Synthesis and Biological Evaluation of Lipophilic 1,4-Naphthoquinone Derivatives against Human Cancer Cell Lines

Abstract

To examine the effect of hydrophobicity on the anticancer activity of 1,4-naphthoquinone derivatives, a series of compounds bearing a 2-O-alkyl-, 3-C-alkyl- or 2/3-N-morpholinoalkyl group were synthesized and evaluated for their anticancer activity against five human cancer cell lines in vitro. The cytotoxicity of these derivatives was assayed against HT-29, SW480, HepG2, MCF-7 and HL-60 cells by the MTT assay. Among them, 2-hydroxy-3-farnesyl-1,4-naphthoquinone (11a) was found to be the most cytotoxic against these cell lines. Our results showed that the effectiveness of compound 11a may be attributed to its suppression of the survival of HT-29. Secondly, in the Hoechst 33258 staining test, compound 11a-treated cells exhibited nuclear condensation typical of apoptosis. Additionally, cell cycle analysis by flow cytometry indicated that compound 11a arrested HT-29 cells in the S phase. Furthermore, cell death detected by Annexin V-FITC/propidium iodide staining showed that compound 11a efficiently induced apoptosis of HT-29 in a concentration-dependent manner. Taken together, compound 11a effectively inhibits colon cancer cell proliferation and may be a potent anticancer agent.

Keywords: 1,4-naphthoquinone; anticancer activity; apoptosis; cell cycle distribution; human colon cancer cells HT-29; terpenoids.

Figure 1

Chemical structures of plumbagin (1); lawsone (2); juglone (3) and shikonin (4).

Scheme 1

Synthesis of compounds 5a,b‒11a,b.

Scheme 2

Synthesis of compounds 13a,b‒19a,b.

Figure 2

The morphological changes of HT-29 cells treated with 0–2.5 μM plumbagin (1) (A) and 11a (B) for 48 h. (magnification, 200×). (A,B) The upper panels showed the cell morphology under phase-contrast microscopy, and the lower panels display the Hoechst 33258-stained nuclear patterns take by fluorescence microscopy (magnification, 200×).

Figure 2

The morphological changes of HT-29 cells treated with 0–2.5 μM plumbagin (1) (A) and 11a (B) for 48 h. (magnification, 200×). (A,B) The upper panels showed the cell morphology under phase-contrast microscopy, and the lower panels display the Hoechst 33258-stained nuclear patterns take by fluorescence microscopy (magnification, 200×).

Figure 3

(A) The effects of the 48 h treatments with 0–2.5 μM plumbagin (1) and 11a on cell cycle distribution of HT-29. After treatment, cells were fixed and stained with PI, and the cell cycle distribution was examined by flow cytometry; (B) Quantitative data of cell cycle analysis of HT-29 treated with 0‒2.5 μM plumbagin (1) and 11a for 48 h; (C) Compared with control, the treated with plumbagin (1) and 11a shows the obvious increase in sub-G1 fraction. Each value represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 vs. control.

Figure 3

(A) The effects of the 48 h treatments with 0–2.5 μM plumbagin (1) and 11a on cell cycle distribution of HT-29. After treatment, cells were fixed and stained with PI, and the cell cycle distribution was examined by flow cytometry; (B) Quantitative data of cell cycle analysis of HT-29 treated with 0‒2.5 μM plumbagin (1) and 11a for 48 h; (C) Compared with control, the treated with plumbagin (1) and 11a shows the obvious increase in sub-G1 fraction. Each value represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 vs. control.

Figure 4

(A) The effects of the 48 h treatments with 0–2.5 μM plumbagin (1) and 11a on apoptotic percentage distribution of HT-29 cells by Annexin V-FITC/PI staining. (B) The apoptosis rate was calculated by flow cytometry and cell apoptosis was defined in early and late apoptosis treatment with 0–2.5 μM plumbagin (1) and 11a for 48 h. Each value represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control.