Cell culture and reagents

The prostate cancer cells (PC3) and fibroblasts (IMR90) purchased from the American Type Culture Collection were grown in the RPMI-1640 and DMEM medium, respectively, supplemented with 2mM L-glutamine, 100Uml⁻¹ penicillin, 100μgml⁻¹ streptomycin, and 10% FBS. The cells were cultured at 37°C and 5% CO₂ in a humidified incubator. AA (Sigma, St Louis, MO, USA) was dissolved in PBS (pH 7.4) immediately before use. α-TOS and VK3 (menadione) (both from Sigma) were dissolved in ethanol and DMSO, respectively, diluted in complete medium to the final concentration and added to cells at 0.1% of solvent (v/v).

Cytoxicity and isobologram analysis

PC3 and IMR90 cells were plated in 96-well flat-bottom tissue culture plates at 10⁴ per well, allowed to attach overnight and incubated for 24, 48 and 72h with α-TOS (10–100μM), VK3 (1–30μM), and AA (0.1–3.2mM) alone or in combination. Cell viability was determined using the MTT assay (Carmichael et al, 1987). Briefly, after the exposure of cells, 10μl of MTT (5mgml⁻¹ in PBS) was added and the plate incubated at 37°C for 3h. After removing the media, 200μl of isopropanol was added and mixed to dissolve the crystals. Absorbance was read at 550nm in an ELISA plate reader and control absorbance was designed as 100%. Survival curves were generated and the IC₅₀ values determined. The effect of combination of the drugs in PC3 and IMR90 cells was estimated both by plotting the percentage of dead cells after treatment with the drug alone or in combination with another drug at its IC₅₀ concentration and by the isobologram analysis carried out as described in detail elsewhere (Tomasetti et al, 2004) using the CalcuSyn1 software.

Soft agar colony-forming assay

Cells (10⁴) were seeded in 24-well plates in the RPMI-1640 medium containing 0.35% low melting point (LMP) agar, overlaid with 0.7% LMP agar, and cultured at 37°C in 5% CO₂ for 30 days. Every 7 days, 0.5ml of fresh medium was added to each well. The colonies of cells were treated with α-TOS (30μM), VK3–AA mixture (3μM VK3 and 0.4mM AA), or α-TOS–VK3–AA combination (30μM α-TOS, 3μM VK3, and 0.4mM AA), and after 7 days the colonies were stained with the crystal violet dye and visualised by optical microscope.

Actin–phalloidine labelling

PC3 cells were placed overnight in 35-mm dishes on glass coverslips. After 6h of incubation with α-TOS or combinations of the agents (VK3-AA and α-TOS–VK3–AA), the cells were washed with PBS, fixed with 4% formaldehyde in PBS, and incubated in the saponin solution (0.05% saponine and 2% FBS in PBS). The cells were then incubated with TRIC-conjugated phalloidine (Sigma) (2μgml⁻¹) at room temperature for 30min, and the coverslips mounted on microscope slides with VectraShield plus DAPI (Vector Laboratories, Burlingame, CA, USA) and inspected in a fluorescence microscope (Zeiss, Axiocam MRc5, Thomwood, NY, USA, magnification × 60).

DNA damage

Damage of DNA was assessed using the alkaline comet assay (Tomasetti et al, 2001). 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 (30μM), VK3–AA mixture (3μM VK3 and 0.4mM AA), α-TOS–VK3–AA combination for increasing time periods, and DNA damage was evaluated. To do so, in brief, cells were sandwiched between thin layers of agarose on a microscope slide, lysed at alkaline pH, electrophoresed, stained with the DAPI dye, and inspected in the fluorescence microscope. The number of strand breaks was scored visually such that 100 randomly selected comets were graded according to the degree of damage into five classes (0–4) to provide an overall score for each gel of 0–400 arbitrary units (AU).

Oxygen consumption assay

The capacity of α-TOS, VK3, and AA alone or in combination to induce reactive oxygen species (ROS) formation was estimated by evaluating the oxygen consumption using the Clark's oxygen electrode. α-TOS (0.3mM), VK3 (30μM), and AA (4mM) were sequentially added into the Clark's oxygen electrode chamber and oxygen consumption assessed.

