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Clinical Cancer Research 13, 2236, April 1, 2007. doi: 10.1158/1078-0432.CCR-06-2308
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular Carcinoma Cell Growth by Suppressing Cyclin D1 Expression through Inhibition of Nuclear Factor {kappa}B Activation

Iwata Ozaki1,2, Hao Zhang1,3, Toshihiko Mizuta1, Yasushi Ide1, Yuichiro Eguchi1, Tsutomu Yasutake1, Toshiyuki Sakamaki4, Richard G. Pestell4 and Kyosuke Yamamoto1

Authors' Affiliations: 1 Division of Hepatology and Metabolism, Department of Internal Medicine, and 2 Health Administration Center, Saga Medical School, Saga University, Nabeshima, Saga, Japan; 3 Second Department of Surgery, China Medical University, Heping District, Shenyang, China; and 4 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, District of Columbia

Requests for reprints: Iwata Ozaki, Health Administration Center, Saga Medical School, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan. Phone: 81-952-34-3215; Fax: 81-952-34-2017; E-mail: ozaki{at}cc.saga-u.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2–induced growth inhibition of HCC cells.

Experimental Design: HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor {kappa}B (NF-{kappa}B) activation was investigated by a NF-{kappa}B reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated I{kappa}B, and an in vitro kinase assay for I{kappa}B kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1.

Results: Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-{kappa}B binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)–, TNF-{alpha}–, and interleukin (IL)-1–induced activation of NF-{kappa}B binding and transactivation. Concomitant with the suppression of NF-{kappa}B activation, vitamin K2 also inhibited the phosphorylation and degradation of I{kappa}B{alpha} and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment.

Conclusion: Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/I{kappa}B/NF-{kappa}B pathway and might therefore be useful for treatment of HCC.

Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors in the world and is especially prevalent in Asian countries (1). Despite many therapeutic approaches, the long-term prognosis of HCC is poor because of the high relapse rate and the frequent incidence of intrahepatic metastasis (1, 2). The altered expression of many genes, the overexpression of oncogenic genes, and/or the down-regulation of tumor suppressor genes has been reported during the development and progression of HCC (3, 4). Alterations of growth-related genes that regulate cell cycle progression such as the cyclins, cyclin-dependent kinases (Cdk), and Cdk inhibitors have been reported in HCC as well as other malignant tumors (3–6). Cyclin D1 is a proto-oncogenic protein that regulates the G1-S transition of the cell cycle by binding to Cdk4 or Cdk6 and by phosphorylating pRb (6). Cyclin D1 overexpression can enhance DNA synthesis and contact-independent growth in several cell types. This overexpression has been shown in several human cancers including HCC (6–8), which indicates that cyclin D1 plays an important role in cell cycle control and carcinogenesis in the liver and could be a potential therapeutic target in HCC. Expression of the cyclin D1 gene is regulated by various factors, and nuclear factor {kappa}B (NF-{kappa}B) is a transcription factor that is presumed to play an important role in this regulation (9–14).

Nuclear factor {kappa}B (NF-{kappa}B) was originally identified as a regulator of immunoglobulin {kappa} light chain gene expression, which controls the inflammatory process (15). NF-{kappa}B transcriptional activity is normally inhibited by I{kappa}B proteins. On stimulation by extracellular inducers such as tumor necrosis factor-{alpha} (TNF-{alpha}) or IL-1ß, I{kappa}B{alpha} is rapidly phosphorylated by the I{kappa}B kinase (IKK) complex on Ser32 and Ser36. The phosphorylation of I{kappa}B leads to the subsequent proteasomal degradation of I{kappa}B{alpha} by ubiquitination, which stimulates the nuclear translocation of NF-{kappa}B and the subsequent activation of genes involved in the immune and inflammatory responses. In addition to its roles in the immune response and inflammation, NF-{kappa}B also influences cell growth and survival by regulating genes involved in cell growth, apoptosis, and metastasis. Due to these roles, NF-{kappa}B has also been implicated in cancer development (13, 16, 17).

Recently, Habu et al. (18) reported that menatetrenone, a vitamin K2 analogue, suppressed the de novo development of HCC in cirrhotic patients, and we showed that the recurrence of HCC after surgical resection or ablation therapy is suppressed by menatetrenone administration in clinical settings (19). Vitamin K is a fat-soluble vitamin that regulates clotting factor production by acting as a coenzyme for a vitamin K–dependent carboxylase that catalyzes the carboxylation of glutamic acid residues to produce {gamma}-carboxyglutamic acid (20). Vitamin K is similarly involved in the regulation of bone metabolism by {gamma}-carboxylation of bone matrix proteins (21). Vitamin K can be divided into two groups: naturally produced vitamin K1 (phytonadione) and vitamin K2 (menaquinone) and chemically synthesized vitamin K3 (menadione). In addition to the physiologic roles of vitamin Ks, vitamin K3 and its derivatives show potent antiproliferative effects against tumor cell lines in vitro (22, 23). However, vitamin K3 is not currently used for cancer treatment in vivo because of its high toxicity. Vitamin K2 and its derivatives also show antiproliferative effects (although less potent than those of vitamin K3) against leukemia and HCC cell lines (24–26). Vitamin K2 also has the ability to induce the differentiation of leukemic cells (24) and has been used for treatment of myelodysplastic syndromes (27).

