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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2008 Jan 17;134(7):803–812. doi: 10.1007/s00432-007-0349-z

Induction of apoptosis in PA-1 ovarian cancer cells by vitamin K2 is associated with an increase in the level of TR3/Nur77 and its accumulation in mitochondria and nuclei

Toshiko Sibayama-Imazu 1, Yukari Fujisawa 1, Yutaka Masuda 2, Toshihiro Aiuchi 3, Shigeo Nakajo 1, Hiroyuki Itabe 1,✉, Kazuyasu Nakaya 1
PMCID: PMC12160726  PMID: 18202854

Abstract

Purpose

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K2 (VK2; menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK2.

Methods

Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment.

Results and conclusions

We found that PA-1 cells were the most sensitive to VK2 (IC50 = 5.0 ± 0.7 μM), while SK-OV-3 cells were resistant to VK2. Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 μM VK2. In the VK2-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK2-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK2 and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

Keywords: Apoptosis, Vitamin K2, TR3, JNK, Ovarian cancer cells

Introduction

Vitamin K2 (VK2) induces growth inhibition (Nishikawa et al. 1995; Wang et al. 1995; Hitomi et al. 2005), differentiation (Sakai et al. 1994; Yaguchi et al. 1997; Miyazawa et al. 2001) and apoptosis in various lines of cancer cells (Yoshida et al. 2003; Iwamoto et al. 2004). We reported previously that VK2 induces apoptosis in human ovarian cancer cells (TYK-nu) and pancreatic cancer cells (MIA-Paca-2) (Shibayama-Imazu et al. 2003) and also induces the production of superoxide, with dissipation of the mitochondrial membrane potential (Shibayama-Imazu et al. 2006). Apoptosis induced by VK2 is almost completely inhibited by cycloheximide, which blocks protein synthesis (Shibayama-Imazu et al. 2003). However, the proteins involved in the induction of apoptosis by VK2 remain to be identified.

TR3, also known as Nur77 and neuron growth factor inducible factor I-B (NGFI-B), was originally isolated as the product of an immediate-early gene that is rapidly expressed in response to stimulation by serum or phorbol ester of quiescent fibroblasts (Hazel et al. 1988; Ryseck et al. 1989; Nakai et al. 1990; Herschman 1991). TR3 is also known as a transcription factor that modulates the expression of genes that are linked to the regulation of cell proliferation and apoptosis (John et al. 1994; Weih et al. 1996; Wu et al. 2002). TR3 is strongly expressed in many different lines of cancer cells (Wu et al. 1997; Uemura and Chang 1998), and the constitutive expression of TR3 results in massive cell death (Weih et al. 1996; Xue et al. 1997). These observations suggest that the level of TR3 might be an important factor with respect to the sensitivity of cancer cells to the induction of apoptosis.

Recently, the translocation of TR3 from nuclei to mitochondria was observed upon exposure of various cancer cells to apoptotic stimuli (Li et al. 2000; Wu et al. 2002; Wilson et al. 2003; Lin et al. 2004b), with subsequent depolarization of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol (Li et al. 2000). In the present study, we compared the sensitivity to VK2 of five lines of human ovarian cancer cells. We found that PA-1 cells were the most sensitive to VK2 with an IC50 value of 5.0 ± 0.7 μM, while VK2 had almost no effect on SK-OV-3 cells. Using these two cell lines, we investigated the mechanism by which VK2 induces apoptosis. We found marked increases in levels of TR3 in lysates of PA-1 cells during VK2-induced apoptosis, whereas there was practically no change in the level of TR3 in SK-OV-3 cells under the same conditions. Furthermore, both the VK2-induced increase in levels of TR3 and induction of apoptosis in PA-1 cells were inhibited by addition of cycloheximide to the culture medium and by transfection of cells with small interfering RNA (siRNA) directed against TR3, indicating that the increased synthesis of TR3 might be associated with the induction of apoptosis by VK2. Furthermore, cellular localization studies indicated that TR3 accumulated in mitochondria and in nuclei in PA-1 cells during treatment with VK2. The results obtained in the present study suggest that the VK2-induced accumulation of TR3 in both mitochondria and nuclei might be associated with the induction of apoptosis in PA-1 ovarian cancer cells.

