Melampodium leucanthum, a source of cytotoxic sesquiterpenes with antimitotic activities

Abstract

A new tricyclic sesquiterpene, named meleucanthin (1), was isolated from an extract of the leaves and branches of Melampodium leucanthum, along with four known germacranolide sesquiterpene lactones, leucanthin-A (2), leucanthin-B (3), melampodin-A acetate (4), and 3α-hydroxyenhydrin (5). The chemical structure of 1 was elucidated by analysis of 1D and 2D NMR and mass spectrometric data. All compounds exhibited antiproliferative and cytotoxic efficacy against PC-3 and DU 145 prostate cancer cells, as well as HeLa cervical cancer cells, with IC50 values ranging from 0.18 to 9 μM. These compounds were effective in clonogenic assays and displayed high cellular persistence. They were also found to be capable of circumventing P-glycoprotein-mediated drug resistance. Mechanism of action studies showed that 4 caused an accumulation of cells in the G2/M phase of the cell cycle, and 2-5 caused the formation of abnormal mitotic spindles. These results suggest the cytotoxic effects of these germacranolides involve inhibition of mitotic spindle function, and it is likely that other mechanisms additionally contribute to cell death. These studies also demonstrate the possibility of isolating new, biologically active compounds from indigenous Texas plants.

Figure 1

Key HMBC (arrows) and COSY (bold) correlations of (1).

Figure 2

Concentration-response curves for growth inhibition of PC-3, DU 145 and HeLa cells by compounds 1–5 or paclitaxel (PTX), measured with the SRB assay. Results represent n = 2–9 independent experiments, with each concentration tested in triplicate.

Figure 3

Effects of a 4 h exposure to 2–5 on colony formation. DU 145 cells were treated with vehicle (DMSO) or a range of concentrations of 2–5 for 4 h before compound removal. Representative images of DU 145 colonies after 9 days of treatment with vehicle or 5 (A). Quantification of colony number after treatment with vehicle or 2– 5 (B). *p < 0.05; ****p < 0.0001 compared to vehicle control; one-way ANOVA with Dunnett’s post-hoc test. Results represent n = 2–9 independent experiments.

Figure 4

Induction of PARP cleavage in HeLa and DU 145 cells by 2–5. Cells were treated with the indicated concentrations of each compound for 24 h prior to lysis, SDS-PAGE and immunoblotting.

Figure 5

Effects of 2 and 4 on cell cycle distribution of PC-3 cells after 18 h of treatment with vehicle, paclitaxel (PTX), 2 or 4. Cells were treated as indicated for 18 h before staining with propidium iodide and analysis by flow cytometry.

Figure 6

Effects of 2–5 on purified tubulin polymerization in vitro. The polymerization of 2 mg/mL porcine tubulin was monitored turbidimetrically (A₃₄₀) in the presence of vehicle, 2–5 or colchicine for 60 min.

Figure 7

Representative immunofluorescence images of HeLa cells after treatment with vehicle (A), 100 nM PLK-1 inhibitor BI2536 (B), 10 µM 2 (C), 7.5 µM 3 (D), 5 µM 4 (E) and 25 µM 5 (F). DNA was labelled with DAPI (blue) and microtubules were labeled with a monoclonal anti-β-tubulin antibody and FITC-conjugated anti-mouse IgG (green).

Figure 8