Cytotoxic effect of α-tocopheryl succinate (α-TOS), vitamin K3 (VK3), and ascorbic acid (AA) on prostate cancer cells and fibroblasts. PC3 and IMR90 cells were seeded into 96-well tissue culture plates at 10⁴ per well and treated with increasing concentration of α-TOS, VK3, and AA. The cells were assessed for viability using the MTT assay following exposure to the agents for 24 h at different concentrations (left panels) or for different times at the concentrations shown (μM) (right panels). The IC₅₀ values were derived from the data in the left panels. All data are expressed as mean±s.d. of the percentage variation with respect to the control (untreated cells) of three independent experiments carried out in duplicate.

Effect of α-tocopheryl succinate (α-TOS) alone or in combination with the vitamin K3 (VK3) and ascorbic acid (AA) on cell-death induction and viability of prostate cancer cells and fibroblasts. (A) PC3 and (B) IMR90 cells were exposed to a sub-apoptotic dose of α-TOS (30 μM) alone or in combination with sub-apoptotic levels of VK3 (3 μM) plus AA (0.4 mM). The cells were then assessed for cell death and viability. The results are expressed as mean±s.d. of percentage and of the percentage variation with respect to the control (untreated cells) of three independent experiments carried out in duplicate.

Reactive oxygen species (ROS) generation and DNA fragmentation induced in prostate cancer cells by α-tocopheryl succinate (α-TOS) or vitamin K3 (VK3) plus ascorbic acid (AA) alone or in combination. (A) The capacity of α-TOS or VK3 plus AA alone or in combination to induce ROS formation was evaluated by assessing the oxygen consumption using the Clark's oxygen electrode. (B) PC3 cells were placed in 96-well flat-bottom tissue culture plate at 10⁴ per well. After overnight incubation, cells were treated with α-TOS (30 μM), VK3 (3 μM), and AA (0.4 mM) alone or in combination, and aliquots of the conditions media were taken at regular intervals and evaluated for the level of hydroperoxides using the d-ROMs assay. Data are expressed as of percentage variation with respect to the control. (C) Intracellular ROS were estimated using the fluorescent dye 2′7′-dichlorofluorescein diacetate (DCFA). PC3 cells were seeded in 24-well flat-bottom plates and 20 μM of DCFA added. After 30 min of incubation, the cells were exposed to α-TOS (30 μM), VK3 (3 μM), and AA (0.4 mM) alone or in combination. After 24 h, the cells were evaluated by flow cytometry. The amount of ROS was detected as fluorescence intensity normalised for the blank (samples without DCFA) and expressed as fold change with respect to the control (untreated cells). (D) DNA fragmentation was analysed using the alkaline comet assay. PC3 cells were seeded in 96-well flat-bottom tissue culture plates at 10⁴ per well, treated after overnight incubation with α-TOS (30 μM), VK3 (3 μM), and AA (0.4 mM) alone or in combination, and evaluated for DNA damage at different time points. The level of DNA strand breaks was assessed using the comet assay that is based on visual scoring with the maximum of 400 arbitrary units (see Material and Methods for detail). The data are expressed as the percentage variation with respect to the control (untreated cells).

Induction of apoptosis by α-tocopheryl succinate (α-TOS) or vitamin K3 (VK3) plus ascorbic acid (AA) alone or in combination in the presence of a lysosomal protease inhibitor. PC3 cells were exposed for 24 h to sub-apoptotic doses of α-TOS (30 μM) or VK3 (3 μM) plus AA (0.4 mM) alone or in combination, and (A) lysosmal and (B) mitochondrial destabilisation were evaluated using the Acridine orange (AO) and MitoTracker Red-580 staining, respectively, in the presence or absence of the lysosomal protease inhibitor E-64d. (C) Cytochrome c release and cell viability after treatments of prostate cancer cells as described above in the presence or absence of lysosomal protease inhibitor E-64d was assessed. The sign ‘^*' denotes significantly different data for treated and control cells with P<0.05.

Induction of cell death in prostate cancer cells exposed to α-tocopheryl succinate (α-TOS) in combination with vitamin K3 (VK3) and ascorbic acid (AA). The effect of α-TOS, VK3, and AA on prostate cancer cells was evaluated on the basis of cell death induced by the drugs alone or in combination (left panels). Synergistic, additive or antagonistic effects of sets of two drugs were assessed by constructing the isobolograms at IC₅₀ based on the results of the MTT assay for PC3 cells treated with individual combinations of two drugs. Each isobologram combines the results of at least three independent experiments carried out in duplicate.

Morphological changes of prostate cancer cells exposed to α-tocopheryl succinate (α-TOS) and vitamin K3 (VK3) plus ascorbic acid (AA) alone or in combination. (A) For the soft-agar colony-forming assay, cells were seeded in 24-well culture plates at 10⁴ per well in the RPMI-1640 medium containing 0.35% low melting point (LMP) agarose overlaid with 0.7% LMP agarose. The cells were maintained at 37°C in 5% CO₂ for 30 days; every 7 days, 500 μl of fresh medium was added to each well. The formed colonies were treated with a sub-lethal dose of α-TOS (30 μM) or VK3 (3 μM) plus AA (0.4 mM), or the three drugs at these doses together, and the cells were stained with crystal violet after 7 days and inspected by optical microscope. (B) PC3 cells were placed overnight in 35-mm dishes on glass coverslips. After a 6-h incubation with α-TOS (30 μM) or VK3 (3 μM) plus AA (0.4 mM), or the three drugs at these concentrations together, the cells were stained with phalloidine–TRIC, and the coverslips mounted on microscope slides with Vectrashield containing DAPI and inspected in a fluorescence microscope.

Effect of α-tocopheryl succinate (α-TOS) or vitamin K3 (VK3) plus ascorbic acid (AA) alone or in combination on the cell death induction and destabilisation of mitochondria and lysosomes. PC3 cells were exposed for 24 h to sub-apoptotic level of α-TOS (30 μM) or VK3 (3 μM) plus AA (0.4 mM) alone or in combination, and cell death evaluated by the (A) annexin V-propidium ioide staining and (B) caspase activation. Lysosomal destabilisation in cells treated as described above was evaluated by the uptake of (C) Acridine orange (AO), (D) mitochondrial perturbation was assessed by cytochrome c release. The sign ‘^*' denotes significantly different data for treated and control cells with P<0.05.