Assessment of hydroperoxide formation

Hydroperoxide levels were evaluated in the conditioned medium using the d-ROMs assay (Vassalle et al, 2006). PC3 cells were placed in 96-well flat-bottom tissue culture plates at 10⁴ per well. After overnight incubation, cells were treated with α-TOS (30μM), VK3 (3μM) AA (0.4μM) VK3–AA mixture (3μM VK3 and 0.4mM AA), or the α-TOS–VK3–AA combination, and aliquots were taken at different time points. Briefly, 3μl of medium was added to the reaction mixture containing N,N-diethyl-para-phenylendiamine and acetate buffer (pH 4.8). Samples were subjected to 20 repeated spectrophotometric readings (520nm). The concentration was automatically calculated from the mean slope (the rate of change in absorbance).

Assessment of generation of intracellular ROS

Intracellular ROS levels were estimated using the fluorescent dye 2′7′-dichlorofluorescein diacetate (DCFA). PC3 cells were seeded in 24-well flat-bottom plates and 20μM of DCFA, a cell-permeable, ROS-sensitive dye added to each well. After 30min of incubation, the florescent probe was removed and the cells exposed to α-TOS (30μM) VK3 (3μM) AA (0.4μM) and vitamin combination (VK3-AA and α-TOS–VK3–AA). After a 24-h incubation, the cells were collected, washed, and resuspended in PBS, and analysed by flow cytometry (FACS Calibur, Becton Dickinson, Palo Alto, CA, USA). The level of ROS was detected as fluorescence intensity and expressed as fold change with respect to the control.

Annexin V–propidium ioide (PI) staining

Apoptosis was quantified using the annexin V-FITC method, which detects phosphatidyl serine (PS) externalised in the early phases of apoptosis (Boersma et al, 1996). Cells were plated at 10⁵ per well in 24-well plates. After an overnight incubation, cells were treated with α-TOS (30μM), VK3–AA mixture (3μM VK3 and 0.4mM AA), and α-TOS–VK3–AA combination. Floating and attached cells were collected, washed with PSBS, resuspended in 100μl binding buffer, incubated for 20min at room temperature with 2μl annexin V-FITC, supplemented with 10μl PI (10μgml⁻¹), and analysed by flow cytometry using channel 1 for annexin V-FITC binding and channel 2 for PI staining.

Western blot analysis

PC3 cells were treated with α-TOS (30μM), VK3–AA mixture (3μM VK3 and 0.4mM AA), α-TOS–VK3–AA combination for 24h. Floating and attached cells were collected, lysed in a buffer containing 250mM NaCl, 25mM Tris–HCl (pH 7.5), 5mM EDTA, 1% Nonidet P-40, and a cocktail of protease inhibitors (2μgml⁻¹ aprotinin, 2μgml⁻¹ leupeptin, 1mM phenylmethyl-sulfonyl fluoride, and 2μgml⁻¹ proteinin), and stored at −80°C until used. The protein levels were quantified using the Bradford assay (Sigma). The protein samples (50μg per lane) were boiled for 5min, resolved using 12.5% SDS–PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked (PBS containing 0.1% Tween and 5% skimmed milk) for 1h, and incubated overnight with anti-caspase-8, anti-caspase-9, anti-caspase-3, or anti-Bid IgG (all Cell Signaling Technology, Danvers, MA, USA). After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualised by ChemiDoc using the Quantity One software (BioRad Laboratories, Hercules, CA, USA). β-Actin was used as a control for protein loading.

Assessment of lysosomal and mitochondrial destabilisation, and inhibition of lysosomes

The integrity of lysosomes and mitochondria was monitored based on the uptake of Acridine orange (AO; Sigma) (Hopkins, 2008) and MitoTracker Red-580 (Molecular Probes, Carlsbad, CA, USA), respectively.

Fluorescent microscopy

PC3 cells were placed in 6-well plates at 3 × 10⁵ per well on glass coverslip. The cells were allowed to attach overnight and then incubated 24h with α-TOS (30μM), or vitamin combination (VK3–AA and α-TOS–VK3–AA). After treatment, cells were resuspended in 2ml RPMI-1640 medium with 5μgml⁻¹ AO or 100nM MitoTracker Red-580, incubated at 37°C for 15min, mounted on slides with Vectashield (Vector Laboratories) and viewed in a fluorescence microscope (Zeiss, Axiocam MRc5, magnification × 60).

Cytofluorimetry

PC3 cells were plated overnight in a 6-well plate at 3 × 10⁵ per well. After 24h of treatment with α-TOS (30μM) or vitamin combination (VK3–AA and α-TOS–VK3–AA), the cells were incubated with 5μgml⁻¹ AO for 15min. Floating and attached cells were collected, resuspended in PBS, and red fluorescence evaluated by flow cytometry. The percentage of cells with low intensity of red fluorescence (pale cells) was used as a marker for the extent of lysosomal destabilisation (impairment of AO uptake).