Several reports have shown that vitamin K2 alters the expression of growth-related genes and inhibits cell cycle progression at the G1-S phase in several cancer cells including HCC. However, the precise mechanisms by which vitamin K2 induces cell cycle arrest and growth inhibition have not been fully elucidated. We therefore studied the effects of vitamin K2 on HCC cell proliferation and investigated the mechanisms responsible for these effects. In this report, we show that vitamin K2 inhibits HCC cell proliferation by regulating cyclin D1 expression through the inhibition of NF-{kappa}B activation.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. The human HCC cell lines HepG2, Hep3B, and Huh7 were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan). The cells were cultured and maintained in DMEM (Gibco BRL, Gaithersburg, MD) containing 10% FCS (Gibco BRL). Menatetrenone, a vitamin K2 analogue, was provided by Eisai Co. (Tokyo, Japan).

Plasmids. Cyclin D1 promoter-luciferase (CD1Luc) reporter constructs were previously described (10, 11). CD1Luc with 1,745 bp of cyclin D1 promoter (–1745CD1Luc), truncated CD1Luc (–964CD1Luc and –66CD1Luc), CD1Luc mutated in activator protein 1 (AP-1)–binding site (–964AP-1mutCD1Luc), and CD1Luc mutated in NF-{kappa}B–binding site (–66NF-{kappa}BmutCD1Luc) were used for the luciferase assay (Fig. 3A). The luciferase reporter gene containing the NF-{kappa}B responsive element was purchased from BD Biosciences Clontech (Palo Alto, CA). Complementary DNA coding full-length cyclin D1 cDNA or p65 NF-{kappa}B subunit was synthesized by reverse transcription PCR (RT-PCR) from human liver mRNA. The oligonucleotides introduced with restriction enzyme sites that were used for the cloning of full-length human cyclin D1 or p65 NF-{kappa}B subunit cDNA were as follows: cyclin D1, forward 5'-CTCGAGATGGAACACCAGCTCCTGTGC-3', reverse 5'-AAGCTTTCCCTTCTGGTATC-3'; p65 NF-{kappa}B subunit, forward 5'-CTCGAGATGGACAGCTGTTCCCCTCATC-3', reverse 5'-GGATCCTTAGGAGCTGATCTGACTC-3'. RT-PCR products were cloned into pT7-Blue-T vector (Novagen, Madison, WI) and subcloned into pcDNA3.1(+) (Invitrogen, San Diego, CA) for construction of the mammalian expression vector driven by the cytomegalovirus promoter.


Figure 3
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  		Fig. 3. Effect of vitamin K2 on cyclin D1 promoter activity. A, the maps of cyclin D1 luciferase plasmids. B, vitamin K2 inhibited cyclin D1 promoter activity in hepatoma cells. The cells were transiently transfected with –1745CD1Luc plasmid and were then treated with vitamin K2. After 24 h, the cells were harvested and assayed for luciferase activity as described in Materials and Methods. Columns, mean obtained from three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with cells untreated with vitamin K2. C, vitamin K2 inhibited cyclin D1 promoter activity in a NF-{kappa}B–dependent manner. HepG2 cells were transiently transfected with cyclin D1 promoter plasmids as described in (B) and were then treated with vitamin K2. After 24 h, the cells were harvested and assayed for luciferase activity as described in Materials and Methods. Columns, mean obtained from three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with cells untreated with vitamin K2. D, overexpression of p65 subunit of NF-{kappa}B inhibited the vitamin K2–induced cyclin D1 promoter suppression. HepG2 cells were transiently cotransfected with –1745CD1Luc and p65 subunit cDNA of NF-{kappa}B as described in (B), treated with vitamin K2 for 24 h, and subjected to luciferase assay. Columns, mean obtained from three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with cells untreated with vitamin K2.

 
Cell proliferation assay. The sensitivity of hepatoma cells to vitamin K2 was determined with the WST-1 proliferation assay kit (Takara, Kyoto, Japan) as previously described (28). The cells were seeded in 24-well culture plates at a density of 1 x 104 per well and incubated at 37°C for 24 h. The cells were subsequently incubated with vitamin K2 at the indicated concentrations for 48 h. The cells were then incubated with WST-1 reagents and the absorbance of formazan products at 450 nm was measured with a CS-9300 microplate reader (Shimadzu, Tokyo, Japan).

