Inhibitory Effects of Glycyrrhetinic Acid on DNA Polymerase and Inflammatory Activities

We investigated the inhibitory effect of three glycyrrhizin derivatives, such as Glycyrrhizin (compound 1), dipotassium glycyrrhizate (compound 2) and glycyrrhetinic acid (compound 3), on the activity of mammalian pols. Among these derivatives, compound 3 was the strongest inhibitor of mammalian pols α, β, κ, and λ, which belong to the B, A, Y, and X families of pols, respectively, whereas compounds 1 and 2 showed moderate inhibition. Among the these derivatives tested, compound 3 displayed strongest suppression of the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in a cell-culture system using mouse macrophages RAW264.7 and peritoneal macrophages derived from mice. Moreover, compound 3 was found to inhibit the action of nuclear factor-κB (NF-κB) in engineered human embryonic kidney (HEK) 293 cells. In addition, compound 3 caused greater reduction of 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced acute inflammation in mouse ear than compounds 1 and 2. In conclusion, this study has identified compound 3, which is the aglycone of compounds 1 and 2, as a promising anti-inflammatory candidate based on mammalian pol inhibition.


Introduction
The human genome encodes at least 15 DNA polymerases (pols) that conduct cellular DNA synthesis [1,2]. Eukaryotic cells contain 3 replicative pols (α, δ, and ε), 1 mitochondrial pol (γ), and at least 11 nonreplicative pols [β, ζ, η, θ, ι, κ, λ, μ, ν, terminal deoxynucleotidyl transferase (TdT) and REV1] [3,4]. Pols have a highly conserved structure, which means that their overall catalytic subunits show little variance among species. Enzymes with conserved structures usually perform important cellular functions, the maintenance of which provides evolutionary advantages. On the basis of sequence homology, eukaryotic pols can be divided into 4 main families, termed A, B, X, and Y [4]. Family A includes mitochondrial pol γ, as well as pols θ and ν. Family B includes 3 replicative pols (α, δ, and ε) and pol ζ. Family X comprises pols β, λ, and μ, as well as TdT, and lastly, family Y includes pols η, ι, and κ, in addition to REV1. We have been screening for selective inhibitors of each pol derived from natural products, in particular from traditional medical plants, food materials and food additives, for more than 15 years [5,6]. Licorice (fabaceae and glycyrrhiza), a well-known herb plant with biological properties, has been widely used in food additives (sweetener 2 Evidence-Based Complementary and Alternative Medicine and flavoring agent), nutraceuticals (liver protection), and the treatment of various inflammatory diseases since ancient times [7]. Licorice root has been used since ancient Egyptian, Greek, and Roman times in the West, and since the Former Han era (the 2nd-3rd century B.C.) in ancient China in the East. In traditional Chinese medicine, licorice is one of the most frequently used drugs. There are many reports about its pharmacological actions and physiological functions, such as detoxification, induction of decreased blood glucose, and antitumor, anti-inflammatory, hypocholesterolemic, antiestrogen, antihistamine, antiallergic hepatitis, antileukemia, anticancer, and antibiotic-like effects [8].
The seed stock of licorice (Glycyrrhiza glabra L.) has 6 varieties, and licorice production is widely distributed over the Eurasian continent, South Europe, Central Asia, China and Russia. In particular, G. inflata, G. uralensis, and G. glabra are utilized in the production of sweeteners, cosmetics, and medicines. The major constituents of licorice root are triterpenoid saponins, such as glycyrrhizin (3-8%). Glycyrrhizin (glycyrrhizic acid and glycyrrhizinic acid) is a sweet-tasting compound, and is 30-50 times as sweet as sucrose. Glycyrrhizin (compound 1) consists of one molecule of glycyrrhetinic acid (compound 3) and two molecules of glucuronic acid (Figure 1), and it is converted to these constituents by acidic hydrolysis [9].
In our studies of pol inhibitors, we have found that pol λ-selective inhibitors, such as curcumin derivatives [10][11][12], have anti-inflammatory activity against 12-Otetradecanoylphorbol-13-acetate-(TPA-) induced inflammation [13][14][15]. Although tumor promoters, such as TPA, are classified as compounds that promote tumor formation [16], they also cause inflammation and are commonly used as artificial inducers of inflammation in order to screen for anti-inflammatory agents [17]. Tumor promoter-induced inflammation can be distinguished from acute inflammation, which is exudative and accompanied by fibroblast proliferation and granulation. The tumor promoter TPA is frequently used to search for new types of anti-inflammatory compound. TPA not only causes inflammation, but also influences mammalian cell growth [18], suggesting that the molecular basis of the inflammation stems from pol reactions related to cell proliferation. This relationship, however, needs to be investigated more closely.
In this study, we have investigated the inhibitory effects of glycyrrhizin (compound 1) and its derivatives, including the potassium salt of compound 1 (dipotassium glycyrrhizate and compound 2) and glycyrrhetinic acid (compound 3) (Figure 1), on mammalian pol activity and inflammatory responses both in vitro and in vivo. In particular, we demonstrate that these compounds exert inhibitory effects against TNF-α production and NF-κB activation in cell culture models of inflammatory response. The relationship between the inhibition of pols and the anti-inflammatory action of the glycyrrhizin derivatives is discussed.

