**7. Evaluation of MKH delivery in HCC cells with MKH esters and the antiproliferative effect**

MKH was susceptible to oxidation, so we measured the levels of menaquinone-4 epoxide (MKO) in HCC cells to assess the function of MKH-bis-DMG as a delivery system for MKH in HCC cells. Concomitant with vitamin K-dependent carboxylation of Glu to Gla by GGCX, MKH is stoichiometrically converted to MKO and that means MKO levels in HCC cells reflect the levels of MKH. The AUCMKH values after MKH—DMG administration in three types of HCC cell lines were 3.5-fold to 15-fold higher than those after MK-4 administration (**Table 3**). Based on these results, it was clearly confirmed that MKH-bis-DMG works as an effective delivery prodrug of MKH into HCC cells. The resultant MKH may exhibit excellent antiproliferative activity against HCC cells despite their DCP-positive and DCP-negative status.

> **AUC0–72h for MK-4**

47.7 ± 7.25 143 ± 13.6

74.5 ± 17.0 25.9 ± 5.04

122 ± 19.5 193 ± 21.5

**Table 3.** Area under the curve for intracellular concentration versus time (AUC) after treatment with MK-4 or MKH–

One of the mechanisms of the antiproliferative effect of MK-4 was thought to involve G1/S cellcycle arrest via reduced protein expression of cyclin D1 and Cdk4, and through suppression of NF-κB activation [10, 12]. We investigated whether the antiproliferative activity of MKHbis-DMG was via cell-cycle arrest in HCC cells using flow cytometry and Western blotting [32]. MKH-bis-DMG-treated PLC/PRF/5 cells showed an increase in G1 phase cells and a decrease in S phase cells in flow cytometric analysis. Treatment of both DCP-positive and DCP-negative HCC cells with MKH-bis-DMG downregulated cyclin D1, cyclin D3, and Cdk4 expression after 24 h, and almost completely removed expression after 48 h. In comparison, the modest downregulation of cyclin D1, cyclin D3, and Cdk4 expression was observed after 48 h of MK-4 treatment in all tested HCC cell lines. NF-κB was downregulated after MKH-bis-DMG treatment in all tested HCC cell lines, but no effect was observed after MK-4 treatment in PLC/

**8. Mechanism of growth inhibition of HCC cells by MKH esters**

**(nmol h mg protein−1)**

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

**AUC0–72h for MKH**

22.2 ± 3.72a 336 ± 37.2b

113 ± 4.87a 397 ± 34.2b

38.4 ± 4.44a 329 ± 35.4b

**(nmol h mg protein−1)**

http://dx.doi.org/10.5772/63343

319

**HCC cell line Compound AUC0–72h**

MKH–DMG

MKH–DMG

MKH–DMG

MKH value after MK-4 administration: MKO.

PRF/5 and SK-Hep-1 cell lines at this dose.

Doses are 25 μM (at near IC50 value).

PLC/PRF/5 MK-4

Hep3B MK-4

SK-Hep-1 MK-4

Adapted from Ref. [32].

DMG in HCC cell lines.

a

b

**for MKO**

22.2 ± 3.72 193 ± 25.1

113 ± 4.87 371 ± 31.0

38.4 ± 4.44 136 ± 14.3

MKH value after MKH–DMG administration: sum of MKO and MK-4.

**(nmol h mg protein−1)**

MKH-bis-DMG inhibited the proliferation of both DCP-positive (PLC/PRF/5, Hep3B) and DCP-negative (SK-Hep-1) HCC cell lines in a time- and dose-dependent manner, and exhibited lower IC50 values (range from 14–37 μmol/L), and a fourfold to 18-fold increase in growthinhibitory activity compared with MK-4. MKH-bis-DMG showed a rapid and strong growthinhibitory effect after only 48 h of treatment. In contrast, MK-4 had little inhibitory effect on cell proliferation, and its effects appeared after 72 h of treatment (**Figure 3**).

**Figure 3.** Inhibitory effects of MKH–DMG and MK-4 on DCP-positive and DCP-negative HCC cell proliferation. MKH– DMG treatment of PLC/PRF/5 (A), Hep3B (C), and SK-Hep-1 (E) cell lines, and MK-4 treatment of PLC/PRF/5 (B), Hep3B (D), and SK-Hep-1 (F) cells. Symbols: ○, 0 μM; ■, 20 μM; △, 40 μM; ▼, 60 μM after MKH–DMG treatment. Symbols: ○, 0 μM; △, 40 μM; ▼, 60 μM; ●, 100 μM after MK-4 treatment. Error bars indicate mean ± SD (n = 3). Adapted from Ref. [32].

MKH was susceptible to oxidation, so we measured the levels of menaquinone-4 epoxide (MKO) in HCC cells to assess the function of MKH-bis-DMG as a delivery system for MKH in HCC cells. Concomitant with vitamin K-dependent carboxylation of Glu to Gla by GGCX, MKH is stoichiometrically converted to MKO and that means MKO levels in HCC cells reflect the levels of MKH. The AUCMKH values after MKH—DMG administration in three types of HCC cell lines were 3.5-fold to 15-fold higher than those after MK-4 administration (**Table 3**). Based on these results, it was clearly confirmed that MKH-bis-DMG works as an effective delivery prodrug of MKH into HCC cells. The resultant MKH may exhibit excellent antiproliferative activity against HCC cells despite their DCP-positive and DCP-negative status.


Doses are 25 μM (at near IC50 value).

a MKH value after MK-4 administration: MKO.

b MKH value after MKH–DMG administration: sum of MKO and MK-4.

Adapted from Ref. [32].

**7. Evaluation of MKH delivery in HCC cells with MKH esters and the**

cell proliferation, and its effects appeared after 72 h of treatment (**Figure 3**).

MKH-bis-DMG inhibited the proliferation of both DCP-positive (PLC/PRF/5, Hep3B) and DCP-negative (SK-Hep-1) HCC cell lines in a time- and dose-dependent manner, and exhibited lower IC50 values (range from 14–37 μmol/L), and a fourfold to 18-fold increase in growthinhibitory activity compared with MK-4. MKH-bis-DMG showed a rapid and strong growthinhibitory effect after only 48 h of treatment. In contrast, MK-4 had little inhibitory effect on

**Figure 3.** Inhibitory effects of MKH–DMG and MK-4 on DCP-positive and DCP-negative HCC cell proliferation. MKH– DMG treatment of PLC/PRF/5 (A), Hep3B (C), and SK-Hep-1 (E) cell lines, and MK-4 treatment of PLC/PRF/5 (B), Hep3B (D), and SK-Hep-1 (F) cells. Symbols: ○, 0 μM; ■, 20 μM; △, 40 μM; ▼, 60 μM after MKH–DMG treatment. Symbols: ○, 0 μM; △, 40 μM; ▼, 60 μM; ●, 100 μM after MK-4 treatment. Error bars indicate mean ± SD (n = 3). Adapt-

**antiproliferative effect**

318 Vitamin K2 - Vital for Health and Wellbeing

ed from Ref. [32].

**Table 3.** Area under the curve for intracellular concentration versus time (AUC) after treatment with MK-4 or MKH– DMG in HCC cell lines.