Fluorescence-activated cell sorting analysis. The fluorescence-activated cell sorting (FACS) analysis of propidium iodide–stained nuclei was done as previously described (29). Briefly, cells were plated at a density of 2 x 105 per well in six-well dishes and were incubated at 37°C for 24 h. The cells were further incubated with the indicated concentrations of vitamin K2 for 48 h. The cells were then harvested by trypsinization, collected by centrifugation, and suspended in hypotonic lysis buffer (0.1% Triton X-100 in PBS), 0.1 mg/mL RNase, and 40 µg/mL propidium iodide. After 30 min at 37°C, the cells were analyzed with a FACSCalibur cytofluorometer (Becton Dickinson, San Jose, CA) and the percentage of cells in different cell cycle was determined.

RNA isolation and semiquantitative RT-PCR. The total RNA was extracted from the cultured HCC cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The concentration of RNA was determined with a spectrophotometer and the integrity of the samples was confirmed by visualizing 28S and 18S rRNA bands under UV light after gel electrophoresis. Semiquantitative RT-PCR was done as previously described (30). Briefly, 1 µg of total RNA was reverse transcribed with reverse transcriptase (Takara) using random primers. Subsequently, each RT reaction mixture was subjected to PCR amplification using Taq Gold polymerase (Perkin-Elmer, Branchburg, NJ) with cycle numbers varying from 15 to 40. Each cycle consisted of heat denaturation (94°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 2 min). The PCR products were size fractionated on a 2% agarose gel and visualized under UV light. The sequences of oligonucleotide primers used for RT-PCR to determine the expression of the target gene are listed, preceded by accession number for GenBank or references, and followed by expected transcript sizes: p27 (NM004064), sense 5'-TCAAACGTGCGAGTGTCTAA-3', antisense 5'-ACGGATCAGTCTTTGGGTCCA-3', 409 bp; p21 (BC001935), sense 5'-CGATGGAACTTCGACTTTGTC-3, antsense 5'-GATTAGGGCTTCCTCTTGG3', 356 bp; cyclin D1 (BC001501), sense 5'-GTGCTGCGAAGTGGAAAC-3', antisense 5'-AAGCTTTCCCTTCTGGTATC-3', 986 bp; glyceraldehyde 3-phosphate dehydrogenase (NM002046), sense 5'-CCACCCATGGCAAATTCCATGGCA-3', antisense 5'-TCTAGACGGCAGGTCAGGTCCACC-3', 593 bp.

Western blotting. The protein expression ofp27, p21, cyclin D1, and IKK{alpha}, I{kappa}B{alpha}phosphorylation, and I{kappa}B{alpha}degradation were investigated by Western blotting. Cells (2 x 106) cultured under various conditions were collected and lysed with extraction buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% SDS, 5 mmol/L EDTA (pH 8.0), 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL trypsin inhibitor, and 50 mmol/L iodoacetamide. After 30 min at 4°C, the cell debris was eliminated by centrifugation at 15,000 rpm for 20 min and the supernatant was collected. After measuring the protein concentration with a protein assay kit (Bio-Rad, Hercules, CA), 40 µg of protein were mixed with SDS sample buffer, separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and blocked with 0.1% Tween 20 and 5% skim milk overnight. The membranes were incubated with anti-p27 (BD Transduction Laboratories, Franklin Lakes, NJ), p21 (Oncogene Research Products, San Diego, CA), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti–phospho-specific I{kappa}B{alpha}, total I{kappa}B{alpha}(New England Biolabs, Beverly, MA), and IKK{alpha} (Santa Cruz Biotechnology) antibodies in PBS with 1% bovine serum albumin for 1 h. Antihuman ß-actin antibodies (Biomedical Technologies, Stoughton, MA) were used as a control. The membranes were washed thrice with 0.1% Tween 20 in PBS and stained with horseradish peroxidase–conjugated secondary antibodies. All immunoblots were detected by the enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom) according to the manufacturer's instructions.

Transfection and luciferase reporter gene assay. The effects of vitamin K2 on cyclin D1 promoter activity and NF-{kappa}B transcriptional activity were detected by luciferase assay using the method described by the supplier (Dual-Luciferase Reporter Assay System, Promega, Madison, WI). Cyclin D1 promoter luciferase reporter plasmids (–1745CD1Luc, –964CD1Luc, –964AP-1mutCD1Luc, –66CD1Luc, and –66NF-{kappa}BmutCD1Luc) and NF-{kappa}B-luciferase reporter plasmid were used for assay. Luciferase expression plasmid pRL-SV40 (Promega) was cotransfected to normalize for transfection efficiency. The HCC cells were seeded onto six-well plates at 1 x 105 per well without antibiotics and incubated until 80% confluent at 37°C. Next, the cells were washed twice with OPTI-MEM I Reduced Medium (Life Technologies, Rockville, MD), followed by the addition of 2 mL of OPTI-MEM I Reduced Medium containing 5 µg of target gene reporter plasmid, 1 µg of pRL-SV40 luciferase plasmid, and 15 µL of Lipofectamine 2000 reagent (Life Technologies). After 6 h of incubation, the medium was changed and the transfected cells were treated with indicated doses of vitamin K2. After 24 h of treatment, the cells were washed twice with PBS and carefully scraped into 1x passive lysis buffer (Promega). The cell extracts were immediately assayed for luciferase activity using a Berthold Luminometer (MLR-100 Micro Lumino Reader, Corona Electric, Ibaragi, Japan).

