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

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 therefore suppressed IκB phosphorylation and NF-κB activation.

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