Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular

Cancer Therapy: Preclinical

Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular Carcinoma Cell Growth by Suppressing Cyclin D1 Expression through Inhibition of Nuclear Factor KBActivation Iwata Ozaki,1, 2 Hao Zhang,1, 3 To s hi hi ko Mi z u t a , 1Ya s u s hi Id e ,1Yuichiro Eguchi,1TsutomuYasutake,1 Toshiyuki Sakamaki,4 Richard G. Pestell,4 and KyosukeYamamoto1

Abstract 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 D1promoter was done under vitamin K2 treatment.The regulation of nuclear factor nB(NF-nB) activation was investigated by aNF-nB reporter gene assay, an electrophoretic mobility shift assay, aWestern blot for phosphorylated InB, and an in vitro kinase assay for InB 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 D1mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-nB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)^, TNF-a^, and interleukin (IL)-1^ induced activation of NF-nB binding and transactivation. Con- comitant with the suppression of NF-nB activation, vitamin K2 also inhibited the phosphorylation and degradation of InBa and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1and p65 became resistant to vitamin K2 treatment. Conclusion: Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1expression through the IKK/InB/NF-nB pathway and might therefore be useful for treatment of HCC.

Hepatocellular carcinoma (HCC) is one of the most common metastasis (1, 2). The altered expression of many genes, the human malignant tumors in the world and is especially overexpression of oncogenic genes, and/or the down-regulation prevalent in Asian countries (1). Despite many therapeutic of tumor suppressor genes has been reported during the approaches, the long-term prognosis of HCC is poor because of development and progression of HCC (3, 4). Alterations of the high relapse rate and the frequent incidence of intrahepatic 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 G -S transition of the cell cycle by binding to Cdk4 or Cdk6 Authors’ Affiliations: 1Division of Hepatology and Metabolism, Department of 1 Internal Medicine, and 2Health Administration Center, Saga Medical School, Saga and by phosphorylating pRb (6). Cyclin D1 overexpression can University, Nabeshima, Saga, Japan; 3Second Department of Surgery, China enhance DNA synthesis and contact-independent growth in Medical University, Heping District, Shenyang, China; and 4Department of several cell types. This overexpression has been shown in Oncology, Lombardi Comprehensive Cancer Center, Georgetown University several human cancers including HCC (6–8), which indicates School of Medicine,Washington, District of Columbia Received 9/18/06; revised 12/16/06; accepted 1/24/07. that cyclin D1 plays an important role in cell cycle control and Grant support: Ministry of Education, Culture, Sports, Science, andTechnology of carcinogenesis in the liver and could be a potential therapeutic Japan grants P-03346 (I. Ozaki) and 16590606 (T. Mizuta) and NIH grants target in HCC. Expression of the cyclin D1 gene is regulated by R01CA70896, R01CA75503, R01CA86072, R01CA93596, and R01CA107382 various factors, and nuclear factor nB (NF-nB) is a transcription (R.G. Pestell). factor that is presumed to play an important role in this 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 regulation (9–14). with 18 U.S.C. Section 1734solely to indicate this fact. Nuclear factor nB (NF-nB) was originally identified as a Note: I. Ozaki and H. Zhang contributed equally to this work. regulator of immunoglobulin n light chain gene expression, Requests for reprints: Iwata Ozaki, Health Administration Center, Saga Medical which controls the inflammatory process (15). NF-nB tran- School, Saga University, 5-1-1Nabeshima, Saga 849-8501, Japan. Phone: 81-952- n 34-3215; Fax: 81-952-34-2017; E-mail: [email protected]. scriptional activity is normally inhibited by I B proteins. On F 2007 American Association for Cancer Research. stimulation by extracellular inducers such as tumor necrosis doi:10.1158/1078-0432.CCR-06-2308 factor-a (TNF-a) or IL-1h,InBa is rapidly phosphorylated by

