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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract:  To assess the ability of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) to promote apoptosis, we investigated the effect of YC-1 on tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the human epithelial ovarian carcinoma cell lines. In OVCAR-3 and SK-OV-3 cell lines, we examined the stimulatory effect of YC-1 on TRAIL-induced apoptosis by monitoring cell death, nuclear damage, changes in apoptosis-related protein levels, activation of caspases and changes in the mitochondrial transmembrane potential. TRAIL induced a decrease in Bid, Bcl-2 and Bcl-xL protein levels, increase in cleaved Bid and Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9 and -3) and an increase in the tumour suppressor p53 levels. YC-1 enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. Results from this study suggest that YC-1 may enhance the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway, leading to caspase activation. YC-1 may confer a benefit in TRAIL treatment of epithelial ovarian adenocarcinoma.

Apoptosis in cancer cells is mediated both by activating cell surface death receptors, leading to caspase-8 activation, and by the mitochondrial signalling pathway, leading to cytochrome c release and subsequent activation of caspases [1,2]. The tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can initiate apoptosis through the activation of death receptors [3,4]. TRAIL induces apoptosis in transformed or tumour cells (but not in normal cells) via binding to its functional death receptors, DR4 and DR5 [4]. Upon ligand binding, the receptor becomes trimerized and activated. The activated receptor recruits adapter proteins such as Fas-associated protein with death domain and pro-caspase-8 to form a death-inducing signalling complex, which causes the autoproteolytic activation and release of caspases [3,4]. Activation of caspase 8 induces the activation of caspases-3 and -7 and activates the Bid protein, which causes mitochondrial cytochrome c release and caspase-9 activation, leading to activation of caspase-3 and apoptosis [5].

YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] is a small molecule that functions as a guanylate cyclase activator by direct binding with the enzyme. YC-1 is used as an anticancer drug, likely due to its inhibitory effect against hypoxia-inducible factor, which is involved in tumour growth, vascularization and metastasis [6–8]. Therefore, YC-1 is considered to be an important lead drug for cancer treatment. YC-1 exhibits antiproliferative effects against various cancer cell lines by inducing cell cycle arrest, apoptosis, anti-angiogenesis and inhibition of matrix metalloproteinases [6,8–10]. Additionally, it has been shown that YC-1 inhibits the growth of human hepatocellular carcinoma cells without significant cytotoxicity [11]. It enhances the chemosensitivity of cancer cells to cisplatin or carboplatin via changes in apoptosis-related protein levels, leading to the activation of caspases [12,13]. Further, YC-1 increases hypoxia-induced cell cycle arrest and death in Hep3B hepatoma cells, Caki-1 renal carcinoma cells and pancreatic cancer cells [9,14]. However, YC-1 prevents oxidized low-density lipoprotein-induced apoptosis in vascular smooth muscle cells [15] and has been shown to inhibit oxygen/glucose deprivation–induced axonal damage [16].

Combined treatments of anticancer drugs may exert synergistic toxicity against tumour cells with less damaging effects on normal cells [17]. TRAIL has been shown to induce apoptosis in transformed or tumour cells but not in normal cells, and YC-1 may enhance the sensitivity of cancer cells to anticancer drugs. However, the combined effects of TRAIL and YC-1 on the viability of epithelial ovarian cancer cells have not been determined. To assess the apoptosis-promoting effect of YC-1, we investigated the effect of YC-1 on TRAIL-induced apoptosis using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials.  The TiterTACSTM colorimetric apoptosis detection kit was purchased from Trevigen, Inc. (Gaithersburg, MD, USA), and the Quantikine® M human cytochrome c and caspase (-8, -9 and -3) assay kits were purchased from R&D systems (Minneapolis, MN, USA). Antibodies [anti-Bid (5C9), anti-cleaved Bid (h71), anti-Bax (6A7), anti-Bcl-2 (10C4), anti-Bcl-xL (H-5), anti-cytochrome c (A-8), anti-p53 (DO-1) and anti-β-actin] were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TRAIL (Apo2L; human recombinant), 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), horseradish peroxidase-conjugated anti-mouse IgG, z-Asp-(OMe)-Gln-Met-Asp(OMe) fluoromethyl ketone (z-DQMD.fmk) and z-Ile-Glu-(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (z-IETD.fmk) were all purchased from EMD-Calbiochem (La Jolla, CA, USA). SuperSignal® West Pico chemiluminescence substrate for cytochrome c detection in western blotting was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), z-Leu-Glu-(O-ME)-His-Asp(O-Me) fluoromethyl ketone (z-LEHD.fmk), 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] and other chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).

