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Indole Derivative Interacts with Estrogen Receptor Beta and Inhibits Human Ovarian Cancer Cell Growth

Ovarian cancer remains the leading cause of mortality among gynecological tumors. Estrogen receptor beta (ERβ) expression has been suggested to act as a tumor suppressor in epithelial ovarian cancer by reducing both tumor growth and metastasis. ERβ expression abnormalities represent a critical step in the development and progression of ovarian cancer: for these reasons, its re-expression by genetic engineering, as well as the use of targeted ERβ therapies, still constitute an important therapeutic approach. 3-{[2-chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-1H-indol-3-yl]methylene}-5-hydroxy-6-methyl-1,3-dihydro-2H-indol-2-one, referred to here as compound 3, has been shown to have cytostatic as well cytotoxic effects on various hormone-dependent cancer cell lines. However, the mechanism of its anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of such an effect in the human ovarian cancer cell line IGROV1. Chromatin binding protein assay and liquid chromatography mass spectrometry were exploited to localize and quantify compound 3 in cells. Molecular docking was used to prove compound 3 binding to ERβ. Mass spectrometry-based approaches were used to analyze histone post-translational modifications. Finally, gene expression analyses revealed a set of genes regulated by the ERβ/3 complex, namely CCND1, MYC, CDKN2A, and ESR2, providing possible molecular mechanisms that underline the observed antiproliferative effects.


Nuclei extraction
The nuclei of IGROV1 cells treated for 2, 6, 12, 24, or 48 h with 3 were extracted according to Amellem et al. [1] while nuclear lipids were extracted according to Folch, Lees, and Stanley [2]. The nuclei extracts (pellets) were added to 100 μL of a mixture of methanol:water:formic acid 50:50:0.1 (v/v), placed in an ultrasonic bath, centrifuged, and the supernatants were injected into the liquid chromatography-mass spectrometry (LC-MS) system.

LC-MS analysis
LC-MS analysis was carried out on a Jasco PU-1585 Liquid Chromatograph (Jasco Corporation, Tokyo, Japan) interfaced with a LCQ-Duo Mass Spectrometer (Thermo Finnigan, San Jose, CA). The mass spectrometer was equipped with an electrospray ionization (ESI) source and operated with an Ion Trap analyzer. ESI system was employed at 4.5 kV (positive polarity) spray voltage and heated capillary temperature of 220 °C. The sheath gas and the auxiliary gas (nitrogen) flow rates were set at 0.75 and 1.2 L/min, respectively. Electrospray ionization was optimized using 3 as the reference compound. The mass chromatograms were acquired in Total Ion Current (TIC) modality from 150 to

Histones treatment and extraction.
IGROV1 cells were cultured with or without 5 μM 3l for 6 h, and the histone fraction was immediately extracted. Cells were harvested using 0.11% trypsin and 0.02% EDTA, washed twice with 10 mM sodium butyrate (NaBU) in PBS, and nuclei were isolated according to Amellem et al. [1]. The nuclear pellet was suspended in 0.1 mL of ice-cold water using a Vortex mixer, and H2SO4 was added to the suspension to give a final concentration of 0.4 N. After incubation at 4 C for 1 h, the suspension was centrifuged for 5 min at 14,000 g, and the supernatant was taken and mixed with 1 mL of acetone. After overnight incubation at −20 °C, the coagulate material was collected by microcentrifugation and air-dried.

LC-ESI-MS analysis.
The histones PTMs were analyzed slightly adapting the analytical workflow previously reported [3] using an Agilent 1200 chromatography system (Agilent, Walbronn, Germany) interfaced with a quadrupole-time of flight (Q-TOF Micro, Micromass, Manchester, UK) hybrid mass analyzer equipped with a Z-spray electrospray (ESI) ion source. Briefly, the chromatographic separation of histones was performed on a C4 (Jupiter Phenomenex 5 mm, 2.0 ×150 mm i.d.) column, assembled with a precolumn SecurityGuardTM HPLC system consisting of a C4 guard cartridge (Phenomenex

Assessment of the acetylation position on histone H4
Chromatographic purification of H4.
LC preparative separation of the histone H4 was performed using a Jasco PU-1585 liquid chromatograph (Jasco Corporation, Tokyo, Japan) with a Rheodyne 7281 injection valve (50-μL sample loop) interfaced with a UV detector (Jasco, UV 1575), fixed at 220 nm. The chromatographic method employed for the histone H4 purification is the same used for the LC-ESI-MS analysis. Repeated injections of histones sample were performed and histones H4 were eluted from the column, collected, and dried under vacuum. The purity of the isolated protein was checked under the same chromatographic conditions.

H4 enzymatic digestion and MALDI-TOF analysis.
Histone H4 was digested with endoproteinase Arg-C (Roche) in 20 mM ammonium bicarbonate buffer pH 8.0 at an enzyme ratio 1:50 at 37 °C overnight. Matrix assisted laser desorbtion ionizationtime of flight (MALDI-TOF) analysis of the Arg-C digested was performed using a Voyager DE Pro (Applied Biosystems, Foster City, CA) equipped with a pulsed N2 laser operating at 337 nm. Positive ion spectra were acquired in reflector mode over an m/z range of 500-4000 amu, using a 20,000-V accelerating voltage, a 14,900-V grid voltage, and an extraction delay time of 150 ns. The spectrum of each spot was obtained by averaging the results of 100 laser shots. Histones digest spectra were internally calibrated on 40-45 and 1-17 theoretical histone digest peptide masses that were sufficiently abundant in the spectra. The analysis was performed by spotting 1 μL of histone H4 digested sample mixed with an equal volume of matrix solution, consisting of CHCA (10 mg/mL) in water-acetonitrile-TFA (50:50:0.1, v/v/v), on the target plate.

Results MALDI-TOF analysis
To confirm that the increased level of the histone H4 acetylation, assessed by LC-MS analysis, was localized at the level of the N-term tail, the enzymatic digestion of the purified histone H4 followed by MALDI-TOF analysis was performed. Endoproteinase Arg-C was used as proteolytic enzyme to obtained peptides of length compatible with the MS detection. Among others, peptides 5-18 were identified as carrying different degrees of acetylation (from 0 to 5; Figure S1). The relative abundance of these forms, derived from the analysis of sample treated or not with 3 for 6 and 24 h, confirmed the results achieved by the analysis on the intact protein. Indeed, 3 was found to induce an increment of the relative abundance of the forms carrying a higher number of acetylation (from 2 to 4) with a concomitant decrease of the less acetylated forms ( Figure S1). Figure S2. The histograms report the relative abundance of the peptide 5-18 derived from the MALDI-TOF analysis of the histone H4 digested with the endoproteinase Arg-C. Histones were extracted from IGROV1 cells after treatment with 3 (red) or from untreated cells (grey) for: 6 h (a); and 24 h (b).