**1. Introduction**

Vitamin K has two types of molecular homologues: phylloquinone (vitamin K1, PK) and menaquinone (vitamin K2, MK-n). These homologues have the same aromatic naphthoquinone

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

"head" but different hydrocarbon "tails": PK with a phytyl tail and MK-n with an unsaturated isoprenoid chain. MK-n can be classified into 14 types based on the length of the unsaturated isoprenoid chain, where "n" quantifies the repeating isoprenyl units. MK-4 is normally synthesized from PK in certain animal tissues by removal of the phytyl tail of PK to produce menadione (vitamin K3, MD) as an intermediate. The intermediate is then condensed with a geranylgeranyl tail. UbiA prenyltransferase domain-containing protein 1 (UBIAD1) converts MD to MK-4 with geranylgeranyl diphosphate [1]. Forthis reason,MK-4 appears to be the most important form of vitamin K.

In addition, Xia et al. [14] reported that the inhibitory effect on NF-κB activity by MK-4 is mediated through the inhibition of protein kinase C α and ε kinase activities, as well as subsequent inhibition of protein kinase D1 activation. Kaneda et al. proposed that MK-4 suppressed HuH7 HCC tumor malignancy via induced Cx32 expression through the reduction of Cx43 expression. Consequently, gap junctional intercellular communication through Cx32 is activated. Normal hepatocytes communicate with neighboring cells via Cx32-containing gap junction communication, a process essential for suppressing tumorigenesis [15]. Yamamoto et al. suggested that regulation of the expression of the hepatoma-derived growth factor gene is one of the crucial mechanisms of MK-4-induced cell growth suppression in HCC. Hepatomaderived growth factor stimulates the proliferation of HCC cells after its translocation to the nucleus by use of bipartite nuclear localization signals [16]. Azuma et al. [17] suggested that activation of steroid and xenobiotic receptors (SXR) by MK-4 contributes to the tumor suppressive effects on HCC cells. Li et al. suggested that MK-4 inhibited the growth of Smmc-7721 HCC cells by induction of apoptosis involving caspase-8 activation and p53. This apoptotic process was not mediated by the caspase-9 pathway [18]. Yao et al. suggested that the mechanism involved induced p53 and increased p21 levels that eventually lead to cell-cycle arrest in the G2 phase. In addition, they suggested that the antitumor effect of MK-4 may be improved

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

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In clinical trials with cirrhotic women, Habu et al. demonstrated that daily doses of 45 mg MK-4 decreased the risk of HCC to about 20% compared with the control group. Twenty-one women were in the treatment group and 19 in the control group [20]. Mizuta et al. reported that a daily dose of 45 mg MK-4 suppressed the recurrence of HCC in HCC patients who had undergone curative resection or percutaneous local ablation therapy. Thirty-two patients were in the treatment group and 29 in the control group [21]. From the results of these small-scale clinical trials, it is expected that MK-4 acts as a chemopreventive agent for HCC. However, a recent larger scale study that enrolled 548 patients at 31 study sites, and included a placebocontrolled, double-blind trial, demonstrated that the efficacy of vitamin K2 in suppressing HCC recurrence could not be confirmed [22]. The poor anticancer activity of MK-4 observed in this Japanese trial may have been a consequence of the large study design and meant that MK-4 could not be developed as an anticancer drug. However, various attempts are being

by silencing BCL-2 expression in SMMC-7721 HCC cells [19].

made to try to improve the anticancer effect of MK-4 in HCC.

**4.1. A novel chemosynthetic vitamin K derivative**

**4. Improvement of the antiproliferative effect of vitamin K2**

Carr et al. demonstrated that a new, chemically synthesized vitamin K analog, compound 5 (Cpd5), inhibited Cdc25A phosphatase activity and particularly reduced HCC cell growth through arrest of the G1/S phase of the cell cycle. Inhibition of Cdc25A by Cpd5 results in

**3. Clinical trials of MK-4 to treat HCC**

Vitamin K (PK and MK-n) plays a major role in the clotting cascade by acting as a coenzyme for a vitamin K-dependent carboxylase. The carboxylase catalyzes the carboxylation of glutamic acid (Glu) residues to produce γ-carboxyglutamic acid (Gla). Vitamin K also appears to play a role in the regulation of bone metabolism through a similar mechanism that involves γ-carboxylation of pro-osteocalcin. Interestingly, MK-4 intake seems to be associated with greater effects of reduced bone resorption compared with PK consumption [2]. Clinically, high doses (45 mg daily) of MK-4 have been used as an approved treatment for osteoporosis in Japan since 1995. Therefore, the safety of long-term administration of MK-4 has been established in Japanese patients with osteoporosis.

Over the last decade, many reports have shown that MK-4 has antioncogenic effects within various cancer cell lines, including leukemia, lung cancer, ovarian cancer, prostate cancer, and hepatocellular carcinoma (HCC) [3–6]. Specifically, numerous articles describe the effects of MK-4 against HCC. This is because des-γ-carboxy prothrombin (DCP, PIVKA-II), an abnormal prothrombin that is not completely carboxylated, is a well-recognized HCC-specific tumor marker, and a predictor of vascular invasion, metastasis, and tumor recurrence [7]. It has been reported that apoptosis, cell-cycle arrest, and autophagy are involved in the antitumor activity of MK-4 [8–10]. Although the possible mechanisms of the antitumor effect of MK-4 have been investigated previously, they remain unclear.