Cell culture.  NIH-OVCAR-3 and SK-OV-3 cell lines (origin: human ovary; cellular morphology: epithelial; histopathology: adenocarcinoma) were obtained from the Korean cell line bank (Seoul, South Korea) and cultured in RPMI medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 units/ml of penicillin and 100 μg/ml of streptomycin, according to directions provided by the cell bank. Cells were washed with RPMI medium containing 1% FBS 24 hr before the experiments and were seeded in 96- and 24-well plates.

Cell viability assay with MTT reduction.  Cell viability was measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases [18]. In this study, the treatment time of TRAIL to cancer cells was based on previous reports [19,20]. Cells (3 × 104) were treated with TRAIL for 24 hr at 37°C. The medium (200 μl) was then incubated with 10 μl of 10 mg/ml MTT solution for 2 hr at 37°C. After centrifugation at 412 × g for 10 min., the culture medium was removed and 100 μl of dimethyl sulphoxide was added to each well to dissolve the formazan. The absorbance was measured at 570 nm using a microplate reader (Spectra MAX 340; Molecular Devices Co., Sunnyvale, CA, USA). Cell viability is expressed as a percentage of the absorbance value determined for the control cultures.

Cell viability assay with neutral red uptake.  Cell viability was determined using the neutral red uptake assay, which is based on the observation that neutral red is accumulated in the lysosomes of live cells [21]. OVCAR-3 cells (2 × 104) were treated with TRAIL for 24 hr at 37°C. The cell suspension (200 μl) was then incubated with 10 μl of 1 mg/ml neutral red solution for 3 hr at 37°C. After centrifugation at 412 × g for 10 min., the culture medium was removed and the dye was extracted with 100 μl of a 1% acetic acid and 50% ethanol solution for 20 min. The absorbance was measured at 540 nm using a microplate reader.

Observation of changes in nuclear morphology.  To clearly define the promoting effect of YC-1 on TRAIL-induced nuclear damage, we investigated the effects at a 16-hr incubation. OVCAR-3 cells (1 × 106 cells/ml) were treated with TRAIL for 16 hr at 37°C, and the changes in nuclear morphology were assessed using Hoechst dye 33258 [22]. Cells were incubated with 1 μg/ml Hoechst 33258 for 3 min. at room temperature, and nuclei were visualized using an Olympus microscope with a WU excitation filter (Tokyo, Japan).

Measurement of oligonucleosomal DNA fragmentation.  To clearly define the promoting effect of YC-1 on TRAIL-induced DNA fragmentation, we investigated the effects at a 16-hr incubation. DNA fragmentation because of the activation of endonucleases was assessed by gel electrophoresis. OVCAR-3 cells (4 × 106 cells/ml) were treated with TRAIL for 16 hr at 37°C and then washed with phosphate-buffered saline (PBS). DNA was isolated with the DNA purification kit, according to the manufacturer’s directions (Wizard® Genomic; Promega Co., Madison, WI, USA). DNA pellets were loaded in a 1.5% agarose gel in Tris-acetate buffer (pH 8.0) and 1 mM EDTA and were separated at 100 V for 2 hr. DNA fragments were stained with ethidium bromide and then visualized using a UV transilluminator.

Quantitative analysis of DNA fragmentation.  DNA fragmentation during apoptosis was assessed using a solid-phase enzyme-linked immunosorbent assay (ELISA). OVCAR-3 cells (3 × 104) were treated with TRAIL for 16 hr at 37°C, washed with PBS and fixed with formaldehyde solution. Deoxynucleotides (dNTPs) were incorporated at the 3′-ends of DNA fragments using terminal deoxynucleotidyl transferase, and the nucleotides were detected using streptavidin-horseradish peroxidase and TACS-Sapphire according to the TiterTACS protocol. Data are expressed as the absorbance at 450 nm.