Clin Cancer Res 2007;13(7) April 1, 20 07 2236 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Inhibition of NF-kB Activation by Vitamin K2 the InBkinase(IKK)complexonSer32 and Ser36.The cyclin D1 cDNA or p65 NF-nB subunit was synthesized by reverse phosphorylation of InB leads to the subsequent proteasomal transcription PCR (RT-PCR) from human liver mRNA. The oligonu- degradation of InBa by ubiquitination, which stimulates the cleotides introduced with restriction enzyme sites that were used for the n nuclear translocation of NF-nB and the subsequent activation of cloning of full-length human cyclin D1 or p65 NF- B subunit cDNA ¶ genes involved in the immune and inflammatory responses. In were as follows: cyclin D1, forward 5 -CTCGAGATGGAACAC- CAGCTCCTGTGC-3¶, reverse 5¶-AAGCTTTCCCTTCTGGTATC-3¶; p65 addition to its roles in the immune response and inflamma- n ¶ n NF- B subunit, forward 5 -CTCGAGATGGACAGCTGTTCCCCTCATC- tion, NF- B also influences cell growth and survival by 3¶, reverse 5¶-GGATCCTTAGGAGCTGATCTGACTC-3¶. RT-PCR products regulating genes involved in cell growth, apoptosis, and were cloned into pT7-Blue-T vector (Novagen, Madison, WI) and metastasis. Due to these roles, NF-nB has also been implicated subcloned into pcDNA3.1(+) (Invitrogen, San Diego, CA) for construc- in cancer development (13, 16, 17). tion of the mammalian expression vector driven by the cytomegalovirus Recently, Habu et al. (18) reported that menatetrenone, a promoter. vitamin K2 analogue, suppressed the de novo development of Cell proliferation assay. The sensitivity of hepatoma cells to vitamin HCC in cirrhotic patients, and we showed that the recurrence of K2 was determined with the WST-1 proliferation assay kit (Takara, HCC after surgical resection or ablation therapy is suppressed Kyoto, Japan) as previously described (28). The cells were seeded in Â 4 by menatetrenone administration in clinical settings (19). 24-well culture plates at a density of 1 10 per well and incubated at 37jC for 24 h. The cells were subsequently incubated with vitamin K2 Vitamin K is a fat-soluble vitamin that regulates clotting factor at the indicated concentrations for 48 h. The cells were then incubated production by acting as a coenzyme for a vitamin K–dependent with WST-1 reagents and the absorbance of formazan products at carboxylase that catalyzes the carboxylation of glutamic acid 450 nm was measured with a CS-9300 microplate reader (Shimadzu, residues to produce g-carboxyglutamic acid (20). Vitamin K is Tokyo, Japan). similarly involved in the regulation of bone metabolism by Fluorescence-activated cell sorting analysis. The fluorescence-activated g-carboxylation of bone matrix proteins (21). Vitamin K can be cell sorting (FACS) analysis of propidium iodide–stained nuclei was divided into two groups: naturally produced vitamin K1 done as previously described (29). Briefly, cells were plated at a density (phytonadione) and vitamin K2 (menaquinone) and chemi- of 2 Â 105 per well in six-well dishes and were incubated at 37jC for cally synthesized vitamin K3 (menadione). In addition to the 24 h. The cells were further incubated with the indicated concentrations physiologic roles of vitamin Ks, vitamin K3 and its derivatives of vitamin K2 for 48 h. The cells were then harvested by trypsinization, collected by centrifugation, and suspended in hypotonic lysis buffer show potent antiproliferative effects against tumor cell lines A in vitro (0.1% Triton X-100 in PBS), 0.1 mg/mL RNase, and 40 g/mL (22, 23). However, vitamin K3 is not currently used for j in vivo propidium iodide. After 30 min at 37 C, the cells were analyzed with cancer treatment because of its high toxicity. Vitamin K2 a FACSCalibur cytofluorometer (Becton Dickinson, San Jose, CA) and and its derivatives also show antiproliferative effects (although the percentage of cells in different cell cycle was determined. less potent than those of vitamin K3) against leukemia and RNA isolation and semiquantitative RT-PCR. The total RNA was HCC cell lines (24–26). Vitamin K2 also has the ability to extracted from the cultured HCC cells using ISOGEN (Nippon Gene, induce the differentiation of leukemic cells (24) and has been Tokyo, Japan) according to the manufacturer’s instructions. The used for treatment of myelodysplastic syndromes (27). concentration of RNA was determined with a spectrophotometer and Several reports have shown that vitamin K2 alters the the integrity of the samples was confirmed by visualizing 28S and 18S expression of growth-related genes and inhibits cell cycle rRNA bands under UV light after gel electrophoresis. Semiquantitative RT-PCR was done as previously described (30). Briefly, 1 Ag of total progression at the G -S phase in several cancer cells including 1 RNA was reverse transcribed with reverse transcriptase (Takara) using HCC. However, the precise mechanisms by which vitamin K2 random primers. Subsequently, each RT reaction mixture was subjected induces cell cycle arrest and growth inhibition have not been to PCR amplification using TaqGold polymerase (Perkin-Elmer, fully elucidated. We therefore studied the effects of vitamin K2 Branchburg, NJ) with cycle numbers varying from 15 to 40. Each cycle on HCC cell proliferation and investigated the mechanisms consisted of heat denaturation (94jC for 1 min), annealing (55jC for responsible for these effects. In this report, we show that 1 min), and extension (72jC for 2 min). The PCR products were size vitamin K2 inhibits HCC cell proliferation by regulating cyclin fractionated on a 2% agarose gel and visualized under UV light. The D1 expression through the inhibition of NF-nB activation. 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 Materials and Methods 5¶-ACGGATCAGTCTTTGGGTCCA-3¶, 409 bp; p21 (BC001935), sense 5¶-CGATGGAACTTCGACTTTGTC-3, antsense 5¶-GATTAGGGCTT- Cell lines and reagents. The human HCC cell lines HepG2, Hep3B, CCTCTTGG3¶, 356 bp; cyclin D1 (BC001501), sense 5¶-GTGCTGC- and Huh7 were obtained from the Japanese Cancer Research Resources GAAGTGGAAAC-3¶, antisense 5¶-AAGCTTTCCCTTCTGGTATC-3¶, 986 Bank (Osaka, Japan). The cells were cultured and maintained in DMEM bp; glyceraldehyde 3-phosphate dehydrogenase (NM002046), sense (Gibco BRL, Gaithersburg, MD) containing 10% FCS (Gibco BRL). 5¶-CCACCCATGGCAAATTCCATGGCA-3¶, antisense 5¶-TCTAGACGG- Menatetrenone, a vitamin K2 analogue, was provided by Eisai Co. CAGGTCAGGTCCACC-3¶, 593 bp. (Tokyo, Japan). Western blotting. The protein expression ofp27, p21, cyclin D1, and Plasmids. Cyclin D1 promoter-luciferase (CD1Luc) reporter con- IKKa,InBaphosphorylation, and InBadegradation were investigated by structs were previously described (10, 11). CD1Luc with 1,745 bp of Western blotting. Cells (2 Â 106) cultured under various conditions were cyclin D1 promoter (À1745CD1Luc), truncated CD1Luc collected and lysed with extraction buffer containing 50 mmol/L Tris (pH (À964CD1Luc and À66CD1Luc), CD1Luc mutated in activator protein 7.5), 150 mmol/L NaCl, 0.1% SDS, 5 mmol/L EDTA (pH 8.0), 1 mmol/L 1 (AP-1)–binding site (À964AP-1mutCD1Luc), and CD1Luc mutated phenylmethylsulfonyl fluoride, 10 Ag/mL trypsin inhibitor, and 50 mmol/L in NF-nB–binding site (À66NF-nBmutCD1Luc) were used for the iodoacetamide. After 30 min at 4jC, the cell debris was eliminated by luciferase assay (Fig. 3A). The luciferase reporter gene containing the centrifugation at 15,000 rpm for 20 min and the supernatant was collected. NF-nB responsive element was purchased from BD Biosciences After measuring the protein concentration with a protein assay kit (Bio-Rad, Clontech (Palo Alto, CA). Complementary DNA coding full-length Hercules, CA), 40 Ag of protein were mixed with SDS sample buffer,