Materials and methods

Reagents

Vitamin K2 (VK2; menaquinone 4) was provided by Eisai Chemical Co. Ltd (Ibaraki, Japan). Sodium-3'-[1-[(phenylamino)carbonyl]-3,4-tetrazo-lium-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), phenazine methosulfate (PMS), cycloheximide (CHX), Hoechst 33342, a cocktail of protease inhibitors, bovine insulin, RPMI 1640 medium, Eagle’s minimum essential medium (MEM) and leptomycin B were obtained from Sigma-Aldrich Co. Ltd (St Louis, MO). Macoy’s 5A medium (modified), fetal bovine serum (FBS) and MEM nonessential amino acids were purchased from Gibco Co. Ltd (Glasgow, UK). SB203580, PD169316 and SP600125 were purchased from Calbiochem (a brand of EMD Biosciences Inc., La Jolla, CA). A Cell-Death Detection ELISAPLUS kit for the detection of released nucleosomes was purchased from Roche Diagnostics GmbH (Mannheim, Germany).

Antibodies

Antibodies against Nur77 (TR3; SC-5569), lamin A/C, was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal antibodies against cytochrome oxidase subunit IV (OX) (A-21347) were obtained from Molecular Probes Inc. (Eugene, OR). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Chemicon International Co. Ltd (Temecula, CA). Monoclonal antibodies against cytochrome c (6H2.B4) were purchased from BD Pharmingen Co. Ltd (San Diego, CA).

Cell lines and cell culture

All lines of cells originated from human ovarian cancer tissues. Both PA-1 cells and TYK-nu cells were provided by the Japan Health Sciences Foundation (Tokyo, Japan). PA-1 cells were cultured in MEM that contained 10% FBS and 1% MEM nonessential amino acids. TYK-nu cells were maintained in MEM with 10% FBS. SK-OV-3 cells, SW626 cells and OVCAR3 cells were purchased from the American Type Culture Collection (Manassas, VA). SK-OV-3 cells were cultured in Macoy’s 5A medium (modified) supplemented with 10% FBS. SW626 cells were cultured in RPMI 1640 supplemented with 10% FBS. OVCAR3 cells were cultured in RPMI 1640 supplemented with 20% FBS and bovine insulin (1.25 μg/ml; Sigma-Aldrich Co.). All cells were cultured in an atmosphere of 5% CO2 in air at 37°C. Confluent cells were detached from the substratum by treatment with 0.25% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS). Cells were transferred to fresh medium at a concentration of 5 × 104 cells/ml every 3 or 4 days to maintain the logarithmic growth of cells. For assays, cells (5 × 104 cells/ml) were transferred to 10-ml dishes or 24-well plates and incubated overnight at 37°C in fresh medium. Then VK2, dissolved in ethanol, was added (or ethanol was added alone, as a control) to the culture medium.

Quantification of cell proliferation

Cell proliferation was quantified by measuring absorbance at 492 nm after exposure of cells to XTT, which is metabolized by mitochondrial dehydrogenase to yield a formazan dye (Roehm et al. 1991), as described in a previous paper (Shibayama-Imazu et al. 2003). In brief, cells were transferred to 96-well plates at 5 × 103 cells/well and then treated with VK2 at concentrations from 0.39 to 100 μM for 96 h, as indicated. Absorbance was measured after a further 4-h incubation at 37°C with a solution of XTT (0.33 mg/ml) that contained 12.5 μM PMS. Cell growth (as a percentage) was calculated from the absorbance at 492 nm of treated cells relative to the absorbance at 492 nm of nontreated cells.

Analysis of the induction of apoptosis with a cell-death detection kit and Hoechst staining

Cells (1 × 105 cells/ml) were treated with VK2 in 24-well plates. Both the culture medium and cells were collected after brief centrifugation at 200×g for 10 min, and pelleted cells were lysed in lysis buffer supplied with the Cell-Death Detection ELISAPLUS kit. After brief centrifugation at 200×g for 10 min, the supernatant was incubated with histone-specific and DNA-specific antibodies, and released nucleosomes were quantified as described in the instruction manual supplied with the kit. Apoptotic cells were assessed by an examination of nuclear morphology after staining with Hoechst 33342. Cells were stained with 10 μM Hoechst 33342 for 15 min on ice and were examined under a fluorescence microscope.