Western blot analysis.  It has been shown that cells start to show signs of cell death after 6-hr treatment of TRAIL [19]. From this report, to assess the promoting effect of YC-1 on TRAIL-induced apoptosis-related protein activation as an early phenomenon, we investigated the effects at a 6-hr incubation. Cell lines (5 × 106 cells/ml) were harvested by centrifugation at 412 × g for 10 min., washed twice with PBS and suspended in lysis buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin and 20 mM HEPES–KOH, pH 7.5). The lysates were homogenized further by successive passages through a 26-gauge hypodermic needle. The homogenates were centrifuged at 100,000 × g for 5–30 min. depending on the protein that was being detected, and the supernatant was used for western blotting and ELISA. The protein concentration was determined by the Bradford method, according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA).

For western blotting, supernatants were mixed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and boiled for 5 min. Samples (30 μg protein/well) were loaded into each lane of a 12% SDS–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (GE Healthcare Chalfont St. Giles, Buckinghamshire, UK). Membranes were blocked for 2 hr in TBS (50 mM Tris–HCl, pH 7.5 and 150 mM NaCl) containing 0.1% Tween 20 and 5% non-fat dried milk. The membranes were incubated with antibodies (Bid, cleaved Bid, Bax, Bcl-2, cytochrome c, p53 and β-actin) overnight at 4°C with gentle agitation. After four washes in TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG for 2 hr at room temperature. The membranes were then incubated with SuperSignal® West Pico chemiluminescence substrate, and the proteins were detected using enhanced chemiluminescence in a Luminescent image analyzer (Lite for Las-1000 plus version 1.1; Fuji Photo Film Co., Tokyo, Japan).

Measurement of cytochrome c level and caspase activity.  For the solid-phase ELISA detection of cytochrome c, cells (5 × 105 cells/ml) were suspended in lysis buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 20 mM HEPES–KOH, pH 7.5) to harvest whole-cell lysates. The supernatants and cytochrome c conjugate were added to 96-well microplates coated with monoclonal antibody specific for human cytochrome c. The procedure was performed according to the manufacturer’s instructions (R&D systems). The absorbance of samples was measured at 450 nm in a microplate reader. A standard curve was constructed by plotting the absorbance values of dilutions of a cytochrome c standard. The amount is expressed in ng/ml.

For quantitative analysis of caspase (-8, -9 and -3) activity, cells (2 × 106 cells/ml) were treated with TRAIL for 6 hr at 37°C, and activities of caspases were determined using the caspase assay kit according to the manufacturer’s directions (R&D systems, Minneapolis, MN, USA). The supernatant obtained after centrifugation of the lysed cells was added to the reaction mixture containing dithiothreitol and caspase substrates (for -8, -9 and -3), and the mixture was incubated for 1 hr at 37°C. The absorbance of the chromophore developed was measured at 405 nm. Data are expressed as arbitrary units.

Flow-cytometric measurement of mitochondrial transmembrane potential.  To clearly define the promoting effect of YC-1 on TRAIL-induced mitochondrial permeability change, we investigated the effects at a 16-hr incubation. Changes in the mitochondrial transmembrane potential during the TRAIL-induced apoptosis in OVCAR-3 cells were quantified by flow cytometry with the cationic lipophilic dye DiOC6(3) [23]. Cells (1 × 106 cells/ml) were treated with TRAIL for 16 hr at 37°C, and then DiOC6(3) (40 nM) was added to the medium and cells were incubated for 15 min. at 37°C. After centrifugation at 412 × g for 10 min., the supernatants were removed and the pellets were suspended in PBS containing 0.5 mM EDTA. For analysis, a FACScan cytofluorometer (BD Biosciences, San Jose, CA, USA) with argon laser excitation at 501 nm was used to assess 10,000 cells from each sample.

Statistical analysis.  Data are expressed as the mean ± S.D. Statistical analysis was performed by one-way analysis of variance. When significance was detected, Duncan’s test for multiple comparisons was performed on the data obtained for experimental groups. A probability value of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

YC-1 enhances TRAIL-induced cell death and DNA damage.