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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 24h of incubation, the cells were collected and stained with propidium iodide, and the DNA content was subsequently analyzed by FA CS. The histograms represent total DNA contents, and the percentages of cells with G1are shown (M1). Representative results from three independent experiments.

Clin Cancer Res 2007;13(7) April 1, 20 07 2238 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Inhibition of NF-kB Activation by Vitamin K2 separated by SDS-PAGE, transferred to a polyvinylidene difluoride Stable transformation of HCC cells. Plasmids encoding human p65 membrane (Bio-Rad), and blocked with 0.1% Tween 20 and 5% skim NF-nB subunit (pcDNA/p65), cyclin D1 (pcDNA/CD1), or pcDNA milk overnight. The membranes were incubated with anti-p27 (BD alone (as a control) were introduced into HepG2 cells using Lipofect- Transduction Laboratories, Franklin Lakes, NJ), p21 (Oncogene Research amine (Gibco BRL) according to the manufacturer’s instructions. The Products, San Diego, CA), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, transfected HepG2 cells were treated with 500 ng/mL G418 for 2 weeks CA), anti–phospho-specific InBa,totalInBa(New England Biolabs, Beverly, before selection. Individual clones of HepG2 cells transduced with MA), and IKKa (Santa Cruz Biotechnology) antibodies in PBS with 1% pcDNA/p65 or pcDNA/CD1 were analyzed. Clones overexpressing p65 bovine serum albumin for 1 h. Antihuman h-actin antibodies (Biomedical or cyclin D1 were selected and subjected to further analysis. Technologies, Stoughton, MA) were used as a control. The membranes were Statistical analysis. Differences were analyzed using Student’s t test, washed thrice with 0.1% Tween 20 in PBS and stained with horseradish and P < 0.05 was considered significant. All experiments were done at peroxidase–conjugated secondary antibodies. All immunoblots were least thrice. The data are shown as the mean F SD. detected by the enhanced chemiluminescence system (Amersham, Buck- inghamshire, United Kingdom) according to the manufacturer’s instruc- Results tions. Transfection and luciferase reporter gene assay. The effects of vitamin Vitamin K2 induces growtharrest in theG phase in HCC n 1 K2 on cyclin D1 promoter activity and NF- B transcriptional activity cells. Three HCC cell lines, HepG2, Hep3B, and Huh7, were were detected by luciferase assay using the method described by the treated with different concentrations of vitamin K2 for 48 h, supplier (Dual-Luciferase Reporter Assay System, Promega, Madison, WI). Cyclin D1 promoter luciferase reporter plasmids (À1745CD1Luc, and their growth was subsequently assayed by WST-1 prolifer- À964CD1Luc, À964AP-1mutCD1Luc, À66CD1Luc, and À66NF- ation assay. As shown in Fig. 1A, vitamin K2 significantly nBmutCD1Luc) and NF-nB-luciferase reporter plasmid were used for inhibited the growth of HCC cells in a dose-dependent manner. assay. Luciferase expression plasmid pRL-SV40 (Promega) was cotrans- The DNA synthesis was also suppressed by vitamin K2 in these fected to normalize for transfection efficiency. The HCC cells were cells as shown by bromodeoxyuridine incorporation assay seeded onto six-well plates at 1 Â 105 per well without antibiotics and (data not shown). To clarify the mechanism of vitamin K2– incubated until 80% confluent at 37jC. Next, the cells were washed induced growth inhibition in HCC cells, we did a FACS analysis twice with OPTI-MEM I Reduced Medium (Life Technologies, Rockville, in these cells treated with vitamin K2. As shown in Fig. 1B, the MD), followed by the addition of 2 mL of OPTI-MEM I Reduced FACS analysis of the vitamin K2–treated cells showed a Medium containing 5 Ag of target gene reporter plasmid, 1 Ag of pRL- significantly increased number of cells in the G phase. SV40 luciferase plasmid, and 15 AL of Lipofectamine 2000 reagent (Life 1 Technologies). After 6 h of incubation, the medium was changed and However, the proportion of pre-G1 cells (presumably apoptotic the transfected cells were treated with indicated doses of vitamin K2. cells) was not significantly changed after vitamin K2 treatment, After 