Fig 1.
Chemical structures of (a) doxorubicin, daunorubicin; (b) oxazolinodoxorubicin and oxazolinodaunorubicin.
Table 1.
The value of IC50 parameters for DOX and O-DOX, or DAU and O-DAU after 72 h incubation.
Fig 2.
Oxazolines induce oxidative stress.
(a) The effect of drugs on ROS production in SKOV-3 and HepG2 cells. (b) The effect of drugs on nitrite level. (c) SKOV-3 cells and (d) HepG2 cells stained with the fluorescence probe H2DFC-DA after 1 h incubation with 80 nM concentrations of investigated compounds: DOX (A), O-DOX (B), DAU (C), O-DAU (D). In experiments with the antioxidant, cells were preincubated with 1 mM NAC for 1 h. Then DOX, DAU or oxazoline derivatives (80 nM) were added and incubation was continued for up to 4 h (scale bar: 50 μm): DOX (A′), O-DOX (B′), DAU (C′), O-DAU (D′).
Fig 3.
Influence of drugs on intracellular levels of Ca2+ in SKOV-3 and HepG2 cells.
The cells were treated with 80 nM concentrations of drugs for 2, 4, 24 or 48 h. In experiments with antioxidant, cells were preincubated with 1 mM NAC, then drugs were added and incubation continued for another 2–48 h.
Fig 4.
Oxazolinoanthracyclines reduced the mitochondrial membrane potential.
Changes in mitochondrial membrane potential of (a) SKOV-3 and (b) HepG2 cancer cells. The fluorescence ratio of JC-1 dimers/JC-1 monomers in the control was assumed to be 100%. (c) SKOV-3 cells and (d) HepG2 cells stained with the fluorescence probe JC-1 after 48 h incubation with 80 nM concentrations of investigated compounds: DOX (A), O-DOX (B), DAU (C), O-DAU (D). In the experiments with antioxidant, the cells were preincubated with 1 mM NAC for 1 h, then DOX, DAU or oxazoline derivatives (80 nM) were added and incubation were continued for up to 48 h (scale bar: 50 μm): DOX (A′), O-DOX (B′), DAU (C′), O-DAU (D′). The right-hand column shows enlarged representative images of single drugs treated cells, scale bar: 10 μm. The cells were stained with JC-1 probe. Yellow-orange fluorescence of JC-1 dimers is present in cell areas with high mitochondrial membrane potential, while green fluorescence of JC-monomers is prevalent in cell areas with low mitochondrial membrane potential. The cells were visualized under an inverted fluorescence microscope (Olympus IX70, Tokyo, Japan). (For interpretation of the colours, the reader is referred to the web version of the article).
Fig 5.
Modified anthracyclines are able to trigger autophagy.
Fraction of autophagic cells in (a) SKOV-3 and (b) HepG2 cancer cell lines. The measurements were carried out in the presence or absence of the inhibitors. The cells were treated with 80 nM concentrations of DOX, O-DOX, DAU or O-DAU and then incubated for 4, 24 or 48 h. Autophagic changes in control cells (without drug treatment) measured after 2, 4, 24 and 48 h incubation were taken as 100%. 3-MA (5 mM) was added to SKOV-3 or HepG2 cell lines in the presence of 80 nM DOX, O-DOX, DAU or O-DAU for a period of 4, 24 and 48 h. (+) p < 0.05—statistically significant differences observed between the investigated probes in comparison to the effect after treatment with the 3-MA inhibitor. (c) Fluorescence images of autophagic SKOV-3 and HepG2 cells after 48 h incubation with 80 nM concentrations of investigated compounds. The left-hand column shows images of cells treated with DOX (A), O-DOX (B), DAU (C), O-DAU (D). Some cells display abnormal morphology: giant cells and elongated cells with protrusions from the plasma membrane (blebbing); cytoplasmic bridges between cells. The right-hand column shows representatives images of single cells with progressive autophagosome formation (autophagy is indicated by bright green staining of autophagic vacuoles–marked by the yellow arrows). (d) Fluorescence images of SKOV-3 and HepG2 cells after 48 h incubation with 80 nM concentrations of investigated compounds. Cells were stained with acridine orange (AO). The left-hand column shows images of cells treated with DOX (A), O-DOX (B), DAU (C), O-DAU (D). The right-hand column shows representative images of single cells with progressive autophagosome formation (autophagy is indicated by bright red staining of autophagosomes–marked by the yellow arrows). (c, d) Cells were analyzed with an inverted fluorescence microscope (Olympus IX70, Tokyo, Japan). Scale bar: 50 μm.
Fig 6.
LC3 protein contents after DOX, DAU and oxazolinoanthracyclines treatment in the SKOV-3 and HepG2 cells.
The measurements were carried out in the presence or absence of the inhibitor, 3-MA (5 mM). (+) p < 0.05—statistically significant differences observed between the investigated probes in comparison to the effect after treatment with the 3-MA inhibitor.
Fig 7.
The effect of drugs on nSMase activity in SKOV-3 and HepG2 cells.
Fig 8.
Oxazolines delay the growth of tumor cells and their migration.
(a) Drugs inhibited colony formation of cancer cells. SKOV-3 (panel left) and HepG2 (panel right) cells were grown in six-well plates (200 cells per well) in triplicates. After 24 h, the culture medium was replaced with fresh medium containing 80 nM of drugs concentration for 4 or 48 h. After treatment each well was washed twice with medium and the experiment continued for 14 days. Surviving colonies were stained (upper panel) and counted (lower panel). (b) The effect of drugs treatment on cells migration. Representative photographs of cell scratch test captured from 0 to 75h (SKOV-3) and to 55h (HepG2) after treatment with investigated compounds (Olympus IX70 with phase-contrast, Japan; 100× magnification.) The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch (marked by yellow line) toward the center of the scratch.
Fig 9.
Interference of oxazoline derivatives with signaling pathways in ovarian and liver cancer cells.
ER, endoplasmic reticulum; nSMase, neutral sphingomyelinase.