci. Res.
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