Electrophoretic mobility shift assay. The NF-{kappa}B binding activity in nuclei isolated from hepatoma cells was determined by electrophoretic mobility shift assay. The nuclear protein extractions and electrophoretic mobility shift assay were done as previously described (31). Briefly, 5 µg of nuclear protein were incubated for 30 min at room temperature with binding buffer [20 mmol/L HEPES-NaOH (pH 7.9), 2 mmol/L EDTA, 100 mmol/L NaCl, 10% glycerol, 0.2% NP40], poly(deoxyinosinic-deoxycytidylic acid), and 32P-labeled double-stranded oligonucleotide containing the NF-{kappa}B binding motif (Promega). The sequence of the double-stranded oligomer used for electrophoretic mobility shift assay was 5'-AGTTGAGGGGACTTTCCCAGGC-3' (only the sense strand is shown). The reaction mixtures were loaded on a 4% polyacrylamide gel and electrophoresed with a running buffer of 0.25x Tris-borate EDTA. After the gel was dried, the DNA-protein complexes were visualized by autoradiography.

IKK assay. The assay for endogenous IKK was done according to a previously method described with minor modifications (32). The cells were treated with vitamin K2 alone or in combination with TPA, IL-1, or TNF-{alpha} and subjected to IKK kinase assay. Cytoplasmic extracts (300 µg) were immunoprecipitated with 2 µg of anti-IKK{alpha} antibody and then treated with 20 µL of protein G-Sepharose (Pierce, Rockford, IL). After 2 h, the beads were washed with lysis buffer and then with the kinase buffer [50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, and 2 mmol/L DTT]. The immune complex was then assayed for kinase activities with the use of kinase assay buffer containing 10 µCi [{gamma}-32P]ATP, 10 µmol/L unlabeled ATP, and 2 µg glutathione S-transferase-I{kappa}B (1–54). After incubation at 30°C for 30 min, the reaction was stopped by boiling the solution in 4x SDS sample buffer. The reaction mixture was then resolved on 12% SDS-PAGE. The gel was dried and exposed to X-ray films (Fuji) at –80°C. The total amount of IKK{alpha} was determined by Western blot analysis.

Stable transformation of HCC cells. Plasmids encoding human p65 NF-{kappa}B subunit (pcDNA/p65), cyclin D1 (pcDNA/CD1), or pcDNA alone (as a control) were introduced into HepG2 cells using Lipofectamine (Gibco BRL) according to the manufacturer's instructions. The transfected HepG2 cells were treated with 500 ng/mL G418 for 2 weeks before selection. Individual clones of HepG2 cells transduced with pcDNA/p65 or pcDNA/CD1 were analyzed. Clones overexpressing p65 or cyclin D1 were selected and subjected to further analysis.

Statistical analysis. Differences were analyzed using Student's t test, and P < 0.05 was considered significant. All experiments were done at least thrice. The data are shown as the mean ± SD.


   Results
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 Materials and Methods
 Results
Discussion
 References
 
Vitamin K2 induces growth arrest in the G1 phase in HCC cells. Three HCC cell lines, HepG2, Hep3B, and Huh7, were treated with different concentrations of vitamin K2 for 48 h, and their growth was subsequently assayed by WST-1 proliferation assay. As shown in Fig. 1A , vitamin K2 significantly inhibited the growth of HCC cells in a dose-dependent manner. The DNA synthesis was also suppressed by vitamin K2 in these cells as shown by bromodeoxyuridine incorporation assay (data not shown). To clarify the mechanism of vitamin K2–induced growth inhibition in HCC cells, we did a FACS analysis in these cells treated with vitamin K2. As shown in Fig. 1B, the FACS analysis of the vitamin K2–treated cells showed a significantly increased number of cells in the G1 phase. However, the proportion of pre-G1 cells (presumably apoptotic cells) was not significantly changed after vitamin K2 treatment, thus suggesting that vitamin K2 suppresses the growth of HCC cells by inducing G1 arrest.


Figure 1
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  		Fig. 1. A, vitamin K2–induced growth inhibition in human hepatoma cell lines. Cells seeded in 24-well plates (104 per well) were incubated with different concentrations of vitamin K2 for 48 h, and the degree of cell growth was evaluated. The results are expressed as the percentage of the growth seen in untreated hepatoma cells. Columns, mean from three independent experiments; bars, SD. B, a FACS analysis of propidium iodide–stained nuclei in hepatoma cells. Cells were treated with the indicated doses of vitamin K2. After 24 h of incubation, the cells were collected and stained with propidium iodide, and the DNA content was subsequently analyzed by FACS. The histograms represent total DNA contents, and the percentages of cells with G1 are shown (M1). Representative results from three independent experiments.