Preparation of cell lysates and immunoblotting

Cells were collected by centrifugation and washed twice with ice-cold PBS. Then cell lysates were prepared as follows. Cells were suspended in a solution of 50 mM HEPES (pH 7.4), 0.2% Triton X-100, 1 mM EGTA, 1 mM Na2VO4, 100 mM NaCl, 10 mM NaF, 1 mM phenylmethylsulfonylfluoride (PMSF) and a cocktail of protease inhibitors (1/100 volume) and kept on ice for 30 min. The suspension was agitated briefly with a vortex mixer and centrifuged at 4°C for 15 min at 17,400×g. The concentration of protein in the supernatant was determined with a BCA kit (Pierce Co. Ltd, Rockford, IL) or a Bio-Rad protein assay kit (Bio-Rad Co. Ltd, Hercules, CA) with bovine serum albumin (BSA) as the standard, and then an aliquot was supplemented with 1/10 volume of 10× sample buffer [10% SDS, 10% β-mercaptoethanol, 10 mM EDTA, 10% sucrose and 30 mM Tris–HCl (pH 6.8)] for SDS-PAGE, boiled for 5 min and then subjected to SDS-PAGE on a 10% polyacrylamide gel (Laemmli 1970). Bands of proteins were transferred electrophoretically to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH) in a transfer buffer that contained 192 mM glycine, 20 mM Tris–HCl (pH 8.2) and 20% methanol, and then the membrane was blocked in a blocking solution of 5% nonfat dry milk in TBS [20 mM Tris–HCl (pH 8.0), 0.85% NaCl] for 30 min. Then the membrane was incubated for 18 h at 4°C with monoclonal or polyclonal antibodies (1:1,000; diluted in blocking solution) and washed three times in TBS that contained 0.05% Tween-20. The membrane was incubated for 1 h with horseradish peroxidase-conjugated goat antibodies against mouse IgG or rabbit IgG (Cell Signaling Technology, Inc., Beverly, MA; 1:3,000; diluted in blocking solution). Then it was washed three times with 0.05% Tween 20 in TBS and finally incubated in Western LightningTM Chemiluminescence Reagent (PerkinElmer Life Science Products, Boston, MA). Immune complexes were detected on O-Max X-ray film (Fuji Film Co. Ltd, Kanagawa, Japan).

Preparation of heavy membrane fraction, cytosol and nuclei

The procedure for isolation of the heavy membrane (HM) fraction, which contained mainly mitochondria, was performed as described by Lin et al. (2004a). In brief, cells (1 × 106 cells) were suspended in 0.5 ml of hypotonic buffer [5 mM Tris–HCl (pH 7.5), 5 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT and a cocktail of protease inhibitors (1/100 volume)] and homogenized 60 strokes with Dounce-homogeneizer on ice. The homogenate was centrifuged at 500×g for 5 min. The resulting supernatant was centrifuged at 10,000×g for 30 min at 4°C to obtain the HM fraction. The supernatant was further centrifuged at 100,000×g for 60 min, and the resulting supernatant was separated as the cytosol. For immunoblotting analysis, HM fraction was suspended in lysis buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 10 mM sodium pyrophosphate, 5 mM NaF, 2 mM Na2VO4, a cocktail of protease inhibitors (1/100 volume)]. After centrifugation of the mixture at 17,400×g for 15 min, the supernatant was supplemented with 1/10 volume of 10× sample buffer for SDS-PAGE and boiled for 5 min. Nuclei were obtained from the pellet after centrifugation of the homogenate at 500×g for 5 min. The pellet was suspended in nuclear isolation solution (0.2% Triton X-100 in hypotonic buffer), and passed several times through a 22G syringe and centrifuged at 500×g for 10 min. The pellet was dissolved in 2× sample buffer, boiled for 5 min and sonicated for 5 s to break the DNA. The concentration of protein was determined with the Protein Assay kit from Bio-Rad with BSA as the standard.