We aimed to examine the combined effect of TRAIL and YC-1 against ovarian cancer cells using the human epithelial ovarian carcinoma cell line NIH-OVCAR-3. Firstly, we examined the effect of TRAIL on cell viability. When cells were treated with 5–100 ng/ml TRAIL for 24 hr, cell viability decreased in a concentration-dependent manner (fig. 1A). The incidence of cell death in OVCAR-3 cells after the treatment with 50 ng/ml TRAIL for 24 hr was approximately 48%.

image

Figure 1.  Effect of YC-1 on TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death. In A and B, OVCAR-3 cells were treated with 5–100 ng/ml TRAIL in the presence or absence of 1–75 μM YC-1 for 24 hr, and then cell viability was determined using a MTT reduction assay. In C and D, SK-OV-3 cells were treated with 5–100 ng/ml TRAIL in the presence or absence of 1–75 μM YC-1 for 24 hr. Data represent the mean ± S.D. (n = 6). +< 0.05 compared with control (percentage of control); *p < 0.05 compared with TRAIL alone.

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Secondly, we examined the effect of YC-1 on cell viability in OVCAR-3 cells. When cells were treated with YC-1 for 24 hr, cell viability decreased in a concentration-dependent manner. The incidence of cell death in both cell lines after treatment with 50 μM YC-1 for 24 hr was approximately 18%. We then examined the effect of YC-1 on the TRAIL-induced cell death. Treatment with 5–25 μM YC-1 significantly enhanced TRAIL (50 ng/ml)-induced cell death (fig. 1B). The combined effect of TRAIL and YC-1 on cell death was much greater than the sum of each independent drug effect. For instance, while the incidences of cell death caused by 10 and 50 μM YC-1 were approximately 3% and 18%, respectively, the addition of 10 and 50 μM YC-1 increased TRAIL-induced cell death (48%) to approximately 60% and 82%, respectively.

To confirm the promoting effect of YC-1 on apoptosis, we further investigated whether YC-1 promoted TRAIL-induced cell death using another ovarian cancer cell line SK-OV-3 (fig. 1C,D). TRAIL and YC-1 reduced viability in SK-OV-3 cells, similar to the results observed in OVCAR-3 cells. Treatment with YC-1 significantly enhanced TRAIL (50 ng/ml)-induced cell death in SK-OV-3 cells (fig. 1D). While the incidence of cell death caused by 50 μM YC-1 was approximately 20%, the addition of 50 μM YC-1 increased TRAIL-induced cell death (45%) to approximately 86%.

Using the neutral red uptake viability assay, we further confirmed the enhanced cell death caused by YC-1. Combination of 10–75 μM YC-1 enhanced a TRAIL-induced cell death in OVCAR-3 and SK-OV-3 cells, and the promoting effect of YC-1 was similar in both cell lines. The combined effect of TRAIL and YC-1 on cell death was much greater than the sum of each independent effect of the two compounds (fig. 2).

image

Figure 2.  YC-1 enhances TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death. OVCAR-3 (A) and SK-OV-3 (B) cells were treated with 50 ng/ml TRAIL in the presence of 10–75 μM YC-1 for 24 hr, and then cell viability was determined using the neutral red uptake assay. Data represent the mean ± S.D. (n = 6). +p < 0.05 compared with control (percentage of control); *p < 0.05 compared with TRAIL alone.

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To clearly define the stimulatory effect of YC-1 on TRAIL-induced apoptosis, we examined the effects at fixed concentrations (50 ng/ml TRAIL and 50 μM YC-1). To assess nuclear damage caused by TRAIL and YC-1, we investigated the changes in nuclear morphology in OVCAR-3 cells. Nuclear staining with Hoechst 33258 revealed that control cells had regular and round-shaped nuclei. In contrast, condensation and fragmentation of nuclei, which is characteristic of apoptotic cells, were observed in cells simultaneously treated with TRAIL and YC-1 (fig. 3A).

image

Figure 3.  Effect of YC-1 on TNF-related apoptosis-inducing ligand (TRAIL)-induced nuclear damage. OVCAR-3 cells were pre-treated with 50 ng/ml TRAIL for 20 min. and then exposed to 50 μM YC-1 for 16 hr. In (A), cells were observed by fluorescence microscopy after nuclear staining with Hoechst 33258. The figure shows the microscopic morphology of control cells (a) and cells treated with TRAIL and YC-1 (b). a and b are representative of four different experiments. In (B), DNA was extracted, separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, untreated cells; lane 2, cells treated with TRAIL; lane 3, cells treated with YC-1; and lane 4, cells treated with TRAIL and YC-1. Data are representative of three independent experiments. In (C), the 3′-ends of DNA fragments were detected as described in the Materials and Methods section. Data represent the mean ± S.D. (n = 5). +p < 0.05 compared with control; *p < 0.05 compared with TRAIL alone.

