Vitamin K2 suppresses malignancy of HuH7 hepatoma cells via inhibition of connexin 43
Introduction
Vitamin K is a family of structurally similar lipophilic 2-methyl-1,4-naphthoquinones, including phylloquinone (K1), menaquinones (K2), and menadione (K3). VK2 is well known as a co-factor of γ-glutamylcarboxylase which converts glutamic acid residues into γ-carboxyglutamyl (Gla) residues of blood coagulation factors and bone matrix proteins [1], [2]. Meta-analysis of randomized controlled trials showed that supplementation with VK2 reduces bone loss [3]. More recently, many studies suggest that VK2 has inhibitory effects against hepatic cancer. In a clinical study, VK2 prevented development of hepatocellular carcinoma (HCC) cells in female patients with liver cirrhosis [4]. In vitro studies indicate that the anti-angiogenic activity and anti-proliferation activity of VK2 might explain the anti-cancer effect of VK2 [5], [6], [7]. However, the precise mechanism by which VK2 inhibits hepatic cancer is not fully understood.
Connexin (Cx) is the component protein of the gap junction (GJ) channel, forming structures which mediate intercellular communication. Each GJ is made up of two docked hemichannels (connexons) from neighboring cells, comprising self-assemblies of six Cx proteins [8]. Small molecules (molecular mass of up to 1.5 kDa), ions, second messengers, and metabolites can be directly transferred between neighboring cells through GJs, thereby making possible gap junctional intercellular communication (GJIC) [9], [10]. In many tissues, GJIC is responsible for tissue homeostasis such as cardiac conduction and maintenance of CNS function [11], [12]. A large number of studies have indicated that Cx genes have a tumor-suppressing effect [13], [14], [15], [16], [17]. More than 20 kinds of connexins have been reported and their distributions are tissue specific. Cx32 is dominantly expressed in hepatocytes. Cx32 knockout mice exhibit an increase in the risk of tumorigenesis [18], [19]. Cx43 also has tumor-suppressing effects [13], [14], [16], [20], [21]; however, it is not expressed in normal hepatocytes and the expression is strongly up-regulated in HCC cells [22], [23].
To elucidate the anti-cancer mechanism of VK2, we focused on the effects of VK2 on GJIC activity and Cx gene expression in HCC. The treatment of HuH7 cells with VK2 inhibited proliferation, and induced a normal liver phenotype. We found that GJIC activity in VK2-treated cell was strongly enhanced. We also showed that VK2 up-regulated Cx32 at the transcriptional level. On the other hand, Cx43 transcription was inhibited by VK2-treatment. Finally, we found that Cx43 over-expression abolished the VK2-induced expression of Cx32 and albumin. These findings suggest that VK2 indirectly up-regulates Cx32 expression via reduction of Cx43 promoter activity in HuH7 cells, resulting in an activation of GJIC and induction of normal liver phenotype.
Section snippets
Plasmid construction
The cloning of Cx43 cDNA was described previously (pcDNA3.1-Cx43) [20]. The first PCR product containing the human Cx43 promoter sequence from upstream −1546 to +59 was generated by LA Taq polymerase (Takara, Shiga, Japan) using the following primers: 5′-GGAAATAGGATAGCCAACAAGTAAAAGCAA-3′ (forward) and 5′-GGAGGATGAAGTAAAATGAAAAGGCAAG-3′ (reverse) as the first primers. Human genomic DNA was used as the template. Then, the primers, 5′-AAAGCTAGCGCCAACAAGTAAAAGCAAAA-3′ (forward) and
Vitamin K2 induced normal liver phenotype in HuH7 cells
VK2 has been noted for its anti-hepatoma effects. We examined the effect of VK2 on cell proliferation, albumin (as a marker of normal hepatocyte function) and α-fetoprotein (α-FP, as a marker of highly malignant HCC) expressions in HuH7 cells. When HuH7 cells were treated with VK2, cell growth was strongly inhibited (Fig. 1a). In our experiment, VK2 did not affect cell viability (data not shown). Interestingly, VK2-treated HuH7 cells exhibited an increase in the albumin expression and a
Discussion
We provide the first evidence that VK2 inhibited expression of Cx43 and consequently enhanced GJIC activity between HuH7 cells. This was achieved via up-regulation of Cx32 and resulted in suppression of HuH7 tumor malignancy.
First, we demonstrated that VK2 has tumor-suppressing activity. Recently, many studies have reported that VK2 suppresses growth [5], [6], [7], and these reports are consistent with our present results. We also showed that VK2 induced expression of albumin, a marker of
Acknowledgments
This work was supported in part by research Grants 16659509, 17209058, 17052342 and the 21st Century COE Program from the Japan Society for the Promotion of Science, Tokyo, Japan.
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