Anticancer activities of polyynes from the root bark of Oplopanax horridus and their acetylated derivatives

Abstract

Six polyynes OH-1~6, some of which are occur naturally in acetylated form, had been isolated and identified from the root bark of Oplopanax horridus (Devil's Club), a natural dietary supplement and medicinal plant in North America. During the evaluation of the polyynes' potential anticancer activities, sixteen more acetylated derivatives OHR-1~16 have synthesized and their anti-proliferation activity on MCF-7, MDA-MB-231, A549, HepG2 and LO2 cells assayed to elucidate their structure-activity relationships. The results showed that OH-1 ((3S, 8S)-falcarindiol) had the most potent anticancer activity, with IC50 values of 15.3, 23.5, 7.7 and 4.7 μM on MCF-7, A549, HepG2 and MDA-MB-231 cells, respectively. For the primary structure-activity relationship, the anticancer activities of polyynes become weaker if their hydroxyl groups are acetylated, the terminal double bonds transformed into single bonds or they contain one more methylene group in the main skeleton chain.

Figure 1

The structures of the isolated and derivative polyynes.

Figure 2

The anti-proliferative effects of the similar polyynes on MCF-7 cells, A549 cells, HepG2 cells and MDA-MB-231 cells. (A) all the natural polyynes; (B) the polyynes with the hydroxyl in the middle of the carbon chain displaced; (C) the polyynes with the hydroxyl near the end of the carbon chain; (D) the polyynes with no hydroxyls.

Figure 3

Detection of apoptosis in MDA-MB-231 cells by Hochest-staining. Treated cells were stained with Hoechst 33342, and then observed under a fluorescent microscope (magnification of ×400). (A) Sovent control treated MDA-MB-231 cells; (B–E) OHR-1 (15 μM), OHR-2 (5 μM), OHR-3 (10 μM) and OHR-5 (15 μM) treated MDA-MB-231 cells for 12h. The yellow arrows indicated classic apoptosis characteristics in cells with morphology change, chromatin condensation and apoptotic bodies.

Figure 4

Effect of OHR-1, -2, -3 and -5 on mitochondrial potential in MDA-MD-231 cells. Treated cells stained by JC–1 dye and observed by fluorescence microscopy (magnification of ×400). (A1, A2) Solvent control-treated cells showed most of cells had stronger J-aggregation stained red fluorescence. (B1-E1, B2-E2) OHR-1 (15 μM), OHR-2 (5 μM), OHR-3 (10 μM) and OHR-5 (15 μM) treated MDA-MB-231 cells show a majority of cells stained green fluorescence due to low △Ψm. (F) Analysis of the △Ψm (ration of green/red fluorescence intensity) in cells measured by flow cytometry. The data were expressed as ratio of Δψm between OHR-1, -2, -3 and -5 treatments and control cells (solvent vehicle set at 100%). * p < 0.05 vs. vehicle control.

Figure 5

Cell cycle analysis of MDA-MB-231 cells after exposure to OHR-1, -2, -3 and -5 for 24 h. After treatment, cells were fixed in 70% ethanol and then analyzed by flow cytometry distribution by propidium iodide/DNA content staining. (A) Solvent control- treated MDA-MB-231 cells; (B1-E1) Cell cycle analysis of cells treated with OHR-1 (7.5 μM), OHR-2 (2.5 μM), OHR-3 (5 μM) and OHR-5 (7.5 μM) for; (B2-E2): Cell cycle analysis of cells treated with OHR-1 (15 μM), OHR-2 (5 μM), OHR-3 (10 μM), OHR-5 (15 μM) treated MDA-MB-231 cells. (F) Cell cycle analysis of MDA-MB-231 cells treated with OHR-1, -2, -3 and -5 measured by flow cytometry. * p < 0.05 vs. vehicle control.