 
Vitamin K2 inhibits the expression of cyclin D1 in HCC cells. Cyclin D1 is required for the progression of cells from the G1 to S phase. Because we observed G1 arrest in vitamin K2–treated HCC cells, we decided to analyze the effect of vitamin K2 on the expression of cyclin D1. The treatment of cells with vitamin K2 significantly down-regulated cyclin D1 expression at both the mRNA (Fig. 2A and B ) and protein levels (Fig. 2C) in all the HCC cell lines. As shown in Fig. 2, the expression levels of the Cdk inhibitors p27 and p21 were also determined in these cells after vitamin K2 treatment. We observed that the responses of p21 and p27 to vitamin K2 varied from cell to cell. The expression of both p27 and p21 was increased after vitamin K2 treatment in HepG2 cells. A slight increase of p27 expression was shown in Huh7 cells whereas the level of p21 remained low. In Hep3B cells, there was an increase in the level of p21 expression after vitamin K2 treatment but p27 expression level was not altered. These results indicate that cyclin D1 and Cdk inhibitor expression is regulated during vitamin K2–induced growth inhibition in HCC cells.


Figure 2
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  		Fig. 2. Effects of vitamin K2 on the expression of cyclin D1, p27, and p21 in hepatoma cells. A, semiquantitative RT-PCR for cyclin D1, p27, and p21 mRNA expression in vitamin K2–treated hepatoma cells. Glyceraldehyde 3-phosphate dehydrogenase expression was used as a control. B, quantification by densitometric analysis. Points, mean obtained from three independent experiments; bars, SD. C, Western blot analysis of cyclin D1, p27, and p21 protein expression in vitamin K2–treated hepatoma cells. The expression of ß-actin was used as a control.

 
Vitamin K2 inhibits cyclin D1 promoter activity in a NF-{kappa}B–dependent manner. To further investigate the effect of vitamin K2 on cyclin D1 expression, the cyclin D1 promoter activity was examined after vitamin K2 treatment. The cyclin D1 promoter luciferase reporter plasmids –1745CD1Luc, –964CD1Luc, –964AP-1mutCD1Luc, –66CD1Luc, and –66NF-{kappa}BmutCD1Luc (Fig. 3A ) were transfected. Vitamin K2 significantly inhibited the activity of –1745CD1Luc, a full-length cyclin D1 promoter luciferase reporter plasmid, in all three cell lines (Fig. 3B). To examine whether vitamin K2–mediated cyclin D1 promoter inhibition depends on NF-{kappa}B activity, HepG2 cells were transfected with cyclin D1 promoter luciferase reporter plasmids, which included either the wild-type, mutant AP-1, or NF-{kappa}B binding site, and the effect of vitamin K2 on their activities was investigated. As shown in Fig. 3C, vitamin K2 inhibited the activities of –1745CD1Luc, –964CD1Luc, –964AP-1mutCD1Luc, and –66CD1Luc in a dose-dependent manner but had no effect on the activity of –66NF-{kappa}BmutCD1Luc, which contained the mutated NF-{kappa}B site. To confirm the role of NF-{kappa}B in cyclin D1 promoter regulation by vitamin K2, HepG2 cells were transfected with –1745CD1Luc and p65 cDNA, a subunit of NF-{kappa}B. As shown in Fig. 3D, vitamin K2 could not inhibit cyclin D1 promoter activity when p65 was cotransfected, indicating that the effect of vitamin K2 on cyclin D1 promoter activity was NF-{kappa}B dependent. These results indicate that NF-{kappa}B plays an important role in the vitamin K2–mediated inhibition of cyclin D1 promoter activity.

Vitamin K2 inhibits the DNA binding activity and transcriptional activity of NF-{kappa}B in hepatoma cells. Because NF-{kappa}B plays an essential role in the regulation of cyclin D1 promoter activity, we evaluated the effect of vitamin K2 on the DNA binding activity of NF-{kappa}B in HCC cells. For the electrophoretic mobility shift assay, the nuclear protein extracts were incubated with a 32P-labeled oligonucleotide containing a conserved NF-{kappa}B binding sequence. As shown in Fig. 4A , Hep3B and Huh7 cells constitutively expressed NF-{kappa}B activity, and vitamin K2 inhibited the basal level of NF-{kappa}B binding activity in these cells. To confirm the specificity of binding, an excess of unlabeled NF-{kappa}B–binding oligonucleotide abolished the NF-{kappa}B binding whereas oligonucleotide containing a NF-{kappa}B mutant binding sequence did not change the NF-{kappa}B binding activity. These results showed that the binding of the NF-{kappa}B probe was specific. In addition, supershift assays indicated that the primary NF-{kappa}B complex regulated by vitamin K2 was the p50-p65 heterodimer.