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During apoptosis, DNA fragmentation is caused by the activation of endonucleases. The effect of YC-1 on TRAIL-induced DNA fragmentation, indicative of nuclear damage, was assessed by agarose gel electrophoresis. DNA extracted from OVCAR-3 cells showed a small increase in the oligonucleosomal cleavage of DNA (lane 1 in fig. 3B). In contrast, incubation with TRAIL for 16 hr increased the DNA laddering in cancer cells (lane 2 in fig. 3B). Treatment with YC-1 increased the TRAIL-induced DNA laddering, which was somewhat greater than with TRAIL alone (lane 4 in fig. 3B). YC-1 alone caused a small increase in the oligonucleosomal cleavage of DNA but was similar to the control (lane 3 in fig. 3B).

We further assessed the damaging effect of TRAIL and YC-1 on the nucleus by performing quantitative analysis of DNA fragmentation. The amount of fragmented DNA was determined by monitoring the binding of dNTP to the 3′-ends of DNA fragments, and the results were measured using a quantitative colorimetric assay. Control OVCAR-3 cells demonstrated an absorbance of 0.064 ± 0.005 (mean ± S.D., n = 5), whereas exposure to TRAIL alone for 16 hr increased the absorbance approximately 2.8 times. YC-1 markedly increased the TRAIL-induced DNA fragmentation to a greater extent than the sum of each independent effect of the two compounds (fig. 3C). Combination treatment increased the DNA fragmentation by 4.1 times compared with the control. YC-1 alone caused approximately 45% of the increase in the DNA fragmentation compared with the control.

YC-1 enhances TRAIL-induced activation of apoptosis-related proteins.

We examined whether the combined effect of TRAIL and YC-1 was mediated by caspase activation using specific caspase inhibitors. As shown in fig. 4, treatment with 30 μM z-IETD.fmk, z-LEHD.fmk or z-DQMD.fmk (cell-permeable inhibitors of caspase-8, -9 or -3, respectively) reduced the incidence of cell death induced by treatment with either TRAIL alone or TRAIL plus 10 μM YC-1. Relative to concentration, the preventive effect of the caspase-8 inhibitor on cell death induced by TRAIL (or in combination with YC-1) was greater than the effect of caspase-9 or -3 inhibitors. Caspase inhibitors alone did not affect cell viability.

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Figure 4.  Effect of caspase inhibitors on TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death. OVCAR-3 cells were pre-treated with 30 μM caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and then exposed to 50 ng/ml TRAIL in the presence (B) or absence (A) of 10 μM YC-1 for 24 hr, and cell viability was determined. Data represent the mean ± S.D. (n = 6). +p < 0.05 compared with control; *p < 0.05 compared with TRAIL alone. TRAIL plus YC-1 is abbreviated as TYC.

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We assessed cell death caused by TRAIL and YC-1 by measuring the amount of apoptosis-related proteins in ovarian carcinoma cell lines. Treatment with 50 ng/ml TRAIL decreased cytosolic Bid, Bcl-2 and Bcl-xL levels, but increased cytosolic cleaved Bid, Bax and cytochrome c levels in OVCAR-3 cells (fig. 5A). Treatment with YC-1 further increased TRAIL-induced alteration in Bid, cleaved Bid, Bax, Bcl-2, Bcl-xL and cytochrome c levels. The changes in the apoptosis-related protein levels in response to the combined treatment were greater than those induced by TRAIL alone (fig. 5A). YC-1 alone exhibited little effect on the levels of the apoptosis-related proteins.

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Figure 5.  Effect of YC-1 on TNF-related apoptosis-inducing ligand (TRAIL)-induced changes in apoptosis-related protein levels. In (A), OVCAR-3 cells were treated with 50 ng/ml TRAIL and 50 μM YC-1 for 6 hr. The levels of Bid, cleaved Bid, Bax, Bcl-2, Bcl-xL, cytochrome c and p53 were analysed by western blotting with their specific antibodies. Data are representative of three independent experiments. In (B), OVCAR-3 or SK-OV-3 cells were treated with 50 ng/ml TRAIL and 50 μM YC-1 for 6 hr, and then the amount of released cytochrome c was measured by ELISA. Data are expressed in ng/ml and represent the mean ± S.D. (n = 6). +p < 0.05 compared with control; *p < 0.05 compared with TRAIL alone.

