Fig 1.
(A) Effect of the test method on the drug-drug interaction results. (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) Discrepancies between CI calculated on the basis of data obtained with MTT and CVS assays at fa 0.9. CI > 1.0 indicates antagonism, CI = 1 indicates additive effects, and CI < 1.0 indicates synergism. * significant difference, p < 0.05.
Fig 2.
Effect of Selol, SFN, 2-oxoheptyl ITC, 2-oxohexyl ITC and alyssin on cell viability determined by MTT and CVS assays after 24, 48 and 72 h of incubation.
Each bar represents the mean ± SD (n = 18). *Statistically significant difference between the growth inhibition effect determined by MTT and CVS assays, p < 0.05.
Table 1.
IC50 values (μM) of ITCs, Selol and 5-fluorouracil obtained by MTT and CVS assays.
Fig 3.
Effects of Selol, SFN and 2-oxoheptyl ITC on total cell numbers and the live/dead cell ratios.
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.
Fig 4.
Effect of Selol on MTT reduction.
(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.
Fig 5.
Effect of SFN on MTT reduction.
(A) Microscope images of formazan crystals in HT-29 cells. Cells were incubated with 4 and 10 μM of SFN for 24 h. Microscope images were recorded after 3 h of reduction of MTT. Black dots indicate formazan crystals. (B) Microscope images of SFN-induced changes in mitochondrial membrane potential (ΔΨm). HT-29 cells were incubated with 4 and 10 μM SFN 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 the monomers (green fluorescence), indicating the presence of depolarized mitochondria). Right image presents fluorescence of the aggregates (red fluorescence), indicating functional, polarized mitochondria. (C) Microscope images of the intracellular reactive oxygen species induction (ROS) by SFN (green fluorescence). HT-29 cells were incubated with 4 and 10 μM SFN for 24 h and stained with the ROS-sensitive dye DHR123. Scale bar = 50 μm.
Fig 6.
Effect of 2-oxoheptyl ITC on MTT reduction.
(A) Microscopic images of formazan crystals. HT-29 cells were incubated with 4 and 10 μM 2-oxoheptyl ITC for 24 h. Microscope images were recorded after 3 h of reduction of MTT. Black dots indicate formazan crystals (dashed line arrow). Needle-shaped formazan crystals larger than the cells are denoted with solid line arrows. (B) Mitochondria of HT-29 cells after 24 h incubation with 2-oxoheptyl ITC. Cells were stained with MitoTracker® Deep Red. (C) HT-29 cells were incubated with 4 and 10 μM 2-oxoheptyl ITC 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 the 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 induction (ROS) by 2-oxoheptyl ITC (green fluorescence). HT-29 cells were incubated with 4 and 10 μM 2-oxoheptyl ITC for 24 and stained with the ROS-sensitive dye DHR123. Scale bar = 50 μm.