Avoiding the Interference of Doxorubicin with MTT Measurements on the MCF-7 Breast Cancer Cell Line.

Carla Luis,Rúben Fernandes,Yuselis Castaño-Guerrero,Raquel Soares,Goreti Sales

doi:10.3390/mps2020029

Abstract

Doxorubicin (DOXO) is an adjuvant chemotherapy agent and is also commonly used in cell biology research. Cytotoxic assays in cell culture are frequently used in order to stablish drug concentrations that are useful for controlling cell proliferation. One common cytotoxic method used is 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). Our present research aims to support future studies in engaging MTT assay using DOXO that exhibits a strong red coloration and fluorescence, and so it is assumed that DOXO may interfere with commonly used colorimetric assays such as MTT. The interference of DOXO in the MTT determination was evaluated in a Breast Cancer cell line Michigan Cancer Foundation-7 (MCF-7). The interference was evaluated by means of spectroscopic methods in particular spectrophometry and fluorescence spectroscopy of MTT and DOXO. We postulate that the medium and the MTT reagent itself can interfere on the metabolic activity method, so in order to achieve better results, DMEM was replaced by a neutral buffer like Phosphate-buffered saline (PBS). This protocol may be extremely useful in future studies involving DOXO.

Highlights

Breast cancer (BrCa) is the most common cancer among women and advances in screening and treatment have improved, it is still the most invasive form of cancer
Aqueous stock solutions of DOXO was prepared using 18.2 MΩ ultrapure water obtained from a Milli-Q Plus Millipore water filtration system; Dulbecco’s Modified Eagle Medium (DMEM)
When comparing the absorbance of DOXO, MTT, Formazan, DMEM and DMEM+MTT it is possible to observe that there is a superposition between DOXO, Formazan, DMEM and DMEM+MTT (Figure 1) that can directly interfere in colorimetric methods

Summary

Introduction

Breast cancer (BrCa) is the most common cancer among women and advances in screening and treatment have improved, it is still the most invasive form of cancer. It is a DNA intercalator that inhibits topoisomerase I and II, causing DNA damage and formation of reactive oxygen species (ROS) that promote caspases activation, and leads to apoptosis [2]. Used alone, it can be administrated in combination with other pharmaceutical agents to treat several types of malignancies including sarcoma, lymphoma, leukemia and breast cancer

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