Comparison of the Usefulness of MTT and CellTiterGlo Tests Applied for Cytotoxicity Evaluation of Compounds from the Group of Polyphenols ^†

Abstract

Cytotoxicity assays are a fundamental tool used in molecular biology, as well as in pharmacy, for drug discovery studies. Most of these assays are based on the measurement of cell viability and proliferation. The reduction of MTT is a widely used colorimetric assay method that determines the viability of cells/cytotoxicity. In recent years, there has been a growing interest in polyphenolic compounds, which exhibit many health-promoting properties and therefore carry enormous therapeutic potential that could be used in the fight against various diseases of civilization (i.e., cancer, atherosclerosis, obesity, and diabetes). The commonly used MTT test may provide incorrect results due to the phenomenon of chemical interaction with plant extracts. More and more assays based on the measurement of intracellular ATP (i.e., CellTiter-Glo) are appearing on the market, and according to many scientists, they are a promising alternative to the tetrazolium salt reduction test. The aim of this study was to compare the utility of the colorimetric MTT assay and the bioluminescent CellTiter-Glo assay for the evaluation of compounds from the polyphenol’s group. Analysis of both methods was performed by comparing the obtained results of the viability of Caco-2 cells treated with a wide range of concentrations of polyphenolic compounds: cichoric acid, caftaric acid, caffeic acid, and cynarin. The results show some discrepancies in the cell survival. Moreover, the overestimation of Caco-2 cell viability using MTT assay confirms the occurrence of interference between MTT assay and analyzed polyphenols. It was confirmed that the CellTiterGlo assay has a higher accuracy of measurement, thus providing more reliable results of cytotoxic analysis of polyphenols compared to the MTT assay.

1. Introduction

The discovery of drugs and the study of their safe effects on the human organism are linked by the need for reliable tests to assess cytotoxicity. All new compounds of potential drug character are obligatorily assessed for their cytotoxic activity prior to being sent to clinical trials. Observation of changes in physiological processes caused by chemical compounds affecting the cell is possible through the use of various measurement techniques, such as colorimetry, fluorimetry, or bioluminescence [1].

An example of substances with undoubtedly great therapeutic potential is polyphenols. Due to their pro-health effects, these compounds are referred to as nutraceuticals. Commonly found in many fruits and vegetables, they are natural components of a balanced diet. So far, many studies have shown that they are powerful antioxidants. Even in low concentrations, they protect cells against oxidative stress, thus preventing the development of various diseases, including cancer. Therefore, it is extremely important to further deepen the knowledge of these compounds and their impact on the human body [2,3]. The aim of the study was to determine the usefulness of the MTT test and the CellTiter-Glo test to assess the cytotoxicity of compounds from the group of polyphenols in relation to human cells of the Caco-2 cell line.

2. Materials and Methods

The influence of cichoric acid and caffeic acid was studied in colorectal adenocarcinoma Caco-2 cell line, which was obtained from American Type Culture Collection (ATCC). Caco-2 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humified atmosphere of 5% CO₂ in air. The cells’ viability in tested cell lines was examined at concentrations of 0.5 µM, 1 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 300 µM, and 500 µM for every studied compound. Cichoric acid and caffeic cytotoxicity were measured with the use of CellTiter-Glo™ 2.0 Assay (Promega) according to the manufacturer’s protocol. Caco-2 cells were seeded in 96-well white plate at a density of 1 × 10⁴ cells/well and after 24 h time those that were intended to attach to the plate surface cells were treated with cichoric acid and caffeic acid in a concentration range from 0.5 µM to 500 µM. After 24 h, cells were subjected to CellTiter-Glo™ 2.0 Assay (Promega). Luminescence was measured with a plate reader GloMax^®-Multi Microplate Multimode Reader. The study was performed in triplicate to ensure consistent results were obtained. Cytotoxicity was also studied according to Jabłońska-Trypuć et al. using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) with the use of microplate reader GloMax^®-Multi Microplate Multimode Reader (Promega Corporation, Madison, WI, USA) [4]. Viability of Caco-2 cells was presented as a percentage of control cells. All the experiments were performed in triplicate.

3. Results

It was observed that cichoric acid did not stimulate the viability of Caco-2 cells (Figure 1). The smallest decrease in cell proliferation was found after the addition of cichoric acid at a concentration of 0.5 µM (MTT) and 5 µM (CellTiter-GloTM), with the viability decreased by 1.86% (MTT) and 0.38% (CellTiter-GloTM) with regard to control. An exceptionally clear increase in the antiproliferative effect of cichoric acid was noted at a concentration of 500 µM, both in the MTT and CellTiter-GloTM tests. Caco-2 cell viability decreased by 32.68% (MTT) and 51.57% (CellTiter-GloTM) compared to control.

Caffeic acid does not stimulate the growth of Caco-2 cells, which was confirmed by both the MTT and CellTiter-GloTM tests (Figure 2). For Caco-2 cells treated with 300 µM caffeic acid, the MTT test showed nearly two times higher results than CellTiterGloTM (79.03% vs. 40.61% survival, respectively). In contrast with the concentration of 200 µM, where both tests provided similar results (difference of 2.33%), the lowest cytotoxic effect of caffeic acid was noted at concentrations of 5 µM (MTT) and 0.5 µM (CellTiter-GloTM), with the cell viability decreased by 3.67% (MTT) and 1.41% (CellTiter-GloTM) in relation to the control. The greatest decrease in the viability of Caco-2 cells was observed at the highest concentration of the tested compound (500 µM), with the MTT test showing 57.15% and the CellTiter-GloTM test 81.88% compared to the control.

4. Discussion

The results obtained in these two independent experiments did not differ significantly from each other; however, no clear correlation between them can be found. Both the MTT test and the CellTiter-Glo test showed the greatest decrease in cell viability at the maximum concentration of each of the tested compounds; however, the latter mentioned in each case provided much lower values of the survival rate. The available literature data confirm that the ATP test (CellTiter-Glo) has a higher level of inhibition of cell growth compared to the MTT test [5]. It can therefore be concluded that it is characterized by greater sensitivity of the measurement, which was confirmed in the literature. According to Riss, the CellTiter-Glo test is able to detect up to 15 cells, where the luminescent signal can be measured just 10 min after adding the reaction reagents [6].

The most noticeable difference between the results obtained from both tests was in the case of caffeic acid. The greatest decrease in viability among all four analyzed compounds was noted for a concentration of 500 µM of this compound (MTT/CellTiter-Glo test). The obtained result was confirmed in the literature data, where it was proven that at lower concentrations, caffeic acid exhibits antioxidant activity; however, in the range of high concentrations, it begins to show a pro-oxidative effect, leading to a decrease in cell viability [7].

5. Conclusions

The comparison of the two tests showed that the CellTiter-Glo test was a more useful tool for phytochemical analysis. It is characterized by lower susceptibility to interference with these compounds, greater sensitivity, and the possibility of multiplexing with other available measurement methods. This enables a comprehensive evaluation of the cellular processes taking place under the influence of the activity of the assessed substances. Choosing the right test to assess the cytotoxicity of polyphenols is an important factor in obtaining high-quality data that will provide answers to emerging research questions about their performance.