24 h of treatment, the cells were washed twice with PBS and thus suggesting that vitamin K2 suppresses the growth of HCC carefully scraped into 1Â passive lysis buffer (Promega). The cell cells by inducing G1 arrest. extracts were immediately assayed for luciferase activity using a Vitamin K2 inhibits the expression of cyclin D1 in HCC Berthold Luminometer (MLR-100 Micro Lumino Reader, Corona cells. Cyclin D1 is required for the progression of cells from Electric, Ibaragi, Japan). the G1 to S phase. Because we observed G1 arrest in vitamin Electrophoretic mobility shift assay. The NF-nB binding activity in K2–treated HCC cells, we decided to analyze the effect of nuclei isolated from hepatoma cells was determined by electrophoretic vitamin K2 on the expression of cyclin D1. The treatment of mobility shift assay. The nuclear protein extractions and electrophoretic cells with vitamin K2 significantly down-regulated cyclin D1 mobility shift assay were done as previously described (31). Briefly, 5 Ag of nuclear protein were incubated for 30 min at room temperature with expression at both the mRNA (Fig. 2A and B) and protein levels binding buffer [20 mmol/L HEPES-NaOH (pH 7.9), 2 mmol/L EDTA, (Fig. 2C) in all the HCC cell lines. As shown in Fig. 2, the 100 mmol/L NaCl, 10% glycerol, 0.2% NP40], poly(deoxyinosinic- expression levels of the Cdk inhibitors p27 and p21 were also deoxycytidylic acid), and 32P-labeled double-stranded oligonucleotide determined in these cells after vitamin K2 treatment. We containing the NF-nB binding motif (Promega). The sequence of the observed that the responses of p21 and p27 to vitamin K2 double-stranded oligomer used for electrophoretic mobility shift assay varied from cell to cell. The expression of both p27 and p21 was was 5¶-AGTTGAGGGGACTTTCCCAGGC-3¶ (only the sense strand is increased after vitamin K2 treatment in HepG2 cells. A slight shown). The reaction mixtures were loaded on a 4% polyacrylamide gel increase of p27 expression was shown in Huh7 cells whereas Â and electrophoresed with a running buffer of 0.25 Tris-borate EDTA. the level of p21 remained low. In Hep3B cells, there was an After the gel was dried, the DNA-protein complexes were visualized by increase in the level of p21 expression after vitamin K2 autoradiography. IKK assay. The assay for endogenous IKK was done according to a treatment but p27 expression level was not altered. These previously method described with minor modifications (32). The cells results indicate that cyclin D1 and Cdk inhibitor expression is were treated with vitamin K2 alone or in combination with TPA, IL-1, or regulated during vitamin K2–induced growth inhibition in TNF-a and subjected to IKK kinase assay. Cytoplasmic extracts (300 Ag) HCC cells. were immunoprecipitated with 2 Ag of anti-IKKa antibody and then Vitamin K2 inhibits cyclin D1 promoter activity in a NF-kB– treated with 20 AL of protein G-Sepharose (Pierce, Rockford, IL). After dependent manner. To further investigate the effect of vitamin 2 h, the beads were washed with lysis buffer and then with the kinase K2 on cyclin D1 expression, the cyclin D1 promoter activity buffer [50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, and 2 mmol/L was examined after vitamin K2 treatment. The cyclin D1 pro- DTT]. The immune complex was then assayed for kinase activities with moter luciferase reporter plasmids À1745CD1Luc, À964CD1Luc, the use of kinase assay buffer containing 10 ACi [g-32P]ATP, 10 Amol/L À964AP-1mutCD1Luc, À66CD1Luc, and À66NF-nBmutCD1Luc unlabeled ATP, and 2 Ag glutathione S-transferase-InB (1–54). After j (Fig. 3A) were transfected. Vitamin K2 significantly inhibited the incubation at 30 C for 30 min, the reaction was stopped by boiling the À solution in 4Â SDS sample buffer. The reaction mixture was then activity of 1745CD1Luc, a full-length cyclin D1 promoter resolved on 12% SDS-PAGE. The gel was dried and exposed to X-ray films luciferase reporter plasmid, in all three cell lines (Fig. 3B). To (Fuji) at À80jC. The total amount of IKKa was determined by Western examine whether vitamin K2–mediated cyclin D1 promoter blot analysis. inhibition depends on NF-nB activity, HepG2 cells were

