Fluorimetric Evaluation of the Effect of Riboxin, Copper Chlorophyllin, Trolox and the Soluble form of Indralin on the Growth Properties of A549 Cells in Culture

Abstract

Currently, cell cultures are most often used as an experimental model system in biological research. However, to correctly plan studies using this model system, many aspects must be taken into account. Thus, to be able to correctly assess the effect of radioprotective drugs on cells, it is first necessary to study the effect of these substances on the properties of cells in culture. And the main property for work using plate readers is the ability of cells to adhere to the bottom of the plate and the rate of cell proliferation. This work is devoted to the study of human riboxin (inosine), copper chlorophyllin, trolox and indralin, the growth properties of cells in a culture based on the A549 cell line - human lung adenocarcinoma. Riboxin, chlorophyllin and trolox are promising compounds that make it possible to study their radioprotective properties. Indralin is a reference classic radioprotector. This process used a water-soluble form containing tartaric acid to allow indralin to dissolve in water. The experiment consisted of incubating A549 cells for 24 hours in a solution of riboxin (inosine), copper chlorophyllin, Trolox or tartaric acid at a concentration of 2 mM or in a mixture of 2 mM tartaric acid and 1.9 mM indralin, followed by assessment of the cell content in the samples compared to cells incubated without the addition of these substances, based on the fluorescence of the Hoechst-33342 dye. An additional experiment with chlorophyllin consisted in incubating cells with chlorophyllin already attached to the bottom of the tablet in the concentration range of 50–500 μM for 2.5 hours, followed by an assessment of the content of the remaining cells in the tablet. All studied substances statistically significantly reduced the cell content in the samples compared to the control. The greatest decrease in cell content was observed in the sample with chlorophyllin, and the least – with riboxin. As a result of additional experience in incubating already attached cells in chlorophyllin solution with concentrations of 50–500 μM, it was shown that this substance dose-dependently inhibits the adhesive properties of cells of the A549 line. At the same time, chlorophyllin already at a concentration of 50 μM statistically significantly reduced the cell content in the sample after washing the wells of the tablet compared with the control sample. The decrease in cell content in the sample containing a mixture of indralin and tartaric acid was statistically significantly more pronounced than that caused by incubation in a solution of tartaric acid alone. That is, the recognized radioprotector indralin demonstrated a pronounced inhibition of the growth properties of the A549 cell line. Based on the information obtained, we can conclude that when planning future studies of the substances studied in this work on a cell culture model, it is necessary to take into account the fact that they inhibit the growth of the cell culture.