Direct interaction of Cucurbitacin E isolated from Alsomitra macrocarpa to actin filament

Abstract

A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed.

Fig. 1

Chemical structures of cucurbitacin E isolated from A. macrocarpa

Fig. 2

Effect of Cuc E on the morphology of LX-2 cells. (a) 0, (b) 6, (c) 24, and (d) 60 min after addition of 30 μg mL⁻¹ methanol extract of A. macrocarpa

Fig. 3

Removal of Cuc E on morphology, filament actin, and tubulin of LX-2 cells. Cells were incubated with Cuc E (20 nM) for 24 h and medium was replaced with drug-free medium. Bright field images for (a) 0, (b) 6, and (c) 24 h after medium change. Alexa-phalloidin staining for (d) 0, (e) 6, and (f) 24 h. Tubulin staining for (g) 0, (h) 6, and (i) 24 h

Fig. 4

Effects of Cuc E on the distribution of DNA content analyzed by flow cytometry. LX-2 cells were treated with Cuc E (a) 0 nM and (b) 20 nM for 24 h, and (c) 20 nM for 24 h treatment following 24 h incubation with drug-free medium

Fig. 5

Time-lapse analysis of the actin cytoskeleton in situ. The image of YFP-actin-expressing NIH-3T3 cells. Drugs were added at time 0. (a) Cuc E (500 nM). White arrow heads indicate G-actin aggregation and small allows indicate F-actin. (b) Jasplakinolide (500 nM)

Fig. 6

Effect of Cuc E on the actin depolymerization. Polymerized actin (10% pyrene-labeled actin, 2 mg mL⁻¹) was diluted 20 times by G-buffer at room temperature (final concentration 4.7 μM). The samples were mixed to give final concentrations of 90 (open circle), 18 (closed circle), 9 (open square), and 0 μM (closed square) of Cuc E