Immunofluorescence analysis

Cells were cultured overnight on glass coverslips (Matsunami, Tokyo, Japan) and then treated with reagents as indicated. The cells were fixed for 20 min in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), permeabilized with 2% Triton X-100 in PBS for 5 min, washed once with PBS and then incubated for 30 min at 37°C in blocking solution (3% normal goat serum and 1% BSA in PBS). For detection of TR3, cells were then incubated overnight at 4°C with TR3-specific rabbit antibodies. After three washes with PBS, cells were incubated for 1 h with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against rabbit IgG (Molecular Probes, Eugene, OR). Cells were washed three times with PBS and mounted. For detection of mitochondria, we used a mouse monoclonal antibody against cytochrome oxidase subunit IV (OX). As second antibody, we used Cy3-conjugated goat antibodies against mouse IgG (Rockland Inc., Gilbertsville, PA). Fluorescent images were collected and analyzed with a laser-scanning confocal microscope (TCS SP2; LEICA Microsystems Wetzler GmbH, Wetzler, Germany)

Treatment of PA-1 cells with siRNA

Small interfering RNA (siRNA) directed against TR3 (siRNA-TR3) was prepared by Qiagen Inc. (Chatsworth, CA). The sequences of the siRNA were as follows (numbers in parentheses indicate nucleotide positions within the open reading frame of the human gene for TR3): 5'-CGCUUCAUGCCAGCAUUAUd(TT)-3' (929–947) and 5'-AUAAUGCUGGCAUGAAGCGd(TT)-3'. Cells were treated with siRNA according to the instructions provided with the RNAiFectTM transfection reagent (Qiagen K. K., Tokyo, Japan) with slight modifications. In brief, PA-1 cells (5 × 104) were treated with 2.5 μg siRNA-TR3 in MEM medium supplemented with 10% FBS in the presence of the RNAiFectTM transfection reagent. After incubation for 24 h at 37°C, the medium was replaced by fresh MEM medium with 10% FBS, and cells were incubated for a further 24 h. Then, VK2 was added to culture medium and incubation was continued for 72 h. VK2-induced apoptosis was monitored by Hoechst staining after cells had been collected by centrifugation, as described above.

Results

Inhibition of growth of human ovarian cancer cells by vitamin K2

We examined the effects of VK2 on the growth of five lines of ovarian cancer cells. As shown in Fig. 1, VK2 inhibited most effectively the growth of PA-1 cells, whereas the growth of SK-OV-3 cells was practically unaffected. The growth of both OVCAR3 cells and SW626 cells was weakly inhibited. The growth of TYK-nu cells was inhibited by VK2 with an IC50 of 73.0 ± 4.5 μM, which is much higher than the value of IC50, 5.0 ± 0.7 μM, for PA-1 cells. Therefore, we used PA-1 cells and SK-OV-3 cells as VK2-sensitive and VK2-resistant lines, respectively.

Fig. 1.

Fig. 1

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Effects of vitamin K2 (VK2) on the growth of various lines of human ovarian cancer cells. Cell proliferation was determined by the XTT assay 96 h after the treatment of VK2, as defined and described in “Materials and methods”. Each value is the mean ± SD of the results from three independent experiments. Open square PA-1, filled circle SK-OV-3, open circle OVCAR3, filled square SW626, filled triangle TYK-nu

Induction of apoptosis in PA-1 cells

We monitored the release of fragmented nucleosomes into the cytosolic fraction with a Cell-Death Detection ELISAPLUS kit. The level of nucleosomes in the cytosolic fraction of PA-1 cells began to increase 24 h after the start of incubation with VK2 and continued to increase until at least 72 h after the start of incubation (Fig. 2a). After 72 h, induction of apoptosis was evident in 35% of PA-1 cells, as determined by counting apoptotic cells with condensed and fragmented nuclei after staining with Hoechst 33342 as described in “Materials and methods”. In a previous report (Shibayama-Imazu et al. 2003), we showed that treatment of TYK-nu cells with 150 μM VK2 for 96 h caused the release of cytochrome c. We examined the release of cytochrome c in VK2-treated PA-1 cells. As shown in Fig. 2b, we observed the release of cytochrome c 48 h after the start of incubation with VK2. After 72 h, the level of cytochrome c in the cytosol increased markedly, suggesting that apoptosis had been induced via the same pathway as previously reported for TYK-nu cells (Shibayama-Imazu et al. 2003).

Fig. 2.