Mammalian Pol Assays.
Pol α was purified from calf thymus by immunoaffinity column chromatography as described by Tamai et al. [19]. The human pol γ catalytic gene was cloned into pFastBac (Invitrogen Japan K.K., Tokyo Japan). Histidine-tagged enzyme was expressed using the BAC-TO-BAC HT Baculovirus Expression System according to the supplier's manual (Life Technologies, MD) and purified using ProBound resin (Invitrogen Japan K.K.) [20]. A truncated form of pol κ (residues 1-560) with a 6xHis tag attached at the C-terminus was overproduced in E. coli and purified as described previously [21]. Recombinant human His-pol λ was overexpressed and purified according to a method described previously [22].
The reaction mixture for calf pol α was described previously [23,24]. The reaction mixture for human pol γ was previously described by Umeda et al. [20]. The reaction mixtures for mammalian pols κ and λ were the same as that for calf pol α. The components of the pol assay were poly(dA)/oligo(dT) 18 (A/T = 2/1) and dTTP as the DNA template-primer and 2 -deoxynucleoside 5 -triphosphate (dNTP) substrate, respectively. The glycyrrhizin derivatives (i.e., compounds 1-3) were dissolved in distilled dimethyl sulfoxide (DMSO) at various concentrations and sonicated for 30 sec. The sonicated samples (4 μL) were mixed with 16 μL of each enzyme (final amount, 0.05 units) in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glycerol, and 0.1 mM EDTA, and kept at 0 • C for 10 min. These inhibitor enzyme mixtures (8 μL) were added to 16 μL of each of standard enzyme reaction mixture (50 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, 1 mM MgCl 2 , 15% glycerol, 10 μM poly(dA)/oligo(dT) 18 , and 10 μM [ 3 H]-dTTP), and incubation was carried out at 37 • C for 60 min. Activity in the absence of inhibitor was considered to be 100%, and the activity remaining at each concentration of inhibitor was determined relative to this value. One unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol dNTP (dTTP) into the synthetic DNA template-primer (poly(dA)/oligo(dT) 18 , A/T = 2/1) in 60 min at 37 • C under normal reaction conditions for each enzyme (scintillation counts: approximately 1 pmol of incorporated radioactive nucleotide = 100 cpm) [23,24].

Animal Experiments.
All animal studies were performed according to the guidelines outlined in the "Care and Use of Laboratory Animals" of Kobe University. The animals were anesthetized with pentobarbital before undergoing cervical dislocation. Female 8-week-old C57BL/6 mice that had been bred in-house with free access to food and water were used for all experiments. All of the mice were maintained under a 12-h light/dark cycle and housed at a room temperature of 25 • C.  (Compounds 1-3) on Mammalian Pol Activity. Initially, we investigated the in vitro biochemical action of glycyrrhizin (compound 1) and its derivatives (compounds 2 and 3). The inhibition of four mammalian pols, namely, calf pol α, human pol γ, human pol κ, and human pol λ, by each compound at 20 and 100 μM was investigated. Pols α, γ, κ, and λ were used as representatives of the B, A, Y, and X families of pols, respectively [1][2][3]. As shown in Figure 2, at 100 μM these compounds inhibited the activity of all mammalian pols tested, because the relative pol activity was less than 50% after incubation with these compounds. At 20 μM, compound 3 also inhibited the activity of these pols, whereas compounds 1 and 2 had no effect; therefore, the inhibitory effect of these compounds on mammalian pols was ranked as follows: compound 3 > compound 1 = compound 2. Compound 3 showed almost the same strength of inhibition among the four mammalian pols tested; that is, the concentration of compound 3 giving 50% inhibition of pols α, γ, κ, and λ was 16.  DNA (i.e., bovine deoxyribonuclease I-treated DNA) and dNTP were used as the DNA template-primer and nucleotide substrate instead of synthesized DNA [poly(dA)/oligo(dT) 18 (A/T = 2/1)] and dTTP, respectively, the inhibitory effects of these compounds did not change (data not shown).