Figure 4
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  		Fig. 4. A and B, vitamin K2 inhibited basal NF-{kappa}B binding activity and TPA-, TNF-{alpha}–, or IL-1ß–induced NF-{kappa}B binding activity in hepatoma cells. The cells were treated with vitamin K2 alone or were treated with TPA, TNF-{alpha}, or IL-1ß in the presence or absence of vitamin K2 for 24 h, and then nuclear protein was extracted. The NF-{kappa}B binding activity was analyzed by electrophoretic mobility shift assay. The specificity of electrophoretic mobility shift assay was verified by cold competitors, NF-{kappa}B mutant probe, and supershift. C, vitamin K2 (VK2) inhibited basal NF-{kappa}B transcriptional activity and TPA-, TNF-{alpha}–, or IL-1ß–induced NF-{kappa}B transcriptional activity in hepatoma cells. HepG2 cells were transiently transfected with NF-{kappa}B-luciferase reporter plasmid and were then treated with vitamin K2 alone or with TPA, TNF-{alpha}, or IL-1ß in the presence or absence of vitamin K2. After 24 h, the cells were harvested and assayed for luciferase activity as described in Materials and Methods. Columns, mean obtained from three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with cells untreated with vitamin K2 or treated with stimuli alone.

 
To determine whether vitamin K2 could inhibit the activation of NF-{kappa}B by various stimulatory agents, HCC cells were treated with TPA, TNF-{alpha}, or IL-1. As expected, NF-{kappa}B was activated after treatment with TPA, TNF-{alpha}, or IL-1ß, as seen by the increased DNA binding activity. The NF-{kappa}B binding activity induced by these stimuli was significantly inhibited when vitamin K2 was simultaneously added to the cells (Fig. 4B). Vitamin K2 also inhibited the basal level of NF-{kappa}B binding activity although the basal NF-{kappa}B level of HepG2 cells was very low. These results indicated that vitamin K2 could inhibit both the basal and induced NF-{kappa}B binding activities in HCC cells.

To further assess whether the inhibition of DNA binding by vitamin K2 affected NF-{kappa}B transcriptional activity, we did a transient assay using a NF-{kappa}B-luciferase reporter plasmid in HepG2 cells. As shown in Fig. 4C, vitamin K2 inhibited NF-{kappa}B transcriptional activity in a dose-dependent manner. Furthermore, the effect of vitamin K2 on NF-{kappa}B transcriptional activity induced by TPA, TNF-{alpha}, or IL-1ßwas also evaluated, and we observed that vitamin K2 significantly inhibited the TPA-, TNF-{alpha}–, or IL-1ß–induced NF-{kappa}B transcriptional activity in HepG2 cells. These results show that vitamin K2 inhibits both NF-{kappa}B DNA binding activity and NF-{kappa}B–dependent promoter activity.

Vitamin K2 inhibits I{kappa}B{alpha} phosphorylation and IKK activity. The degradation of I{kappa}B{alpha} and the subsequent release of NF-{kappa}B require the prior phosphorylation of Ser32 and Ser36 residues. Therefore, to investigate whether the inhibitory effect of vitamin K2 is mediated by the alteration of I{kappa}B{alpha} phosphorylation, we treated the HepG2 cells with vitamin K2 and examined their protein extracts for phospho-I{kappa}B{alpha} and I{kappa}B{alpha} expression. As shown in Fig. 5A , vitamin K2 dose-dependently inhibited both the basal I{kappa}B{alpha} phosphorylation and the TPA-, TNF-{alpha}–, or IL-1ß–induced I{kappa}B{alpha} phosphorylation in HepG2 cells. In addition, the degradation of total I{kappa}B{alpha}was restored by vitamin K2 treatment.


Figure 5
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  		Fig. 5. A, vitamin K2 inhibited basal I{kappa}B{alpha} phosphorylation and TPA-, TNF-{alpha}–, or IL-1ß–induced I{kappa}B{alpha} phosphorylation in hepatoma cells. HepG2 cells were treated with vitamin K2 alone or with TPA, TNF-{alpha}, or IL-1ß in the presence or absence of vitamin K2 for 24 h. Western blot analysis was done with anti–phospho-specific antibodies to detect I{kappa}B{alpha} phosphorylation. The expression of total I{kappa}B{alpha} was also examined using the expression of ß-actin as a control. B, vitamin K2 inhibited basal I{kappa}B{alpha} kinase activity and TPA-, TNF-{alpha}–, or IL-1ß–induced I{kappa}B{alpha} kinase activity in hepatoma cells. HepG2 cells were treated with vitamin K2 alone or with TPA, TNF-{alpha}, or IL-1ß in the presence or absence of vitamin K2 for 12 h. Cytoplasmic extracts (300 µg) were immunoprecipitated with antibodies against IKK{alpha}. An immune complex kinase assay was subsequently done as described in Materials and Methods. To examine the effect of vitamin K2 on the expression of IKK{alpha} protein, Western blot analysis was done with anti-IKK{alpha} antibodies.