Fig. 2

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Induction of apoptosis in PA-1 cells by vitamin K2 (VK2). PA-1 cells were treated with 30 μM VK2 for up to 72 h. a Apoptosis was monitored, in terms of the release of nucleosomes into the cytosolic fraction, as described in the text. White columns nontreated cells, gray columns VK2-treated cells. b Time-dependent release of cytochrome c in the cytosol prepared from PA-1 cells after treatment with VK2. Cells were treated with 30 μM VK2 and lysed as described in “Materials and methods”. Cytosol obtained from the cells at various time points were subjected to SDS-PAGE on a 15% polyacrylamide gel followed by transferring it to a nitrocellulose membrane. Cytochrome c was detected with specific antibody, as described in the text. Levels of GAPDH confirm the loading of equivalent amounts of protein in each lane

Significant increases in levels of TR3 in PA-1 cells after treatment with VK2

We compared the levels of TR3 in cell lysates of PA-1 cells and SK-OV-3 cells. To our surprise, the level of TR3 in PA-1 cells was four times higher than that in SK-OV-3 cells (compare bands of TR3 at 0 h in Fig. 3). The lysates of both lines of cells also included a small but significant amount of TR3 that generated a band that migrated more slowly than the bulk of TR3 during SDS-PAGE. The more slowly migrating band of TR3 was identified previously as phosphorylated TR3 (Katagiri et al. 2000; Pekarsky et al. 2001).

Fig. 3.

Fig. 3

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The level of TR3 increased gradually and time-dependently in PA-1 cells upon treatment with vitamin K2 (VK2). Cell lysates were prepared from PA-1 and SK-OV-3 cells 0, 24, 48 and 72 h after the start of treatment with 30 μM VK2. TR3 in the cell lysates (10 μg protein each) was detected by immunoblotting using rabbit TR3-specific polyclonal antibody as described in “Materials and methods”. Levels of GAPDH were confirmed by loading an equal amount of proteins in each lane

When PA-1 cells were treated with 30 μM VK2, the level of TR3 in PA-1 cells increased time-dependently. By contrast, the band of TR3 from SK-OV-3 cells was barely visible and the level of TR3 had increased only slightly 72 h after the start of treatment with VK2, and even then did not reach the level of TR3 in untreated PA-1 cells.

Effects of TR3 synthesis and VK2-induced apoptosis

Cycloheximide (CHX) blocks the synthesis of proteins, including those required for signal transduction. When PA-1 cells were treated with VK2 in the presence of 100 nM CHX, subsequent accumulation of TR3 by VK2 was completely inhibited and the level of TR3 was practically unchanged (Fig. 4a). Furthermore, the extent of the induction of apoptosis of PA-1 cells by VK2 was reduced to almost 50% of the control level in the presence of cycloheximide (Fig. 4b). These results suggest that the increase in the TR3 level in PA-1 cells by VK2 treatment is due to the increase in TR3 synthesis and not due to inhibition of its degradation and that TR3 synthesized during treatment with VK2 is required for the induction of apoptosis in PA-1 cells by VK2.

Fig. 4.

Fig. 4

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Effects of CHX on the synthesis of TR3 and induction of apoptosis upon treatment of PA-1 cells with vitamin K2 (VK2). a After PA-1 cells had been incubated under the conditions indicated for 72 h, cell lysates were prepared, fractionated and immunoblotted to detect TR3 as described in the legend of Fig. 3. b After cells were treated under the same conditions as described in a, apoptotic cells that contained condensed and fragmented chromatin were counted after staining with Hoechst 33342. Lane 1 PA-1 cells incubated without VK2, lane 2 PA-1 cells incubated with 30 μM VK2, lane 3 PA-1 cells incubated with 100 ng/ml CHX, lane 4 PA-1 cells incubated with 100 ng/ml CHX for 30 min before the addition of 30 μM VK2 and subsequent incubation for 72 h in the presence of VK2 and CHX. The results shown are typical of results of two experiments. Values are means ± SD of results from five assays

We examined whether siRNA-TR3 treatment might block the induction of apoptosis in PA-1 cells by VK2. When PA-1 cells were transfected with siRNA-TR3, the levels of TR3 in cell lysates fell significantly but small amounts of TR3 were still present (Fig. 5a, lane 2). However, the marked increase in the level of TR3 caused by treatment with VK2 for 72 h was almost completely abolished after transfection of cells with siRNA-TR3 (compare lane 3 with lane 4 in Fig. 5a). Induction of apoptosis by VK2 was also significantly inhibited by siRNA-TR3 (compare columns 3 and 4 in Fig. 5b). Apoptosis was not induced in cells treated with siRNA-TR3 only (column 2 in Fig. 5b). These results suggest that an increase in level of TR3 might be responsible for the induction of apoptosis in PA-1 cells by VK2.

Fig. 5.