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Fig. 2. Effects of vitamin K2 on the expression of cyclin D1, p27, and p21in hepatoma cells. A, semiquantitative RT-PCR for cyclin D1, p27, and p21mRNA 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 p21protein expression in vitamin K2^ treated hepatoma cells.The expression of h-actin was used as a control. transfected with cyclin D1 promoter luciferase reporter plas- mobility shift assay, the nuclear protein extracts were incubated mids, which included either the wild-type, mutant AP-1, or with a 32P-labeled oligonucleotide containing a conserved NF-nB binding site, and the effect of vitamin K2 on their NF-nB binding sequence. As shown in Fig. 4A, Hep3B and activities was investigated. As shown in Fig. 3C, vitamin K2 Huh7 cells constitutively expressed NF-nB activity, and vitamin inhibited the activities of À1745CD1Luc, À964CD1Luc, K2 inhibited the basal level of NF-nB binding activity in these À964AP-1mutCD1Luc, and À66CD1Luc in a dose-dependent cells. To confirm the specificity of binding, an excess of manner but had no effect on the activity of À66NF- unlabeled NF-nB–binding oligonucleotide abolished the nBmutCD1Luc, which contained the mutated NF-nB site. To NF-nB binding whereas oligonucleotide containing a NF-nB confirm the role of NF-nB in cyclin D1 promoter regulation by mutant binding sequence did not change the NF-nB binding vitamin K2, HepG2 cells were transfected with À1745CD1Luc activity. These results showed that the binding of the NF-nB and p65 cDNA, a subunit of NF-nB. As shown in Fig. 3D, probe was specific. In addition, supershift assays indicated that vitamin K2 could not inhibit cyclin D1 promoter activity when the primary NF-nB complex regulated by vitamin K2 was the p65 was cotransfected, indicating that the effect of vitamin K2 p50-p65 heterodimer. on cyclin D1 promoter activity was NF-nB dependent. These To determine whether vitamin K2 could inhibit the results indicate that NF-nB plays an important role in the activation of NF-nB by various stimulatory agents, HCC cells vitamin K2–mediated inhibition of cyclin D1 promoter were treated with TPA, TNF-a, or IL-1. As expected, NF-nB was activity. activated after treatment with TPA, TNF-a, or IL-1h, as seen by Vitamin K2 inhibits the DNA binding activity and transcrip- the increased DNA binding activity. The NF-nB binding activity tional activity of NF-kB in hepatoma cells. Because NF-nB plays induced by these stimuli was significantly inhibited when an essential role in the regulation of cyclin D1 promoter vitamin K2 was simultaneously added to the cells (Fig. 4B). activity, we evaluated the effect of vitamin K2 on the DNA Vitamin K2 also inhibited the basal level of NF-nB binding binding activity of NF-nB in HCC cells. For the electrophoretic activity although the basal NF-nB level of HepG2 cells was very