Lysosmal inhibition

Cells were incubated overnight in the presence of 20μM E-64d, a broad-spectrum cathepsin and calpain inhibitor, treated with α-TOS or vitamin combination (VK3-AA and α-TOS-VK3- AA) for 24h, and assessed for viability, mitochondrial and lysosomel integrity, and cytochrome c release.

Cytochrome c release

PC3 cells (3 × 10⁵ per well in 6-well plates) were treated with α-TOS or vitamin combination (VK3–AA and α-TOS–VK3–AA) for 24h. Cells were then harvested and the pellet resuspended in the digitonin cell permebilization buffer. After incubation on ice for 5min, the cells were centrifuged at 1000g for 5min at 4°C. The supernatant containing the cytosolic fraction of cytochrome c was collected. The remaining pellet was resuspended in the RIPA cell lysis buffer, vortexed, and incubated on ice for 30min. The lysate was then centrifuged at 10000g for 10min at 4°C. The cytosolic and mitochondrial fractions were then assessed using the enzyme immunometric assay kit (Assay Designs, Hines Drive Ann Arbor, MI, USA). The results were quantified as pgml⁻¹ and expressed as percentage of cytochrome c in each fraction respect to the total.

Statistical analysis

Data are presented as mean±s.d. Comparisons between groups were carried out using the Mann–Whitney U-test for unpaired samples and Kruskall–Wallis analysis for multiple comparisons. Statistical calculations were carried out using the SPSS statistical package version 12.0F. Statistical differences of at least P<0.05 were considered statistically significant.

•  Other Sections▼
  • Abstract
  • Materials and methods
  • Results
  • Discussion
  • References

Results

αTOS, VK3, and AA exert different toxicity towards prostate cancer cells and fibroblasts

Treatment of PC3 cells with α-TOS and VK3 resulted in dose-dependent cytotoxicity, whereas the cells were completely resistant to AA. Figure 1 (left panel) shows the viability curves of PC3 cells with IC₅₀ value ranging from 30 to 40μM for α-TOS and 4–5μM for VK3. The cells were completely resistant to AA treatment up to 3.2mM, exerting its cytotoxic effect at prolonged exposure times (Figure 1, right panel). The dose- and time-dependent plots show that low doses of α-TOS and AA were not cytotoxic within the initial 24h of drug exposure, whereas cell death was observed at prolonged time points. No effect of VK3 below 3μM occurred. It should be noted that non-malignant cells such as fibroblasts were resistant to α-TOS and AA at concentrations up to 100μM and 3.2mM, respectively. VK3 was found toxic for the IMR90 fibroblasts at concentrations over 10μM.

Figure 1 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 (more ...)

Effect of the combination of α-TOS, VK3, and AA on prostate cancer cells

To study the combined effects of α-TOS, VK3, and AA, PC3 cells were exposed to increasing concentrations of individual drugs alone or in combination, and cell death was assessed. The data were then used to carry out isobologram analysis. The IC₅₀ values for one drug <1 were plotted against corresponding IC₅₀ values for the other drug. Distribution of individual points along the diagonal connecting the values of 1 suggests an additive effect of the two drugs, whereas the points below or above the line indicate their synergism and antagonism, respectively (Figure 2, right panel).

Figure 2 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 (more ...)

Antagonistic effects on cell death induction were found for the combination of α-TOS and VK3, and the presence of α-TOS markedly reduced VK3-induced cell death (Figure 2, left panel). No cytotoxic interaction was observed for the α-TOS–AA combination, whereas a synergistic effect was found for the VK3–AA combination. The cytotoxic effect of VK3 was synergistically enhanced by the addition of increasing doses of AA (Figure 2).

αTOS and VK3–AA in combination exert selective cooperative effect in prostate cancer cells

The effect of α-TOS on cytotoxicity induced by the combination of VK3 and AA was evaluated using the PC3 prostate cancer cells and the IMR90 fibroblasts. Cell death induced by the VK3–AA combination was found to be enhanced by α-TOS, and the effect was synergistic/additive (Figure 3A). Combining sub-lethal dose of α-TOS with a VK3 plus AA that alone does not induce cell death caused induction of cell death in PC3 cells after 24h of exposure to the agents. This effect was selective for the cancer cells, as no cell death was observed when the combination of the three compounds was used to challenge the non-malignant fibroblasts (Figure 3B).

Figure 3 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 (more ...)

