Initially, we investigated the in vitro biochemical action of VK₂ (MK-4), VK₃ and their chemically synthesized derivatives, MK-3, MK-2 and MK-1. The inhibition of four mammalian pols, namely calf pol α, human pol γ, human pol κ and human pol λ, by 50 μM of each compound was investigated. Pols α, γ, κ and λ were used as representatives of the B, A, Y and X families of pols, respectively [1–3]. As shown in Figure 2, MK-3, MK-2 and MK-1, which are intermediates between VK₂ and VK₃, inhibited the activity of mammalian pols α, κ and λ, whereas MK-4 (VK₂) had no effect on pol activity. VK₃ selectively inhibited pol γ among the mammalian pols tested. The inhibitory effect of these compounds on pols α, κ and λ ranked as follows: MK-2 > MK-1 > MK-3 > MK-4 = VK₃; and the inhibitory effect of these compounds on pol γ ranked as follows: VK₃ > MK-1 > MK-2 > MK-3 > MK-4. The IC₅₀ values of MK-2 against pols α, γ, κ and λ were 27.6, 68.8, 35.3 and 24.6 μM, respectively. When activated 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).

Among the five compounds investigated, MK-2 displayed the strongest inhibitory effect on mammalian pols (Figure 2) and was therefore the focus of this section. As described briefly in the introduction, we have succeeded in obtaining ten eukaryotic pol species including pols α, β, γ, δ, ɛ, ι, η, κ and λ, and TdT; however, pols ζ, θ, μ and ν, and REV1 are not yet available (Table 1). Currently, eukaryotes are thought to express at least 15 species of pols [1,2], and we are still in an era when most pols are very difficult to obtain in their purified form in a laboratory. Table 1 shows the inhibitory effect (IC₅₀ value) of MK-2 against various pol species including the ten eukaryotic pols that can be obtained. This compound inhibited the activity of all of the pols from mammals, and 50% inhibition of the A, B, X and Y families of pols was observed at a dose of 68.8, 27.6–29.1, 24.6–31.0 and 35.3–39.0 μM, respectively; therefore, the strength of the inhibitory effect of MK-2 on mammalian pol families can be ranked as follows: B-family pols = X-family pols > Y-family pols > A-family pol. MK-2 showed the strongest inhibition of pol λ activity among the pols investigated, with an IC₅₀ value of 24.6 μM. This compound also suppressed the activity of the animal pols from fish (cherry salmon) and insect (fruit fly) at almost the same concentrations as it inhibited the activity of mammalian pols.

By contrast, MK-2 had no effect on plant (cauliflower) pol α or prokaryotic pols, such as E. coli pol I, Taq pol or T4 pol (Table 1). The three-dimensional structures of eukaryotic pols are likely to differ greatly from those of prokaryotic pols. MK-2 did not inhibit the activity of other DNA metabolic enzymes, such as calf primase pol α, T7 RNA polymerase, T4 polynucleotide kinase, or bovine deoxyribonuclease I. These results suggest that MK-2 may be a selective inhibitor of animal pols, especially the B and X families of pols containing pol λ.

To test whether MK-2 is an intercalating agent that distorts DNA and subsequently inhibits enzyme activity, we measured the thermal transition of DNA in the presence or absence of this compound. The thermal transition profile of DNA was the same with or without the compound (data not shown). Therefore, the inhibition of pols by MK-2 is not due to DNA distortion, but seems to be due to a direct effect of this compound on the enzymes themselves.

In previous a pol inhibitor study, we found that there is a relationship between pol λ inhibitors and TPA-induced acute anti-inflammatory activity [6,19,20]. Thus, using the mouse ear inflammatory test, we examined the anti-inflammatory activity of the intermediates between VK₂ and VK₃. 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 3, pretreatment with MK-2 and MK-1 suppressed inflammation, and the effect of MK-2 was stronger than that of MK-1. By contrast, MK-4, MK-3 and VK₃ had a weak effect on the level of inflammation. Therefore, the anti-inflammatory effect of these compounds correlated with their inhibitory effect on mammalian pols including pol λ, which was strongly inhibited by MK-2 (Figure 2). These results suggest that inhibition of pol λ activity has a positive correlation with the anti-inflammatory activity observed.