Clin Cancer Res 2007;13(7) April 1, 20 07 224 0 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Inhibition of NF-kB Activation by Vitamin K2 low. These results indicated that vitamin K2 could inhibit both TNF-a–, or IL-1h–induced InBa phosphorylation in HepG2 the basal and induced NF-nB binding activities in HCC cells. cells. In addition, the degradation of total InBawas restored by To further assess whether the inhibition of DNA binding by vitamin K2 treatment. vitamin K2 affected NF-nB transcriptional activity, we did a The phosphorylation of InBais controlled by IKK. An in vitro transient assay using a NF-nB-luciferase reporter plasmid in kinase assay using immunoprecipitated IKK from HepG2 cells HepG2 cells. As shown in Fig. 4C, vitamin K2 inhibited NF-nB and the glutathione S-transferase-InBa as substrate showed transcriptional activity in a dose-dependent manner. Further- that vitamin K2 inhibited the basal and the TPA-, TNF-a–, or more, the effect of vitamin K2 on NF-nB transcriptional activity IL-1h–induced IKK activities (Fig. 5B). However, a Western induced by TPA, TNF-a, or IL-1hwas also evaluated, and we blot analysis of the cell extracts showed no significant change in observed that vitamin K2 significantly inhibited the TPA-, TNF- the protein level of IKKa expression. These results indicated a–, or IL-1h–induced NF-nB transcriptional activity in HepG2 that vitamin K2–mediated IKKa inhibition was responsible for cells. These results show that vitamin K2 inhibits both NF-nB the inhibition of NF-nB activation by vitamin K2. DNA binding activity and NF-nB–dependent promoter activity. Overexpression of p65 and cyclin D1 in HCC cells causes Vitamin K2 inhibits IkBa phosphorylation and IKK activity. resistance to vitamin K2–induced growthinhibition. To further The degradation of InBa and the subsequent release of NF-nB identify the role of NF-nB and cyclin D1 in vitamin K2– require the prior phosphorylation of Ser32 and Ser36 residues. induced growth inhibition in HCC cells, HepG2 cells were Therefore, to investigate whether the inhibitory effect of stably transfected with full-length p65 or cyclin D1 cDNA and vitamin K2 is mediated by the alteration of InBa phosphory- the cells overexpressing p65 or cyclin D1 were selected. A lation, we treated the HepG2 cells with vitamin K2 and Western blot analysis showed significantly more p65 or cyclin examined their protein extracts for phospho-InBa and InBa D1 protein in the p65- or cyclin D1–transfected clones than in expression. As shown in Fig. 5A, vitamin K2 dose-dependently the parental or mock-transfected cells (Fig. 6A). In comparison inhibited both the basal InBa phosphorylation and the TPA-, with the results seen in the parental and mock-transfected cells,