Inhibitory Effect of Glycyrrhizin Derivatives (Compounds 1-3) on LPS-Induced Inflammatory Responses in Cultured
Cells. Next, we investigated whether the three glycyrrhizin derivatives could inhibit both the reduction of TNF-α production and the nuclear translocation of NF-κB caused by LPS stimulation in cultured cells. The inflammatory cytokine TNF-α activates the NF-κB signaling pathway by binding to the TNF-α receptor (TNFR) and thereby initiates an inflammatory response, resulting in various inflammatory diseases [27]. In cultured macrophage RAW264.7 cells, no compound showed cytotoxicity at 25 to 250 μM ( Figure 3); therefore, the LD 50 values of compounds 1-3 were >250 μM. These compounds also had no effect on the cell proliferation of peritoneal macrophages (data not shown). As shown in Figure 4(a), RAW264.7 cells produced 693 pg/mL of TNFα after LPS treatment. Compounds 1-3 slightly suppressed this LPS-stimulated production of TNF-α, showing dosedependent inhibition at 100 and 200 μM. The suppression of TNF-α production by compound 3 was stronger than that by compounds 1 and 2. Figure 4 macrophages derived from mice by the glycyrrhizin derivatives. The inhibitory effect of compounds 1-3 showed almost the same tendency in peritoneal macrophages as in the macrophage cell line RAW264.7 although compound 3 significantly suppressed the production of TNF-α in peritoneal macrophages. From these results, the strength of the inhibitory effect of these compounds can be ranked as follows: compound 3 > compound 1 = compound 2. NF-κB is known to be the rate-controlling factor in inflammatory responses. We, therefore, examined the inhibitory effect of compounds 1-3 on the LPS-induced nuclear translocation of NF-κB in RAW264.7 cells ( Figure 5). In this experiment, we used HEK-Blue hTLR4 cells, which are HEK293 cells that have been engineered to report TLR4-linked NF-κB activation. In brief, this cell line is transfected with TLR4 and an NF-κB-inducible alkaline phosphatase reporter gene system. On interaction with the appropriate ligand, TLR4 transduces a signal that results in NF-κB activation. In this assay system, the amount of NF-κB undergoing nuclear translocation in the cells after LPS stimulation was significantly reduced by compound 3 at 100 and 200 μM. By contrast, compounds 1 and 2 had no effect on the LPS-stimulated nuclear translocation of NF-κB; thus, the ranking was compound 3 compound 1 = compound 2. These results demonstrate that compound 3 can strongly suppress the nuclear translocation of NF-κB by inhibiting the production of TNF-α. The effects of glycyrrhizin derivatives on the molecular mechanism underlying inflammatory responses will be addressed in future studies. (Compounds 1-3) on TPA-Induced Anti-Inflammatory Activity. In a previous study of pol inhibitors, we found that there is a relationship between pol λ inhibitors and TPA-induced acute antiinflammatory activity [6,13,14]. Thus, using the mouse ear inflammatory test, we examined the anti-inflammatory activity of the glycyrrhizin derivatives. Application of TPA (0.5 μg) to the mouse ear induced edema, resulting in a 241% increase in the weight of the ear disk 7 h after application. As shown in Figure 6, pretreatment with compounds 1-3 dose-dependently suppressed inflammation, and the effect of these compounds was ranked as follows: compound 3 > compound 1 = compound 2. Thus, these in vivo data from the mouse ear study showed almost the same trend as the LPS-induced inflammatory response data from cultured cells ( Figure 5). Furthermore, the anti-inflammatory effect of these compounds showed the same tendency as their inhibitory effect on mammalian pols including pol λ, which was strongly inhibited by compound 3 (Figure 2). These results suggest that inhibition of pol λ inhibitory activity has a positive correlation with the anti-inflammatory activity observed.