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The tumour suppressor p53 plays a critical role in the induction of apoptosis in cells exposed to anticancer drugs [24]. We examined whether the combined effect of TRAIL and YC-1 was mediated by changes in p53 expression. TRAIL induced an increase in p53 levels in OVCAR-3 cells (fig. 5A). Addition of YC-1 further increased p53 levels. The increase in p53 levels in response to the combined treatment of TRAIL and YC-1 was greater than that caused by TRAIL alone.

We confirmed the effect of YC-1 on TRAIL-induced cytochrome c release by performing ELISA-based quantitative analysis. Treatment with TRAIL induced the release of cytochrome c in OVCAR-3 and SK-OV-3 cells (fig. 5B). The amounts of released cytochrome c induced by the combined treatment of TRAIL and YC-1 in both cell lines were greater than the sum of each independent drug effect.

Next, the change in the activity of apoptotic caspases in ovarian carcinoma cell lines exposed to TRAIL was analysed. Treatment with TRAIL increased the activities of caspase-8, -9 and -3 in OVCAR-3 and SK-OV-3 cells (fig. 6). YC-1 enhanced TRAIL-induced caspase activation in both cell lines. YC-1 at these concentrations alone had little effect on the activation of caspases. The combined effect of TRAIL and YC-1 was greater than the sum of each independent drug effect.

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Figure 6.  Effect of YC-1 on TNF-related apoptosis-inducing ligand (TRAIL)-induced activation of caspases. OVCAR-3 or SK-OV-3 cells were treated with 50 ng/ml TRAIL and 50 μM YC-1 for 6 hr, and activities of caspase-8, -9 and -3 were measured with an analysis kit. Data are expressed as arbitrary units (a.u.) and represent the mean ± S.D. (n = 6). +p < 0.05 compared with control; *p < 0.05 compared with TRAIL alone.

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Opening of the mitochondrial permeability transition pore causes the release of cytochrome c from mitochondria into the cytosol and subsequent activation of caspases [2]. Changes in the mitochondrial transmembrane potential in OVCAR-3 cells treated with TRAIL were quantified by flow cytometry with the dye DiOC6(3). Treatment with TRAIL for 16 hr increased the percentage of cells with depolarized mitochondria (characterized by low values of the transmembrane potential) in OVCAR-3 cells. The effect of TRAIL was inhibited by the addition of 0.5 μM cyclosporin A, the mitochondrial membrane permeability transition inhibitor. YC-1 enhanced TRAIL-induced loss of mitochondrial membrane potential. YC-1 at this concentration alone had little effect on the mitochondrial membrane potential (fig. 7). When subtracted the control value, data showed that the combined effect of TRAIL and YC-1 was greater than the sum of each independent drug effect.

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Figure 7.  Effect of YC-1 on TNF-related apoptosis-inducing ligand (TRAIL)-induced loss of the mitochondrial transmembrane potential. OVCAR-3 cells were treated with 50 ng/ml TRAIL in the presence or absence of compounds [50 μM YC-1 or 0.5 μM cyclosporin A (CsA)] for 16 hr. Data are expressed as the percentage of cells with depolarized mitochondria for the mitochondrial membrane potential and represent the mean ± S.D. (n = 4). +p < 0.05 compared with control; *p < 0.05 compared with TRAIL alone and #p < 0.05 compared with TRAIL plus YC-1 (abbreviated as TYC).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

