Apoptosis/necrosis ratio in THP-1 cell line (human monocytic leukemia cells) exposed to esters of 2,3-dihydroazete series.

Objective: The failure of many anti-cancer chemotherapeutics is due to their inability to induce apoptosis at the cellular level. Many cancers can develop pro-survival adaptations and anti-apoptotic signaling pathways, leading to drug resistance. Development of prodrugs that induce targeted necrosis is one strategy to circumvent apoptosis-resistance. However, we hold the opinion that necrosis should be the natural outcome of primary and fully developed apoptosis, in order to maximally avoid side effects with healthy tissues at the stage of preclinical and clinical studies. Recently we developed two two-step procedures for the synthesis of thermally and hydrolytically stable 2,3-dihydroazete-2,3-di-/2,2,3-tricarboxylates (2,3-dihydroazetes) [in press]. The objective of the present research was to assess their pro-apoptotic and/or pro-necrotic abilities with cell suspension culture of human monocytic leukemia cells (THP-1), along with their general cytotoxic effect. \nMethods: Cultivation of the THP-1 cell line was in supplemented RPMI-1640 medium. For assessment of cell viability, two DNA binding dyes - YO-PRO-1 and propidium iodide (PI) - were used (‘normal’cells, not stained; cells at early stages of apoptosis, YO-PRO-1 positive; cells at later stages, YO-PRO-1 and PI positive). \nResults: 2,3-Dihydroazetes tested over the range 10-5 to 2×10-4 M showed significant differences in their ability not only to induce the cell death, but also in the way they do so by inducing predominantly apoptosis or necrosis. To adequately describe and quantitatively assess their apoptotic and/or necrotic potential, on one hand, and their general cytotoxic potential, on the other hand, we took the difference between the two areas under the curves, apoptotic and necrotic, over the concentration range. Dihydroazete 2a exhibited the maximal apoptotic/necrotic difference with the THP-1 cell line (Figs. 1, 2); 2m,n,k showed a lower and non-significant difference due to higher necrotic potential at nearly the same cytotoxicity as 2a; dihydroazetes 2b,e and 8a showed a lower though significant difference due to higher necrotic potential coupled with a lower cytotoxicity; 2h had both a very low difference and a very low cytotoxicity, and dihydroazetes 8g,c exhibited a negative difference due to predominantly necrotic cell death at a moderate to high level of cytotoxicity. \n \t \nFigure 1. The quantitative estimates of the apoptotic/necrotic difference (AND) of the dihydroazetes 2a,b,e,h,k,m,n and 8a,c,g exhibited \nwith the THP-1 cell line. *) Р<0.05\tFigure 2. Concentration dependency (C = mole × L-1) of apoptosis and necrosis induction. Percentage of dead cells as determined by DNA binding dyes YO-PRO-1 and PI via flow cytometry after treatment of THP-1 cells for 24 h with different concentrations of 2a. \n \nConclusion: The newly synthesized thermally stable 2,3-dihydroazetes greatly differ in their ability to induce apoptosis and/or necrosis, and could be good candidates for further studies of their anti-cancer activities.