 
The phosphorylation of I{kappa}B{alpha}is controlled by IKK. An in vitro kinase assay using immunoprecipitated IKK from HepG2 cells and the glutathione S-transferase-I{kappa}B{alpha} as substrate showed that vitamin K2 inhibited the basal and the TPA-, TNF-{alpha}–, or IL-1ß–induced IKK activities (Fig. 5B). However, a Western blot analysis of the cell extracts showed no significant change in the protein level of IKK{alpha} expression. These results indicated that vitamin K2–mediated IKK{alpha} inhibition was responsible for the inhibition of NF-{kappa}B activation by vitamin K2.

Overexpression of p65 and cyclin D1 in HCC cells causes resistance to vitamin K2–induced growth inhibition. To further identify the role of NF-{kappa}B and cyclin D1 in vitamin K2–induced growth inhibition in HCC cells, HepG2 cells were stably transfected with full-length p65 or cyclin D1 cDNA and the cells overexpressing p65 or cyclin D1 were selected. A Western blot analysis showed significantly more p65 or cyclin D1 protein in the p65- or cyclin D1–transfected clones than in the parental or mock-transfected cells (Fig. 6A ). In comparison with the results seen in the parental and mock-transfected cells, vitamin K2 failed to inhibit the proliferation of cells that overexpressed p65 or cyclin D1. Our findings showed that these p65- or cyclin D1–transfected clones possessed a significant resistance to vitamin K2–induced growth inhibition (Fig. 6B).


Figure 6
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  		Fig. 6. A, Western blot analysis of p65, cyclin D1, and ß-actin protein expression in parental, mock-, and p65- or cyclin D1–transfected HepG2 cells. HepG2/mock, mock-transfected HepG2 cells; HepG2/p65#1 to #3, representative individual clones of HepG2 cells stably transfected with full-length p65 cDNA; HepG2/CD1#1 to #3, representative individual clones of HepG2 cells stably transfected with full-length cyclin D1 cDNA. B, p65 or cyclin D1 overexpression protects HepG2 cells from vitamin K2–induced growth inhibition. The cells were incubated with different concentrations of vitamin K2 for 48 h. Cell growth is expressed as the percentage of cells relative to the number of untreated cells in parental, mock-transfected, and three representative p65- or cyclin D1–transfected HepG2 clones. Columns, mean obtained from three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with parental or mock-transfected HepG2 cells treated with vitamin K2.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
A growing body of literature has linked NF-{kappa}B activity to the onset and progression of tumorigenesis (13, 33). The aberrant activation of NF-{kappa}B has been reported in many malignant neoplasms such as human leukemias, lymphomas, and solid tumors. Moreover, oncogenic viruses, including T-cell leukemia virus 1 and EBV, activate NF-{kappa}B during the transformation process (34–36). In the liver, NF-{kappa}B activity is involved in hepatocyte regeneration after liver injury, indicating its critical role in hepatocyte growth regulation (37). The constitutive activation of NF-{kappa}B was reported in HCC and is considered to be involved in hepatocarcinogenesis (38, 39). HCC development is closely associated with chronic infection by the hepatitis B or C virus (1). Recent reports indicated that components of hepatitis viruses such as the hepatitis B virus X antigen or hepatitis C virus core protein were able to modulate NF-{kappa}B activity, suggesting that NF-{kappa}B activation plays a role in hepatocarcinogenesis (40, 41). Other recent studies have shown the association of NF-{kappa}B activity with the progression of HCC as well as the association between the inhibition of NF-{kappa}B activation and the suppression of HCC progression (42–44). Many cytokines such as TNF-{alpha} and interleukins induce NF-{kappa}B activation and are involved in chronic inflammation induced by hepatitis viruses. This chronic inflammation is linked to hepatocarcinogenesis (reviewed in ref. 45). In this study, we have shown that vitamin K2 inhibited cytokine-induced NF-{kappa}B activation by suppressing IKK activity, and this action of vitamin K2 might contribute to the suppression of HCC development in clinical settings (18, 19). Because many cases of HCC are related to hepatitis virus infection (1) and hepatitis viral antigens are shown to induce NF-{kappa}B activation (40, 41), it will be interesting to examine the effects of vitamin K2 on the interaction of these viral antigens and NF-{kappa}B activation. However, the effects of vitamin K2 on hepatitis viral antigens are not currently known and future studies are needed to examine whether vitamin K2 affects the interaction of hepatitis viral proteins and NF-{kappa}B activation. One mechanism by which transcription factor NF-{kappa}B regulates cell proliferation is through the control of cyclin D1 expression (11–13). Cyclin D1 promotes the G1-S phase of the cell cycle and is frequently overexpressed in many human cancers including HCC (5–8). The expression of cyclin D1 is one of the prognostic factors of HCC patients (9), and the down-regulation of cyclin D1 inhibits HCC growth in animal models (46). Therefore, both NF-{kappa}B and cyclin D1 are regarded as potential therapeutic targets for the treatment of HCC.