Fig. 5

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Effect of inhibition of the TR3 synthesis on the induction of apoptosis. a PA-1 cells were incubated under the conditions indicated for 72 h, and cell lysates were prepared, fractionated and immunoblotted with TR3-specific antibody. b Apoptotic cells were counted after staining with Hoechst 33342 as described in the legend of Fig. 4. Lane 1 PA-1 cells incubated for 72 h, lane 2 PA-1 cells incubated with siRNA-TR3 (2 μg) for 48 h, lane 3 PA-1 cells treated with 30 μM VK2 for 72 h, lane 4 PA-1 cells incubated with siRNA-TR3 (2 μg) for 48 h and then treated with 30 μM VK2 for 72 h. Values are means ± SD of results from five assays

Increases in levels of TR3 in the mitochondrial fraction upon treatment of cells with VK2

Figure 6a shows that the level of TR3 in the HM fraction increased dramatically 48 and 72 h after the start of the treatment of PA-1 cells with VK2. The level of TR3 in the HM fraction 72 h after the start of treatment of PA-1 cells with VK2 was seven times higher than that of TR3 in the HM fraction of control cells that had not been treated with VK2. In parallel with the increase in the level of TR3, the percentage of apoptotic cells was also increased time-dependently by treatment of PA-1 cells with VK2 (Fig. 6b). By contrast, neither the level of TR3 in the HM fraction nor the percentage of apoptotic cells was practically unchanged by treatment of SK-OV-3 cells with VK2 (Fig. 6c, d).

Fig. 6.

Fig. 6

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Changes in the levels of TR3 in HM fractions of PA-1 and SK-OV-3 cells after treatment with vitamin K2 (VK2). Both PA-1 and SK-OV-3 cells were treated with VK2 and fractionated to yield an HM fraction after incubation for 0, 24, 48 and 72 h. a and c The HM fractions were immunoblotted with TR3 specific-antibody and OX-specific antibody. b and d Ratio of apoptotic cells was quantitated after staining with Hoechst 33342, as described in the legend of Fig. 4. The intensities of bands of OX shown in each lane confirmed that equal amounts of mitochondria had been loaded in each lane. a,b PA-1 cells; c,d SK-OV-3 cells

Immunofluorescence staining of PA-1 and SK-OV-3 cells during VK2-induced apoptosis

Using an indirect immunofluorescence-staining method, we studied the changes in the distribution of TR3 in both PA-1 and SK-OV-3 cells during treatment with VK2 (Fig. 7). In control PA-1 cells, endogenous TR3 was observed in nuclei. Moreover, no translocation of TR3 from nuclei was evident and instead, the amount of TR3 in nuclei increased 48 and 72 h after the start of treatment with VK2 (Fig. 7). Localization of TR3 on mitochondria, as identified by immunostaining with antibody against OX, was also evident after treatment with VK2 for 48 and 72 h (Fig. 7). By contrast, TR3 was barely detectable in SK-OV-3 cells. Neither an increase in the amount of detectable TR3 nor the aggregation of TR3 was observed at any time after the start of treatment of SK-OV-3 cells with VK2 (Fig. 7).

Fig. 7.

Fig. 7

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Changes in the distribution of TR3 in PA-1 cells during induction of apoptosis by vitamin K2 (VK2). PA-1 and SK-OV-3 cells were treated with 30 μM VK2 for 0, 24, 48 and 72 h. After fixation of cells as described in the text, cells were subjected to indirect immunofluorescence staining. The TR3-specific polyclonal antibodies reacted with FITC-conjugated antibody against rabbit IgG (green color) and the OX-specific monoclonal antibody (delineating mitochondria) reacted with Cy3-conjugated antibodies against mouse IgG (red color). White arrows indicate typical aggregated mitochondria and TR3. Yellow coloration suggests the localization of TR3 in mitochondria. No changes in the distribution of TR3 were evident in SK-OV-3 cells during treatment with VK2. The images are representative of results from three experiments. The scale bar represents 20 μm

Changes in the distribution of TR3 during VK2-induced apoptosis

As shown in Fig. 8, TR3 in nuclei of PA-1 cells increased 48 and 72 h after the start of treatment of VK2. This result was well coincident with the photographs of the immunofluorescent staining (Fig. 7). The level of TR3 in the cytosol fraction of PA-1 cells was low, but it was also upregulated during induction of apoptosis (Fig. 8). The increase of TR3 in the cytoplasm of PA-1 cells suggests that the synthesis of TR3 in PA-1 cells may be increased by the treatment with VK2.