Fig. 3. Effect of vitamin K2 on cyclin D1promoter activity. A, the maps of cyclin D1luciferase plasmids. B, vitamin K2 inhibited cyclin D1promoter activity in hepatoma cells. The cells were transiently transfected with À1745CD1Luc 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 D1promoter activity in a NF-nB ^ dependent manner. HepG2 cells were transiently transfected with cyclin D1 promoter plasmids as described in (B) and were then treated with vitamin K2. After 24h, 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-nB inhibited the vitamin K2^ induced cyclin D1promoter suppression. HepG2 cells were transiently cotransfected with À1745 CD1L u c and p65 subunit cDNA of NF-nB as described in (B), treated with vitamin K2 for 24h, 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.

www.aacrjournals.org 2241 Clin Cancer Res 2007;13(7) April1,2007 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical vitamin K2 failed to inhibit the proliferation of cells that Discussion overexpressed p65 or cyclin D1. Our findings showed that these p65- or cyclin D1–transfected clones possessed a significant A growing body of literature has linked NF-nB activity to the resistance to vitamin K2–induced growth inhibition (Fig. 6B). onset and progression of tumorigenesis (13, 33). The aberrant

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

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Fig. 5. A, vitamin K2 inhibited basal InBa phosphorylation and TPA-,TNF-a ^,orIL-1h ^ induced InBa phosphorylation in hepatoma cells. HepG2 cells were treated with vitamin K2 alone or withTPA,TNF-a,orIL-1h in the presence or absence of vitamin K2 for 24h.Western blot analysis was done with anti ^ phospho-specific antibodies to detect InBa phosphorylation. The expression of total InBa was also examined using the expression of h-actin as a control. B, vitamin K2 inhibited basal InBa kinase activity and TPA-,TNF-a ^,orIL-1h ^ induced InBa kinase activity in hepatoma cells. HepG2 cells were treated with vitamin K2 alone or withTPA,TNF-a,orIL-1hin the presence or absence of vitamin K2 for12h. Cytoplasmic extracts (300 Ag) were immunoprecipitated with antibodies against IKKa. An immune complex kinase assay was subsequently done as described in Materials and Methods.Toexamine the effect of vitamin K2 on the expression of IKKa protein,Western blot analysis was done with anti-IKKa antibodies. activation of NF-nB has been reported in many malignant affects the interaction of hepatitis viral proteins and NF-nB neoplasms such as human leukemias, lymphomas, and solid activation. One mechanism by which transcription factor NF- tumors. Moreover, oncogenic viruses, including T-cell leukemia nB regulates cell proliferation is through the control of cyclin virus 1 and EBV, activate NF-nB during the transformation D1 expression (11–13). Cyclin D1 promotes the G1-S phase of process (34–36). In the liver, NF-nB activity is involved in the cell cycle and is frequently overexpressed in many human hepatocyte regeneration after liver injury, indicating its critical cancers including HCC (5–8). The expression of cyclin D1 is role in hepatocyte growth regulation (37). The constitutive one of the prognostic factors of HCC patients (9), and the activation of NF-nB was reported in HCC and is considered to down-regulation of cyclin D1 inhibits HCC growth in animal be involved in hepatocarcinogenesis (38, 39). HCC develop- models (46). Therefore, both NF-nB and cyclin D1 are regarded ment is closely associated with chronic infection by the as potential therapeutic targets for the treatment of HCC. hepatitis B or C virus (1). Recent reports indicated that Earlier research showed that vitamin K was a cofactor of components of hepatitis viruses such as the hepatitis B virus g-carboxylation and was necessary for producing functional X antigen or hepatitis C virus core protein were able to proteins involved in blood coagulation reactions. Recent modulate NF-nB activity, suggesting that NF-nB activation plays studies have revealed that vitamin Ks are involved in the a role in hepatocarcinogenesis (40, 41). Other recent studies g-carboxylation of a wide variety of proteins besides clotting- have shown the association of NF-nB activity with the related proteins, and g-carboxylation–independent effects of progression of HCC as well as the association between the vitamin Ks have been observed, indicating that vitamin Ks inhibition of NF-nB activation and the suppression of HCC possess both g-carboxylation–dependent and –independent progression (42–44). Many cytokines such as TNF-a and actions (47). If vitamin K–dependent proteins are involved in interleukins induce NF-nB activation and are involved in HCC cell growth regulation, warfarin (which inhibits g- chronic inflammation induced by hepatitis viruses. This carboxylation of vitamin K–dependent proteins and is known chronic inflammation is linked to hepatocarcinogenesis to increase des-r-carboxy-prothrombin in hepatic cells) might (reviewed in ref. 45). In this study, we have shown that increase the growth of hepatoma cells. However, warfarin has vitamin K2 inhibited cytokine-induced NF-nB activation by been reported to suppress the growth of several types of cells suppressing IKK activity, and this action of vitamin K2 might (48) and the growth of HCC cells was suppressed by warfarin contribute to the suppression of HCC development in clinical treatment.5 The involvement of vitamin K–dependent proteins settings (18, 19). Because many cases of HCC are related to in hepatoma cell growth should therefore be examined further. hepatitis virus infection (1) and hepatitis viral antigens are Vitamin K2 is also involved in bone metabolism via the shown to induce NF-nB activation (40, 41), it will be interesting g-carboxylation of bone matrix proteins and the regulation of to examine the effects of vitamin K2 on the interaction of these bone reabsorption. It is often used for the treatment of viral antigens and NF-nB 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 5 Zhang et al., unpublished data.