TRAIL has been shown to induce apoptosis in various cancer cells by activating cell surface death receptors and by the mitochondrial-mediated pathway [3,4,25]. In this study, we investigated the induction of apoptosis by TRAIL on cancer cells using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced nuclear damage, decreased Bid, Bcl-2 and Bcl-xL protein levels, increased cleaved Bid and Bax levels, induced cytochrome c release and activated caspase-8, -9 and -3 in both cell lines. TRAIL-induced apoptosis in OVCAR-3 cells was demonstrated by the condensation and fragmentation of nuclei and an increase in both the release of cytochrome c and caspase-3 activation. Caspase-9 induces caspase-3 activation through formation of an apoptosome complex with the released cytochrome c from mitochondria [1,26]. Caspase-8 activation followed by the activation of cell surface death receptor increases the mitochondrial membrane permeability through the cleavage and activation of the apoptosis initiator Bid and directly activates caspase-3 [5]. Cleavage of p22 Bid to the p15 form during apoptosis is involved in mitochondria-mediated cell death [27,28]. Cleaved Bid translocates to mitochondria and induces the release of mitochondrial cytochrome c, the activation of caspase-3 and the fragmentation of DNA [27]. Anticancer drugs induce Bax activation, leading to the release of cytochrome c and apoptosis [29]. The inhibitory effect of specific caspase inhibitors and the activation of caspases further suggest that TRAIL induces apoptosis in OVCAR-3 cells by activating the caspase-8- and Bid-dependent pathways as well as the mitochondria-mediated cell death pathway, which leads to the activation of caspase-9 and -3.

Formation of the mitochondrial membrane permeability transition induces mitochondrial membrane potential loss and cytochrome c release, leading to caspase activation [30,31]. The inhibitory effect of cyclosporin A suggests that TRAIL induces apoptosis in OVCAR-3 cells by causing change in the mitochondrial membrane permeability that leads to cytochrome c release and subsequent activation of caspase-3. The inhibitory effect of specific caspase inhibitors further suggests that TRAIL induces apoptosis in the ovarian cancer cell lines OVCAR and SK-OV-3 via the mitochondria-mediated cell death pathway, leading to caspase-3 activation.

It has been shown that YC-1, a guanylate cyclase activator, suppresses cell growth and induces apoptosis in human prostate adenocarcinoma PC-3 and human lung cancer NCI-H226 cells [32,33]. In contrast, YC-1 inhibited cell growth in the human hepatocellular carcinoma cell lines HA22T and Hep3B without significant cytotoxicity [11]. Furthermore, YC-1 may prevent the oxidized low-density lipoprotein-induced or oxygen/glucose deprivation–induced apoptosis [15,16]. In the present study, YC-1 at higher concentrations may cause significant cell death in epithelial ovarian carcinoma cell lines through cell death receptor activation and the mitochondrial pathway.

As previously described, treatment with a combination of anticancer drugs may exhibit a synergistic toxicity to tumour cells with less damaging effects on normal cells. We assessed the effect of the YC-1 concentration that showed little cytotoxicity in the case of TRAIL-induced cell death. To clarify the combined effect of these drugs on the levels of apoptosis-related proteins, we examined the effect of YC-1 at a fixed concentration (50 ng/ml) with TRAIL. The present study showed that YC-1 enhanced the TRAIL-induced decrease in cytosolic Bid levels, decrease in cytosolic Bcl-2 levels, increase in cytosolic cleaved Bid and Bax levels and loss of the mitochondrial membrane potential, leading to the mitochondrial cytochrome c release and activation of caspase-8, -9 and -3. The results suggest that YC-1 may enhance TRAIL-induced apoptosis in OVCAR-3 cells by activating the caspase-8- and Bid-dependent pathways as well as the mitochondria-mediated cell death pathway, leading to the activation of caspase-9 and -3.

The tumour suppressor and transcription factor p53 modulates cellular stress responses, and activation of p53 can trigger apoptosis [24,34]. p53 stimulates either mitochondria-mediated cell death or the death receptor pathway and mediates apoptosis induced by various insults, including DNA damage and oxidative stress [33–35]. p53 acts as a direct transcriptional activator of the Bax gene. We examined whether the combination of TRAIL and YC-1-induced cell death was mediated by p53 activation. In this study, YC-1 further enhanced TRAIL-induced increases in p53 levels in OVCAR-3 cells. Therefore, YC-1 may enhance the apoptotic effect of TRAIL on ovarian carcinoma cell lines by potentiating the increase in p53 levels.

Overall, the results show that YC-1 may enhance the apoptotic effect of TRAIL in human epithelial ovarian carcinoma cell lines by increasing the activation of caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway, leading to the activation of caspase-9 and -3. YC-1 may confer a benefit in TRAIL treatment of epithelial ovarian adenocarcinoma.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A085138).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References