We then verified the morphological changes of PC3 cells treated with the agents alone or in combination. The efficacy of α-TOS to induce cell damage in combination with VK3 plus AA was first evaluated in vitro using the soft-agar colony-forming assay. As shown in Figure 4A, PC3 cells gave rise to numerous and large colonies in soft agar. The colonies were then treated with the drugs alone or in combination. Translucent and pycnotic cells were observed after 1 week of incubation when the agents were added together.

Figure 4 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 (more ...)

Phalloidin staining for actin was used to assess the effect of treatment with α-TOS, VK3–AA, and α-TOS in combination with VK3 plus AA on morphology of the cells. After 6-h exposure, cells treated with the combination of the three agents underwent morphological alterations such as formation of cytoplasmic blebs (Figure 4B). These blebs are associated with the preservation and diminution of cell size and suggest that those cells loose pieces through a mechanism of self-excision. Further, the treated cells display a pleiomorphism that makes them elongated, enormous, or smaller than the control tumour cells. The nuclei of the treated cells are not showing condensations typical of apoptotic stages and are enlarged. They also appear with an enhanced inner envelope with excessive phalloidin contrast and show enlarged nucleoli. Nuclei remain unbroken and are not part of the pieces shed by the cells as it would be in apoptotic bodies. Hence, the cell demise appears to demonstrate the characteristics of autoschizis cell death as previously described (Gilloteaux et al, 1998, 2001, 2005).

α-TOS and VK3 plus AA cause generation of ROS

The ability of α-TOS, VK3, and AA, and their combination to induce ROS formation has been assessed using the Clark's oxygen electrode. The agents were sequentially added into the oxygen electrode chamber and consumption of oxygen evaluated. As shown in Figure 5A, VK3 itself did not induce oxygen consumption, which was observed to occur following the addition of AA. Similarly, no oxygen consumption occurred both in the presence of α-TOS and VK3, whereas it was observed in the presence of AA. We next tested generation of ROS induced by incubation of PC3 cells with sub-apoptotic doses of the three agents alone or in combination. The presence of extracellular ROS has been evaluated by the determination of hydroperoxide levels in the conditioned medium of the treated PC3 cells. The levels of ROS were found to increase after 1h of incubation with VK3 plus AA in the presence of α-TOS (Figure 5B). It should be noted that α-TOS itself was responsible for the observed induction of intracellular ROS formation, whereas VK3 plus AA did not increase the intracellular ROS production any further (Figure 5C).

Figure 5 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 (more ...)

The efficacy of the three agents to induce DNA damage, which was assessed as single strand break (SSB) formation (Figure 5D), was evaluated. VK3 plus AA induced generation of SSBs, which was observed after a 1-h treatment. SSBs induced by VK3 and AA were not repaired and persisted in the cells for up to 24h. It is interesting to note that α-TOS did not increase the SSB formation observed when the cells were exposed to VK3 plus AA, neither did it affect the DNA repair.

Combination of αTOS, VK3, and AA induces cell death by lysosomal and mitochondrial destabilisation

Incubation of PC3 cells with a combination of αTOS, VK3, and AA at a dose at which the individual compounds alone do not induce cell death was found to cause detachment of cells (data not shown) and phosphotidylserine externalisation (Figure 6A). Typically, 50–60% cells were annexin-V positive. However, no PI uptake (data not shown) and no sign of caspase activation (Figure 6B) were observed. We therefore investigated the potential role of the lysosomal/endosomal system, which has a major role in intracellular protein degradation and recycling, as it has been suggested to promote cell death, as shown, for example, for α-TOS (Neuzil et al, 1999, 2002). Therefore, a possible release of lysosomal proteases and mitochondrial cytochrome c to the cytosol have been tested in PC3 cells exposed to α-TOS or VK3 plus AA, as well as to α-TOS with VK3 plus AA. Some 50–60% pale cells (Figure 6C) and cytochrome c release (Figure 6D) were observed in cells treated with the combination of α-TOS, VK3, and AA. Control cells exhibited a punctuated red fluorescence pattern of AO and MitoTracker Red-580, suggesting that the two dyes accumulated in lysosomes and mitochondria, respectively (Figure 7A and B, upper panel). The combination of α-TOS, VK3, and AA caused appearance of cells with substantially decreased red fluorescence due to the loss of lysosomal and mitochondria integrity. Pre-treatment of cells with E-64d reduced neither lysosomal damage nor mitochondrial permeabilisation (Figure 7A and B, lower panel), coinciding with the appearance of cytochrome c in the cytosol (Figure 7C) and cell death induction (Figure 7D).

Figure 6 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 24h to sub-apoptotic (more ...)

Figure 7 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 24h to sub-apoptotic doses (more ...)