Fig. 8.

Fig. 8

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Localization of TR3 in both nuclei and cytosol of PA-1 cells during the induction of apoptosis by treatment of vitamin K2 (VK2). PA-1 cells were treated with VK2 for various times and then fractionated to yield nuclear and cytosolic fractions. Each fraction was analyzed by immunoblotting with TR3-specific antibodies. The intensities of the bands of lamin and GAPDH confirm that equal amounts of the nuclear or cytosolic fractions were loaded

Effects of inhibitors of p38 and JNK on VK2-induced apoptosis

Signaling through the JNK and p38 MAP kinase cascades is frequently associated with the induction of apoptosis (Davis 2000, Tournier et al. 2000; MacFarlane et al. 2000; Holmes et al. 2002, 2003a; Li et al. 1998; Kolluri et al. 2003; Han et al. 2006). Therefore, we examined whether the induction of apoptosis in PA-1 cells by VK2 could be blocked by inhibitors of p38 and JNK. When PA-1 cells were pretreated with SP600125, an inhibitor of JNK, for 60 min, the subsequent induction of apoptosis by VK2 is markedly inhibited in a dose-dependent manner (Fig. 9). In contrast to the effect of JNK inhibitor on VK2-induced apoptosis in PA-1 cells, inhibitors of p38 such as PD169316 or SB203580 had no effect on the induction of apoptosis in the same cell by VK2 (results not shown).

Fig. 9.

Fig. 9

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Effects of a JNK inhibitor (SP600125) on the induction of apoptosis by vitamin K2 (VK2). PA-1 cells were incubated with 7.5 or 10 μM SP600125 for 1 h before addition of 30 μM VK2. After following incubation of PA-1 cells for 72 h, cells were stained with Hoechst 333452 and apoptotic cells were counted. White columns show percentages of apoptotic cells after treatment without VK2 (0.1% ethanol was added as vehicle). Gray columns show percentages of apoptotic cells after treatment with 30 μM VK2 and SP600125. The black column shows the percentage of apoptotic cells after treatment with 30 μM VK2 alone. Values are means ± SD of results from five assays

Discussion

In the present study, we found that PA-1 ovarian cancer cells were extremely sensitive to the induction of apoptosis by VK2, while ovarian cancer SK-OV-3 cells were resistant. Levels of TR3 in various lines of cells rise upon exposure of cells to apoptotic stimuli, such as 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) (Li et al. 1998), tetradecanoyl phorbol-1,3-acetate (TPA), VP-16 (Li et al. 2000), and butyrate (Wilson et al. 2003). In harmony with these results, levels of TR3 in PA-1 cells rose significantly upon treatment of cells with VK2, whereas they did not change at all in SK-OV-3 cells under the same conditions. Furthermore, we found that CHX and siRNA-TR3 inhibited the VK2-induced synthesis of TR3 and apoptosis. From our results, we can conclude that an increase in the synthesis of TR3 is associated with the induction of apoptosis in PA-1 cells by VK2.

When the lysate of PA-1 cells was analyzed by immunoblotting with TR3-specific antibodies, two bands were observed (Figs. 3–5). One is the major band and the other is the more slowly migrating minor band. The more slowly migrating band of TR3 was reported to be phosphorylated TR3 (Katagiri et al. 2000; Pekarsky et al. 2001). As shown in Fig. 4, the intensity of the more slowly migrating band of TR3 was practically unchanged by treatment of PA-1 cells with VK2. By contrast, the intensities of the more slowly migrating bands increased evidently by the VK2-treatment as shown in Figs. 3 and 5a, although their degrees of intensification were different between these figures. These results suggest that the degree of phosphorylation of TR3 after its synthesis upon treatment of PA-1 cells with VK2 might be different in spite of the identical experimental conditions. Further studies are required to clarify the mechanism of VK2-induced activation of TR3 phosphorylation.