Earlier research showed that vitamin K was a cofactor of {gamma}-carboxylation and was necessary for producing functional proteins involved in blood coagulation reactions. Recent studies have revealed that vitamin Ks are involved in the {gamma}-carboxylation of a wide variety of proteins besides clotting-related proteins, and {gamma}-carboxylation–independent effects of vitamin Ks have been observed, indicating that vitamin Ks possess both {gamma}-carboxylation–dependent and –independent actions (47). If vitamin K–dependent proteins are involved in HCC cell growth regulation, warfarin (which inhibits {gamma}-carboxylation of vitamin K–dependent proteins and is known to increase des-r-carboxy-prothrombin in hepatic cells) might increase the growth of hepatoma cells. However, warfarin has been reported to suppress the growth of several types of cells (48) and the growth of HCC cells was suppressed by warfarin treatment.5 The involvement of vitamin K–dependent proteins in hepatoma cell growth should therefore be examined further.

Vitamin K2 is also involved in bone metabolism via the {gamma}-carboxylation of bone matrix proteins and the regulation of bone reabsorption. It is often used for the treatment of osteoporosis in Japan (49). The mechanism by which vitamin K2 inhibits osteoclastic activity involves the receptor activator of NF-{kappa}B (RANK)/RANK ligand signaling pathway that links to NF-{kappa}B activation. Takeuchi et al. (50) reported that vitamin K2 suppressed RANK mRNA expression and osteoclastic activity in murine bone marrow cells, suggesting the involvement of NF-{kappa}B signaling mediated by the RANK/RANK ligand system. However, the direct evidence of NF-{kappa}B activity modulation by vitamin K2 was not shown. Cyclin D1 is a downstream target of RANK/RANK ligand signaling (51) and recent studies have shown that cyclin D1 also regulates RANK expression (52). Therefore, cyclin D1 functions both downstream and upstream of the RANK signaling pathway.

Recently, several studies have shown that vitamin K2 modulates transcriptional activity in bone-derived (53) and HCC cells (54). Tabb et al. (53) showed that vitamin K2 binds to steroid and xenobiotic receptor (SXR) and mediates gene expression in the osteosarcoma cell line. It has been reported that SXR is expressed abundantly in normal liver tissue (55); however, the expression of SXR in human HCC has not been studied. We examined the expression of SXR in several human HCC cell lines and found that three of five HCC cell lines expressed SXR mRNA and protein. Vitamin K2 suppressed all five HCC cell lines irrespective of SXR expression and did not affect SXR expression in these cells.6 Therefore, SXR is unlikely to be involved in vitamin K2–induced HCC cell growth suppression. Otsuka et al. (54) recently reported that vitamin K2 inhibited the growth and invasiveness of HCC cells via protein kinase A activation and Rho kinase inhibition. However, the molecular mechanisms of protein kinase A–mediated growth inhibition in HCC cells have not been fully elucidated. We treated several HCC cells with protein kinase A stimulator, but we could not induce the inhibition of cyclin D1 gene expression and cell proliferation (data not shown). Therefore, although protein kinase A might be partially involved in vitamin K2–induced inhibition of HCC cell growth and invasion, mechanisms other than protein kinase A also seem to play important roles in vitamin K2–induced HCC growth suppression. Besides vitamin K2, other vitamin K analogues are reported to inhibit HCC cell growth and induce apoptosis. Carr et al. (47) showed that a new synthetic vitamin K analogue, compound 5, inhibited Cdc25A phosphatase activity and reduced HCC cell growth. However, the mechanism by which vitamin K2 inhibits HCC cell growth seems to be different from that of compound 5 because vitamin K2 does not affect Cdc25A phosphatase activity (47) and does not induce apoptosis.

In this study, we revealed that vitamin K2 inhibited IKK kinase activity and therefore suppressed I{kappa}B phosphorylation and NF-{kappa}B activation. We do not yet know the specific target of vitamin K2 action functioning upstream of IKK. Recent studies have shown that several kinases that phosphorylate serine residue could possibly regulate an IKK/I{kappa}B/NF-{kappa}B pathway, and these candidate kinases might therefore be the target of vitamin K2. However, further research is needed to specifically determine the target of vitamin K2 antineoplastic activity.


   Footnotes
 
Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan grants P-03346 (I. Ozaki) and 16590606 (T. Mizuta) and NIH grants R01CA70896, R01CA75503, R01CA86072, R01CA93596, and R01CA107382 (R.G. Pestell).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: I. Ozaki and H. Zhang contributed equally to this work.

5 Zhang et al., unpublished data. Back

6 Ozaki et al., unpublished data. Back

Received 9/18/06; revised 12/16/06; accepted 1/24/07.


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 Results
 Discussion
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