Diastereoselective Michael additions to α,β-unsaturated α-sulfinyl phosphonates in the thiolane series

International audienceA chiral racemic 2-phosphono-2,3-didehydrothiolane sulfoxide was used as a Michael acceptor in the reactions with several nucleophiles, in particular thiols. In most cases the reactions were fully diastereoselective. The relative configuration of the resulting adducts was determined

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions

<div><p>Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound.</p></div

Effects of Selol, SFN and 2-oxoheptyl ITC on total cell numbers and the live/dead cell ratios.

<p>Cells were incubated with compounds for 24, 48 and 72 h and stained with FDA/PI. Data analysis was performed using Fluoview500 software (version 5.0) and ImageJ program. * significant difference, p < 0.05.</p

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions - Fig 1

<p><b>(A) Effect of the test method on the drug-drug interaction results.</b> (a) Selol and SFN, (b) Selol and 2-oxoheptyl ITC, (c) Selol and 2-oxohexyl ITC, (d) Selol and alyssin. Combination index (CI) plots present CI as a function of effect (fa = fraction affected, defined as percentage inhibition/100) from 0.05 to 0.95 (5–95% cells killed). CI > 1.0 indicates antagonism, CI = 1 indicates additive effects, and CI < 1.0 indicates synergism. x, experimental data obtained with MTT assay. +, experimental data obtained with CVDE assay. Dashed (for MTT) and solid (for CVS) lines = computer simulation for Fa-CI plot. <b>(B) Discrepancies between CI calculated on the basis of data obtained with MTT and CVS assays at fa 0.9.</b> CI > 1.0 indicates antagonism, CI = 1 indicates additive effects, and CI < 1.0 indicates synergism. * significant difference, p < 0.05.</p

Effect of Selol on MTT reduction.

<p>(A) Microscope images of formazan crystals in HT-29 cells. Cells were incubated with 32 and 63 μM Selol for 24 h. Microscope images were recorded after 3 h of reduction of MTT. Black dots indicate formazan crystals. The arrows indicate a cell without formazan. (B) HT-29 cell mitochondria after 24 h incubation with Selol. Cells stained with the MitoTracker® Deep Red. (C) Microscope images of Selol-induced changes in mitochondrial membrane potential (ΔΨm). HT-29 cells were incubated with 32 and 63 μM Selol and stained with MitoLight dye, which stains mitochondria in a membrane potential-dependent fashion. Changes in the mitochondrial membrane potential ΔΨm were detected by confocal microscopy. Left image presents the detection of monomers (green fluorescence), indicating the presence of depolarized mitochondria. Right image presents the fluorescence of the aggregates (red fluorescence), indicating functional, polarized mitochondria. (D) Microscope images of the intracellular reactive oxygen species (ROS) induction by Selol (green fluorescence). HT-29 cells were incubated with 32 and 63 μM Selol and stained with the ROS-sensitive dye DHR123. (E) On the left: Microscope images of formazan crystals in HT-29 cells incubated with 63 μM Selol for 24 h. On the right: FDA/PI staining of HT-29 cells incubated with 63 μM Selol for 24 h. Microscope images were recorded after 3 h of reduction of MTT. Black dots indicate formazan crystals, green fluorescence denotes living cells stained with FDA, and red fluorescence denotes dead cells stained with PI. Scale bar = 50 μm.</p