The level of TR3 in the mitochondrial fraction of PA-1 cells increased markedly 48 h after the start of treatment with VK2. This result is consistent with observations that TR3 accumulates in the HM fraction during the apoptosis that is induced by various inducers of apoptosis, such as TPA and MM11453 (Li et al, 2000). Furthermore, immunofluorecsence studies showed that TR3 accumulated in mitochondria 48 and 72 h after the start of treatment with VK2. It seems, therefore, that TR3 might be one of proteins that disrupt the functions of mitochondria, with the resultant release of cytochrome c. Lin et al. (2004a) demonstrated that elevated levels of TR3 in mitochondria induce the depolarization of mitochondrial membranes by interfering with the antiapoptotic properties of Bcl-2 that are the result of a conformational change. We observed previously that depolarization of the mitochondrial membrane was induced by treatment of TYK-nu cells with VK2 (Shibayama-Imazu et al. 2006). In our present study, we observed the accumulation of TR3 in the HM fraction during VK2-induced apoptosis. Our results suggest that the accumulation of TR3 in mitochondria might be a critical event in the induction of apoptosis.

Li et al. (2000) proposed that TR3 might induce apoptosis via its translocation from the nucleus to the mitochondria, with the resultant release of cytochrome c. In OV-CA-3 cells, TR3 dose move from nuclei and becomes associated with mitochondria upon treatment of cells with the synthetic retinoid, CD437, whereas apoptosis is induced without similar translocation of TR3 upon treatment of the same cells with another synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR; Holmes et al. 2003b). Thus, the translocation of TR3 from nuclei is thought to be dependent on reagent. In our present study, treatment of PA-1 cells with VK2 did not result in translocation of TR3 from nuclei into the cytoplasm. Indeed, TR3 was found to have accumulated in nuclei 48 and 72 h after the start of treatment with VK2. This is the first report, to our knowledge, regarding treatment of TR3 accumulating in both mitochondria and nuclei during the induction of apoptosis.

In this report, we could not show why TR3 did not translocate from the nuclei to the cytoplasm during the VK2-induced apoptosis of PA-1 cells. Leptomycin B, which is an inhibitor of the chromosomal region maintenance 1 (CRM1)-dependent nuclear export process (Kudo et al. 1999), has been used to block both the translocation of TR3 from nuclei and the induction of apoptosis (Li et al. 2000; Katagiri et al. 2000; Cao et al. 2004). However, the VK2-induced apoptosis of PA-1 cells was not inhibited by 0.05–0.2 nM leptomycin B (results not shown). This observation supports our conclusion that TR3 did not move from the nucleus to the cytosol during the VK2-induced apoptosis of PA-1 cells.

We demonstrated that the VK2-induced apoptosis in ovarian cancer PA-1 cells was inhibited by SP600125, an inhibitor of JNK, but not by inhibitors of p38 such as PD169316 and SB203580 (Fig. 9). JNK is known to regulate the induction of apoptosis (Davis 2000; Tournier et al. 2000). We reported recently that VK2 induces the accumulation of reactive oxygen species (ROS), in particular, superoxide radicals and H2O2, in cells (Shibayama-Imazu et al. 2006). ROS are potent activators of JNK, acting via the oxidative inactivation of endogenous inhibitors of JNK, such as JNK phosphatases and glutathione S-transferase (Chen et al. 2001; Bernardini et al. 2000). Superoxide induces the activation of JNK and cell death in hepatocytes (Lo et al. 1996; Conde de la Rosa et al. 2006). Furthermore, activated JNK-1 efficiently phosphorylates TR3, eliminating its ability to bind to DNA in lung cancer cells (Kolluri et al. 2003). The present study demonstrates a possibility that VK2 might cause accumulation of TR3 in mitochondria directly from ribosomes without translocation from nuclei. Further studies are needed to examine whether VK2 might activate JNK and whether phosphorylation of TR3 by JNK might contribute to the accumulation of TR3 in mitochondria.

In conclusion, we have demonstrated that ovary cancer PA-1 cells are very sensitive to VK2 and that accumulation of TR3 in mitochondria, as well as in nuclei, might be associated in the induction of apoptosis by VK2. This finding suggests that VK2 may have therapeutic efficacy for the clinical treatment of ovary cancer.

Abbreviations

BSA

Bovine serum albumin

CHX

Cycloheximide

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HM

Heavy membrane

JNK

c-Jun N-terminal kinase

NGFI-B

Nerve growth factor induced clone B

OX

Cytochrome oxidase subunit IV

PBS

Phosphate-buffered saline

PMSF

Phenylmethylsulfonyl fluoride

ROS

Reactive oxygen species

siRNA

Small interfering RNA

TBS

Tris-buffered saline

TPA

Tetradecanoylphorbol-1,3-acetate

VK2

Vitamin K2

XTT

Sodium-3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate

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