Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular
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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 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 phosphor- ylated 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 man- ner 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  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  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.