Anti-tumor effects of novel 5-O-acyl plumbagins based on the inhibition of mammalian DNA replicative polymerase activity

Abstract

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins enhanced mammalian pol inhibition and their cytotoxic activity. Plumbagin conjugated with chains consisting of more than C18-unsaturated fatty acids strongly inhibited the activities of calf pol α and human pol γ. Plumbagin conjugated with oleic acid (C18:1-acyl plumbagin) showed the strongest suppression of human colon carcinoma (HCT116) cell proliferation among the ten synthesized 5-O-acyl plumbagins. The inhibitory activity on pol α, a DNA replicative pol, by these compounds showed high correlation with their cancer cell proliferation suppressive activity. C18:1-Acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. This compound inhibited the proliferation of various human cancer cell lines, and was the cytotoxic inhibitor showing strongest inhibition towards HT-29 colon cancer cells (LD50 = 2.9 µM) among the nine cell lines tested. In an in vivo anti-tumor assay conducted on nude mice bearing solid tumors of HT-29 cells, C18:1-acyl plumbagin was shown to be a promising tumor suppressor. These data indicate that novel 5-O-acyl plumbagins act as anti-cancer agents based on mammalian DNA replicative pol α inhibition. Moreover, the results suggest that acylation of plumbagin is an effective chemical modification to improve the anti-cancer activity of vitamin K3 derivatives, such as plumbagin.

Figure 1. Structure of plumbagin (1), 5-O-acyl plumbagins (2), vitamin K₃ (3), juglone (4), and 5-O-acyl juglones (5).

“R” represents a saturated or unsaturated alkyl group in 5-O-acyl plumbagins (2) and 5-O-acyl juglones (5).

Figure 2. Synthesis of 5-O-acyl plumbagins (2a–j).

(A) Synthesis of 5-O-acyl plumbagin (2a). (B) Synthesis of 5-O-acyl plumbagins (2b–f). (C) Synthesis of 5-O-acyl plumbagins (2g–j).

Figure 3. Stability of C18:1-acyl plumbagin (2f) and C18:1-acyl juglone (5f) under basic conditions.

C18:1-acyl plumbagin (2f) (A) and C18:1-acyl juglone (5f) (B) were treated with 1 equivalent of Triton B in 1,4-dioxane and MeOH. The mixtures were monitored by UV-vis spectroscopy over time. Conditions: 1.1×10⁻³ M, 25°C, light path length = 1 mm.

Figure 4. Inhibitory effects of plumbagin (1) and 5-O-acyl plumbagins (2a–j) on the activity of mammalian pols.

Each compound (10 µM) was incubated with calf pol α (B-family pol), human pol γ (A-family pol), human pol κ (Y-family pol), and human pol λ (X-family pol) (0.05 units each). Pol activity in the absence of the compound (control) was taken as 100%, and the relative activity is shown. Data are shown as the mean ± SD (n = 3). ** P<0.01 and * P<0.05 vs. controls.

Figure 5. Effect of plumbagin (1) and 5-O-acyl plumbagins (2a–j) on the proliferation of HCT116 human colon carcinoma cells.

Each compound (10 and 100 µM) was added to cultured HCT116 cells. The cells were incubated for 48 h, and the rate of proliferation inhibition was determined by MTT assay. Cell proliferation inhibition of the cancer cells in the absence of the compound (control) was taken as 100%. Data are shown as the mean ± SD (n = 5). ** P<0.01 and * P<0.05 vs. controls.

Figure 6. Relationship between mammalian pol inhibitory activities versus human cancer cell proliferation inhibition by plumbagin (1) and 5-O-acyl plumbagins (2a–j).

X-axis indicates mammalian pol relative activity at 10 µM compound. (A) Calf pol α, (B) human pol γ, (C) human pol κ, and (D) human pol λ. Y-axis indicates rate of HCT116 human colon carcinoma cell proliferation at 100 µM compound. These data are based on Figs. 4 and 5; correlation coefficient values are shown in each panel.

Figure 7. Effect of C18:1-acyl plumbagin (2f) on the thermal transition of dsDNA.

Control (open-square), C18:1-acyl plumbagin (2f) (100 µM, closed-circle), and EtBr (15 µM, open-diamond) were incubated with 6 µg/mL of calf thymus dsDNA in 0.1 M Na-phosphate buffer (pH 7.0). Data are shown as the mean ± SD (n = 3). ** P<0.01 and * P<0.05 vs. controls.

Figure 8. In vivo anti-tumor effects of C18:1-acyl plumbagin (2f).

Nude mice bearing HT-29 solid tumors were administered with PBS (control), vitamin K₃ (3), juglone (4), C18:1-acyl juglone (5f), and C18:1-acyl plumbagin (2f) at a dose of 5 mg/kg. (A) Inhibitory effect on tumor volume in nude mice. (B) Body weight changes of nude mice; data, means ± SE (n = 6). * P<0.05 vs. controls.