Synthesis, spectral characterization, and in vitro cellular activities of metapristone, a potential cancer metastatic chemopreventive agent derived from mifepristone (RU486)

Abstract

Mifepristone (RU486) is marketed and used widely by women as an abortifacient, and experimentally for psychotic depression and anticancer treatments. After administration, metapristone is found to be the most predominant metabolite of mifepristone. We hypothesized that adhesion of circulating tumor cells (CTCs) to vascular endothelial bed is a crucial starting point in metastatic cascade, and that metapristone can serve as a cancer metastatic chemopreventive agent that can interrupt adhesion and invasion of CTCs to the intima of microvasculature. In the present study, we modified the synthesis procedure to produce grams of metapristone, fully characterized its spectral properties and in vitro cellular activities, including its cytostatic effects, cell cycle arrest, mitochondrial membrane potential, and apoptosis on human colorectal cancer HT-29 cells. Metapristone concentration dependently interrupted adhesion of HT-29 cells to endothelial cells. Metapristone may potentially be a useful agent to interrupt metastatic initiation.

Fig. 1

Synthesis scheme and spectral characterization of metapristone. a the synthetic route for metapristone; b UV–visible spectra of metapristone and the solvent effects: b1, metapristone in alcohol (b1) and methanol (b2) solvents; the UV–vis absorption maxima of metapristone decreased when the carbon chain of alcohol as well as the percentage of water in methanol increased. c representative HPLC analysis (c1) and infrared spectra (c2); d the ¹H-NMR (d1) and mass (d2) spectra of metapristone

Fig. 2

The anti-proliferative and anti-migration effects of metapristone. a In vitro activity of metapristone and RU486 (expressed as IC50 (μM)) against A-375, HepG2, HT-29, and HELF cell lines; b effect of metapristone on migration of HT-29 cells incubated in the cell wound scratch assay for 24 h

Fig. 3

Inhibition by metapristone of HT-29 adhesion to HUVECs (a, b) and Matrigel (c). a representative microscopic observation of the inhibition by metapristone at 0, 10, 50, and 100 μM (a, b, c, and d) on adhesion of HT-29 to HUVECs; b quantitative analysis of the inhibition by metapristone on the adhesion of HT-29 to HUVECs; c quantitative MTT analysis of the inhibition by metapristone on the adhesion of HT-29 to Matrigel

Fig. 4

Investigation of anti-proliferative mechanisms for metapristone. a effects of metapristoe on cell cycle distribution in HT-29 cells; b effects of metapristone on ΔΨm in HT-29 cells; results are expressed as percentage change in metapristone treatment compared to the untreated control (*p < 0.05, **p < 0.01); c flow cytometric analysis of PE Annexin V/7-AAD staining for metapristone in HT-29 cells; d detection of caspase-3 activity by flow cytometric analysis of apoptosis in HT-29 cells, HT-29 cells were preincubated with the following: no inhibitor (upper left and bottom left panels), 20 μM Z-DEVD-FMK (upper center and bottom center panels) or 20 μM of a negative control inhibitor Z-FA-FMK (upper right and bottom right panels) for 30 min, and then either left untreated (bottom row) or treated with 80 μM of metapristone for 24 h (top row)

Fig. 5

The molecular docking of mifepristone and metapristone with glucocorticoid receptors (PDB ID: 1NHZ). The purple structures representing either mifepristone (2-methyl) or metapristone (1-methyl) embedded in the active centers of the receptors linked by the key hydrogen bonds (dash lines)