Next, we investigated whether the intermediates between VK₂ and VK₃ can inhibit both the reduction in TNF-α production and the nuclear translocation of NF-κB p65 induced by LPS stimulation in cultured mouse macrophage RAW264.7 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 [30]. In RAW264.7 cells, no compound showed cytotoxicity at 50 μM, because the LD₅₀ values of these five compounds were >100 μM. As shown in Figure 4, MK-2 at 10 and 50 μM, and MK-1 at 50 μM significantly suppressed the LPS-stimulated production of TNF-α, although MK-4, MK-3 and VK₃ displayed a moderate inhibitory effect on TNF-α production. The inhibitory effects of MK-2 and MK-1 were, respectively, the first and second strongest among these compounds. The effect of the compounds on the suppression of LPS-evoked TNF-α production showed almost the same tendency as their inhibitory effect on mammalian pols including pol λ. These results suggest that VK₂ and VK₃ intermediates, such as MK-2, inhibit the activities of mammalian pols, and then prevent the TNF-α production in the LPS-induced macrophages, but not affect the cell growth.

NF-κB is known to be the rate-controlling factor for inflammatory responses. We therefore examined the inhibitory effect of MK-2 on the LPS-induced nuclear translocation of NF-κB in RAW264.7 cells (Figure 5). By Western blot analysis, it was revealed that the amount of NF-κB nuclear translocation in RAW264.7 cells was 4.47-fold greater after LPS treatment, and that 10 μM MK-2 was sufficient to inhibit the LPS-stimulated nuclear translocation of NF-κB to 2.04-fold. These results demonstrate that this compound can strongly suppress the nuclear translocation of NF-κB by inhibiting the production of TNF-α. The effects of MK-2 on the molecular mechanism of anti-inflammation will be addressed in future studies.

To assess in vivo the anti-inflammatory effects of MK-2, which was the strongest inhibitor of animal pols and strongest suppressor of TPA-induced inflammation and LPS-evoked TNF-α production among the VK₂ and VK₃ intermediates, we investigated the inhibitory activity of this compound against LPS-induced acute inflammation (Figure 6). Treatment with 250 μg/kg BW of LPS considerably increased the serum TNF-α level (5 ng/mL), and intraperitoneal injection of 100 mg/kg BW of MK-2 greatly decreased this LPS-induced TNF-α production by 83.2%. Thus, the in vivo data obtained in this mouse study showed the same trend as the data obtained from cultured mouse macrophage cells (Figures 4 and 5).

VK₂ (MK-4) and VK₃ were obtained from Sigma-Aldrich (St. Louis, MO, U.S.), and these structures are shown in Figure 1. Each compound was purified to more than 99% purity. Chemically synthesized DNA templates such as poly(dA) and nucleotides such as [³H]-deoxythymidine 5′-triphosphate (dTTP) (43 Ci/mmol) were purchased from GE Healthcare Bio-Sciences (Little Chalfont, U.K.). The oligo(dT)₁₈ DNA primer was customized by Sigma-Aldrich Japan K.K. (Hokkaido, Japan). LPS was purchased from Sigma-Aldrich. For Western blot analysis, anti-NF-κB p65 antibody, anti-β-actin antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (i.e., secondary antibody) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.) and Thermo Scientific (Kanagawa, Japan), respectively. All other reagents were of analytical grade and purchased from Nacalai Tesque Inc. (Kyoto, Japan).

The VK₂ and VK₃ derivatives (i.e., MK-3, MK-2 and MK-1) were chemically synthesized according to a procedure reported by Mayer and Isler with slight modification [49]. The structures of these compounds were confirmed by comparison of the spectral data with the reported data, and are shown in Figure 1.

Pols from mammals, a fish (cherry salmon), an insect (fruit fly) and a plant (cauliflower) were purified, and prokaryotic pols and other DNA metabolic enzymes, such as T7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease I, were purchased as described in our previous report [50]. The activities of all pols and other DNA metabolic enzymes were measured as described in previous reports [50–53].

The components of the pol assay were poly(dA)/oligo(dT)₁₈ and dTTP as the DNA template-primer and 2′-deoxyribonucleoside 5′-triphosphate (dNTP) substrate, respectively. VK₂, VK₃, and their synthesized derivatives (i.e., MK-3, MK-2 and MK-1) were dissolved in dimethyl sulfoxide (DMSO) at various concentrations and sonicated for 30 s. The sonicated samples (4 μL) were mixed with 16 μL of each pol 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 standard enzyme reaction mixture (50 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, 1 mM MgCl₂, 15% glycerol, 10 μM poly(dA)/oligo(dT)₁₈ and 10 μM [³H]-dTTP), and incubation was carried out at 37 °C for 60 min, except for Taq pol, which was incubated at 74 °C for 60 min. Activity without the 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 nucleotides = 100 cpm) [51,52].

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.