Discussion
We have shown here that glycyrrhetinic acid (compound 3) was the strongest inhibitor of mammalian pols α, γ, κ, and λ (Figure 2), and this compound prevented the inflammatory response among the three glycyrrhizin derivatives (compounds 1-3) tested (Figures 4 to 6). Compound 3 is the agly- cone of compound 1, and the pentacyclic triterpenoid structure must be important for the inhibitory activity shown by these compounds. Glycyrrhizin (compounds 1) and dipotassium glycyrrhizate (compound 2), which is the dipotassium salt of compound 1, are triterpenoid saponin glycosides, and they both have two molecules of glucuronic acid (Figure 1). Compound 2 is a major sweet-tasting food additive, and it is 5-fold sweeter than compound 1 [7]. The inhibitory effect of compound 1 on pol activity and inflammation showed the same tendency as that of compound 2; therefore, the salt form had no effect on the inhibitory activities of glycyrrhizin. Eukaryotic cells reportedly contain 15 pol species belonging to four families: namely, family A (pols γ, θ, and ν), family B (pols α, δ, ε, and ζ), family X (pols β, λ, and μ and TdT) and family Y (pols η, ι, and κ and REV1) [3,4]. As reported previously, the phenolic compound curcumin, which is a known anti-inflammatory agent, is a pol λ-specific inhibitor [6,13,14]. Intriguingly, on the basis of compound 3, the principle molecular target of the glycyrrhizin derivatives is also pol λ. Among the X family of pols, pol λ has an unclear biochemical function, although it seems to work in a similar way to pol β [28]. Pol β is involved in the short-patch base excision repair (BER) pathway [29][30][31][32], as well as playing an essential role in neural development [33]. Recently, pol λ was found to possess 5 -deoxyribose-5phosphate (dRP) lyase activity, but no apurinic/apyrimidinic (AP) lyase activity [34]. Pol λ is able to substitute for pol β during in vitro BER, suggesting that pol λ also participates in BER. Northern blot analysis indicated that transcripts of pol β are abundantly expressed in the testis, thymus, and brain in rats [35], whereas pol λ is efficiently transcribed mostly in the testis [36]. Bertocci et al. reported that mice in which pol λ expression is knocked out are not only viable and fertile, but also display a normal hypermutation pattern [37].
As well as causing inflammation, TPA influences cell proliferation and has physiological effects on cells owing to its tumor-promoting activity [18]. Therefore, anti-inflammatory agents are expected to suppress DNA replication/repair/recombination in nuclei in relation to the action of TPA. Because pol λ is a repair/recombination-related pol [28], our finding-that pol λ is the molecular target of glycyrrhizin derivatives-is in good agreement with this expected mechanism of anti-inflammatory agents. As a result, any pol λ inhibitor might also be an inhibitor of inflammation. Compound 1 has been reported to possess various pharmacological properties such as anti-inflammatory activity [38], inhibition of prostaglandin E2 production in rat macrophages [39], antiallergic activity [40], antiviral activity [41,42], and induction of interferon-γ [43]. In Japan, a preparation of compound 1, Stronger-Neo Minophagen C, has been used extensively to treat chronic hepatitis for more than 30 years. Compound 3 is also known to have wide pharmacological effects such as anti-inflammatory [44,45], antitumor [46], and antihepatotoxic [47] activities, and inhibition of the growth of mouse melanoma [48]. In 1989, it was reported that compound 3 strongly inhibits renal 11βhydroxysteroid dehydrogenase in rat [49]; this inhibition has been regarded as a cause of the pseudoaldosteronism that is occasionally induced by the administration of a compound 3 preparation or Carbenoxolone. However, the mechanisms underlying the therapeutic effects of the glycyrrhizin derivatives remain unknown.
In this study, therefore, we investigated the inhibitory effect of the glycyrrhizin derivatives on mammalian pols, which are responsible for DNA replication leading to cell proliferation and DNA repair/recombination, as well as the relationship between the degree of the suppression of LPSevoked TNF-α production and anti-inflammatory activity. The molecular mechanism that links the LPS-induced in flammatory response and anti-inflammatory activity in the model of TPA-induced ear edema is unknown. Because activated NF-κB has been observed in a model of TPAinduced ear edema [50], the anti-inflammatory effects of compound 3 may be, at least in part, dependent on the inhibition of NF-κB activation. Our study indicates that compound 3 is useful as an NF-κB inhibitor and may be a potent chemopreventive agent against inflammation. As a result, we found a positive correlation between the pol inhibitory activity and anti-inflammatory activity of compound 3. The relationship between these activities, namely, pol λ inhibition and anti-inflammatory action, may be useful as a new and convenient in vitro assay to screen for novel anti-inflammatory compounds.

Conclusions
Our study is the first to demonstrate that glycyrrhetinic acid (compound 3), which is the aglycone of glycyrrhizin (compounds 1), potently inhibited the activity of mammalian pols including pol λ. Compound 3 also reduced TNF-α production and NF-κB activation and suppressed mouse ear inflammation stimulated by TPA. Thus, compound 3 could be an anti-inflammatory agent based on pol λ inhibition.