Anthracenedione derivatives as anticancer agents isolated from secondary metabolites of the mangrove endophytic fungi

Abstract

In this article, we report anticancer activity of 14 anthracenedione derivatives separated from the secondary metabolites of the mangrove endophytic fungi Halorosellinia sp. (No. 1403) and Guignardia sp. (No. 4382). Some of them inhibited potently the growth of KB and KBv200 cells, among which compound 6 displayed strong cytotoxicity with IC(50) values of 3.17 and 3.21 microM to KB and KBv200 cells, respectively. Furthermore, we demonstrate that the mechanism involved in the apoptosis induced by compound 6 is probably related to mitochondrial dysfunction. Additionally, the structure-activity relationships of these compounds are discussed.

Keywords: anthracenedione derivatives; anticancer; apoptosis; mangrove endophytic fungi; structure-activity relationship.

Figure 1

Compound 6 showed potent cytotoxicity to KB and KBv200 cells. Cytotoxicity was measured by 3-(4,5-Dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The cells having grown for 24 h were exposed to a full range of concentrations of compound 6 for 68 h. After subsequent staining with MTT for 4 h, cell viability was assessed by Model 550 Microplate reader Results were shown as means ± SD of at least triplicate determinations. Each experiment was performed in three replicate wells.

Figure 2

Compound 6 induced cell apoptosis in KB and KBv200 cells. Compound 6 induced cell apoptosis in KB and KBv200 cells detected by Annexin V-FITC/PI double staining and flow cytometer assay. The right bottom quadrant represented cells stained mainly by Annexin-V (early apoptotic cells) and the top right quadrant represented cells stained by both PI and Annexin-V (late apoptotic/necrotic secondary necrosis). The top left quadrant represented cells stained mainly by PI and viable cells negative for both Annexin-V and PI appeared in the left bottom quadrant. (A) KB cells exposed to 0 and 12 μM compound 6, respectively. (B) of KBv200 cells exposed to 0 and12 μM compound 6, respectively. (C) statistical analysis of the above results. The apoptosis rate is showed in the bar graph. The results represent the data of three assays (means ± SD). * P < 0.05 and **P < 0.01 versus the control.

Figure 3

Compound 6 induces morphological changes representative of apoptosis. Cell apoptosis induced by compound 6 was examined by Hoechst 33258 staining and observed under fluorescence microscope at 400 × magnification. KB cells and KBv200 cells were treated with compound 6 of 12.0 μM for 48 h. The apoptotic cells detected by the fluorescence microscopy displayed typical changes of apoptosis including staining bright of condensed or fragmented nucleus. Herein, treatment with adriamycin (0.1 μM for KB cells and 7.0 μM for KBv200 cells) for 48 h was included as the positive control.

Figure 4

Compound 6 treatment results in the loss of ΔΨ_m and release of cytochrome c. After cells were exposed to 0, 6.0 and 12.0 μM of compound 6 for 48 h, the ΔΨ_m was determined by flow cytometry. Loss of ΔΨ_m in KB and KBv200 cells and release of cytochrome c were observed in a dose-dependent manner. (A) the decrease of ΔΨ_m in KB and KBv200 cells; also, ΔΨ_m levels expressed as units of MFI were calculated as percentage of control. Results are mean ± SD of three determinations. * P < 0.05 and ** P < 0.01 vs. the control. (B) the release of cytochrosome c after treatment with compound 6 for the indicated time in KB cells and KBv200 cells, respectively. GAPDH detection was used to confirm equal protein loading.

Figure 5

Compound 6 does not intercalate into DNA. After 10.0, 20.0, 40.0, 80.0 μM ADR and 12.0, 24.0, 48.0, 96.0 μM compound 6 at 37 °C for 48 h, the mixture was subjected to electrophoresis on a 1.2% agarose gel stained with 1.5 μM EB at 60 V for 40 min in 1 × TAE buffer and photographed under ultraviolet light.