The mouse inflammatory test was performed according to Gschwendt’s method [53]. In brief, an acetone solution of the VK₂ and VK₃ intermediates (250 or 500 μg in 20 μL) or 20 μL of acetone as a vehicle control was applied to the inner part of the mouse ear. Thirty minutes after the test compound was applied, a TPA solution (0.5 μg/20 μL of acetone) was applied to the same part of the ear. To the other ear of the same mouse, methanol, followed by TPA solution, was applied as a control. After 7 h, a disk (6 mm diameter) was obtained from the ear and weighed. The inhibitory effect (IE) is presented as a ratio of the increase in weight of the ear disks: IE = [ ( TPA   only ) − ( tested   compound   plus   TPA ) ] / [ ( TPA   only ) − ( vehicle ) ] × 100

A mouse macrophage cell line, RAW264.7, was obtained from American Type Culture Collection (ATCC) (Manassas, VA, U.S.). The cells were cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 4.5 g of glucose per liter plus 10% fetal calf serum, 5 mM L-glutamine, 50 units/mL penicillin and 50 units/mL streptomycin. The cells were cultured at 37 °C in standard medium in a humidified atmosphere of 5% CO₂–95% air.

RAW264.7 cells were placed in a 12-well plate at 5 × 10⁴ cells/well and incubated for 24 h. The cells were pretreated with the VK₂ and VK₃ intermediates (final concentrations of 10 and 50 μM) for 30 min before the addition of 100 ng/mL of LPS. After stimulation with LPS for 24 h, the cell culture medium was collected to measure the amount of TNF-α secreted. The concentration of TNF-α in the culture medium was quantified by using a commercially available enzyme-linked immunosorbent assay (ELISA) development system (Bay Bioscience Co., Ltd., Kobe, Japan) in accordance with the manufacturer’s protocol.

RAW264.7 cells on a 6-well plate at 5 × 10⁵ cells/well were incubated with 10 μM of MK-2 or DMSO (1 μL/mL) as a vehicle control for 30 min, followed by treatment with 100 ng/mL of LPS for 30 min. After treatment, cells were harvested with lysis buffer consisting of 10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂ and 0.5 mM DTT containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 5 μg/mL of leupeptin and 5 μg/mL of aprotinin) and phosphatase inhibitors (10 mM NaF and 1 mM Na₃VO₄) and stood on ice for 15 min with occasional mixing. The mixture was centrifuged at 1000 × g for 10 min at 4 °C. The precipitate was suspended in extraction buffer consisting of 20 mM Hepes (pH 7.6), 20% (v/v) glycerol, 500 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1.0 mM DTT and 0.1% (v/v) Nonidet P-40 containing the same protease and phosphatase inhibitors. The suspension was then gently mixed on a rotary device for 1 h at 4 °C before being centrifuged at 15,000 × g for 20 min at 4 °C. The resulting supernatant was used as a nuclear extract. The protein concentration was measured by using a bicinchoninic acid (BCA) assay kit in accordance with the manufacturer’s protocol. The nuclear proteins were subjected to Western blotting to evaluate the nuclear translocation of NF-κB.

The nuclear proteins (30–50 μg of protein) were boiled in a quarter-volume of sample buffer (1 M Tris-HCl [pH 7.5], 640 mM 2-mercaptoethanol, 0.2% bromphenol blue, 4% SDS and 20% glycerol) and then separated on 10% SDS polyacrylamide gels. Each gel was then electroblotted onto a PVDF membrane. The membrane was blocked with 1% skimmed milk in Tris-buffered saline TBS-T (10 mM Tris-HCl, 100 mM NaCl and 0.5% Tween-20) and probed with anti-NF-κB p65 antibody (1:500) and anti-β-actin antibody (1:5000), before being reacted with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 and 1:20,000, respectively). The protein-antibody complex was visualized with ChemiLumiONE (Nacalai Tesque, Kyoto, Japan) and detected using an Image Reader (LAS-3000 Imaging System, Fuji Photo Film, Tokyo, Japan). The intensity of each band was analyzed using ImageJ, which was developed at the National Institute of Health.

Mice were intraperitoneally injected with 200 μL of MK-2 dissolved in corn oil at 100 mg/kg body weight (BW), or 200 μL of corn oil as a vehicle control. After 30 min, the mice were intraperitoneally injected with 200 μL of 250 μg/kg BW LPS dissolved in PBS or 200 μL of PBS as a vehicle control. After 1 h, the mice were killed, and blood samples were collected. The blood serum was separated by centrifugation at 15,000 × g for 10 min at 4 °C, and the TNF-α level in the serum was measured by ELISA.

All data are expressed as the means ± SE of at least three independent determinations for each experiment. Statistical significance between each experimental group was analyzed using Student’s t-test, and a level of probability of 0.01 and 0.05 was used as the criterion of significance.