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Fig. 6. A, Western blot analysis of p65, cyclin D1, and h-actin protein expression in parental, mock-, and p65- or cyclin D1^ transfected HepG2 cells. HepG2/mock, mock-transfected HepG2 cells; HepG2/p65#1to #3, representative individual clones of HepG2 cells stably transfected with full-length p65 cDNA; HepG2/CD1#1to #3, representative individual clones of HepG2 cells stably transfected with full-length cyclin D1cDNA. B, p65 or cyclin D1overexpression 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.

osteoporosis in Japan (49). The mechanism by which vitamin K2 inhibited the growth and invasiveness of HCC cells via K2 inhibits osteoclastic activity involves the receptor activator protein kinase A activation and Rho kinase inhibition. of NF-nB (RANK)/RANK ligand signaling pathway that links to However, the molecular mechanisms of protein kinase A– NF-nB activation. Takeuchi et al. (50) reported that vitamin K2 mediated growth inhibition in HCC cells have not been fully suppressed RANK mRNA expression and osteoclastic activity in elucidated. We treated several HCC cells with protein kinase A murine bone marrow cells, suggesting the involvement of stimulator, but we could not induce the inhibition of cyclin D1 NF-nB signaling mediated by the RANK/RANK ligand system. gene expression and cell proliferation (data not shown). However, the direct evidence of NF-nB activity modulation by Therefore, although protein kinase A might be partially vitamin K2 was not shown. Cyclin D1 is a downstream target of involved in vitamin K2–induced inhibition of HCC cell growth RANK/RANK ligand signaling (51) and recent studies have and invasion, mechanisms other than protein kinase A also shown that cyclin D1 also regulates RANK expression (52). seem to play important roles in vitamin K2–induced HCC Therefore, cyclin D1 functions both downstream and upstream growth suppression. Besides vitamin K2, other vitamin K of the RANK signaling pathway. analogues are reported to inhibit HCC cell growth and induce Recently, several studies have shown that vitamin K2 apoptosis. Carr et al. (47) showed that a new synthetic vitamin modulates transcriptional activity in bone-derived (53) and K analogue, compound 5, inhibited Cdc25A phosphatase HCC cells (54). Tabb et al. (53) showed that vitamin K2 binds activity and reduced HCC cell growth. However, the mecha- to steroid and xenobiotic receptor (SXR) and mediates gene nism by which vitamin K2 inhibits HCC cell growth seems to expression in the osteosarcoma cell line. It has been reported be different from that of compound 5 because vitamin K2 does that SXR is expressed abundantly in normal liver tissue (55); not affect Cdc25A phosphatase activity (47) and does not however, the expression of SXR in human HCC has not been induce apoptosis. studied. We examined the expression of SXR in several human In this study, we revealed that vitamin K2 inhibited IKK HCC cell lines and found that three of five HCC cell lines kinase activity and therefore suppressed InB phosphorylation expressed SXR mRNA and protein. Vitamin K2 suppressed all and NF-nB activation. We do not yet know the specific target five HCC cell lines irrespective of SXR expression and did not of vitamin K2 action functioning upstream of IKK. Recent affect SXR expression in these cells.6 Therefore, SXR is unlikely studies have shown that several kinases that phosphorylate to be involved in vitamin K2–induced HCC cell growth serine residue could possibly regulate an IKK/InB/NF-nB suppression. Otsuka et al. (54) recently reported that vitamin 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 6 Ozaki et al., unpublished data. activity.

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Iwata Ozaki, Hao Zhang, Toshihiko Mizuta, et al.

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