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

"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

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 estab-

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

Otsuka et al. reported that MK-4 inhibited the growth and invasion of HCC via activation of protein kinase A (PKA) and the subsequent inhibition of Rho activation. It has been reported that PKA induces cell-cycle arrest at the G1 phase and the G2/M phase [11]. Hitomi et al. [12] reported that MK-4-induced G1 arrest of the cell cycle via significantly reduced protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not the p16INK4a Cdk inhibitor in PLC/PRF/5 HCC cells *in vivo*. Cyclin D1 promotes the G1/S phase of the cell cycle and is frequently overexpressed in many human cancers. Matsumoto et al. [13] reported that MK-4-induced arrest of the G1 phase of the cell cycle and apoptosis via activated extracellular signal-regulated kinase ½ (ERK1/2) in a mitogen-activated ERK-regulating kinase-dependent manner in Hep3B HCC cells. Ozaki et al. [10] reported that MK-4 inhibited the growth of HCC cells via suppression of cyclin D1 expression through inhibited IκB kinase activity, and

**2. Mechanisms of growth inhibition of HCC cells by MK-4**

therefore suppressed IκB phosphorylation and NF-κB activation.

important form of vitamin K.

312 Vitamin K2 - Vital for Health and Wellbeing

lished in Japanese patients with osteoporosis.

investigated previously, they remain unclear.

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