Chemistry
Melting points were determined on a Büchi 530 melting point apparatus and are uncorrected. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical shifts are reported in δ (ppm) units relative to the internal reference tetramethylsilane (Me4Si). EIMS spectra were recorded with a Fisons Trio 1000 spectrometer; only molecular ions (M+) and base peaks are given. All compounds were routinely checked by thin layer chromatography (TLC), 1H NMR, and 13C NMR spectra. TLC was performed on aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots visualized by UV light. All solvents were reagent grade and, when necessary, were purified and dried by standard methods. The concentration of solutions after reactions and extractions involved the use of a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. Elemental analysis has been used to determine the purity of the described compounds that is > 95%. Analytical results are within ± 0.40% of the theoretical values. All chemicals were purchased from Sigma-Aldrich, Milan (Italy) or Alfa Aesar, Karlsruhe (Germany) and were of the highest purity.
Benzyl (4-(4-(quinolin-4-ylamino) benzamido) phenyl)carbamate (MC3353)
Triethylamine (0.37 mmol, 0.05 mL) and benzyl chloroformate (0.28 mmol, 0.04 mL) were slowly added to a cooled (0 °C) solution of N-(4-aminophenyl)-4-(quinolin-4-ylamino)benzamide 1 [20] (0.1 g, 0.28 mmol) in tetrahydrofuran (5 mL), and the resulting mixture was stirred at room temperature for 1.5 h. Then, the reaction was quenched by water and extracted by dichloromethane (3 × 10 mL), washed with brine (3 × 10 mL), then dried with anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The oily residue was purified by column chromatography (SiO2 eluting with ethyl acetate/methanol 10/1) to provide pure MC3353. Melting point: 220–222 °C; recrystallization solvent: acetonitrile/methanol; yield: 72%; 1H NMR (DMSO-d6, 400 MHz, δ; ppm) δ 5.16 (s, 2H, -CH2Ph), 7.20–7.49 (m, 10H, benzene protons), 7.58–7.60 (t, 1H, quinoline proton), 7.67–7.76 (m, 3H, benzene protons and quinoline proton), 7.92–8.01 (m, 3H, benzene protons), 8.37–8.39 (d, 1H, quinoline proton), 8.57–8.59 (d, 1H, quinoline proton), 9.24 (bs, 1H, -NH), 9.72 (bs, 1H, -NHCOOBn), 10.09 (bs, 1H, -NHCOPh) ppm; 13C NMR (DMSO-d6, 100 MHz, δ; ppm) δ 66.8, 111.4 (2C), 112.8, 121.6, 121.8 (4C), 124.2 (2C), 125.7, 127.1 (2C), 127.6, 128.9 (2C), 129.2, 129.6, 130.2 (2C), 133.5, 133.6, 136.1, 138.7, 149.3, 149.7, 151.6, 153.8, 164.7 ppm; MS (EI), m/z [M]+ C30H24N4O3 calculated 488.1848, found 488.1852. Elemental analysis: calculated %: C, 73.76; H 4.95; N 11.47. Found %: C, 73.88; H, 5.06; N, 11.20.
Dissolution of compounds
5-AZA (Sigma-Aldrich, Milan, Italy) was solubilized in a HOAc:H2O (1:1) solution at 200 mM. All other compounds including RG108 (synthetized as previously described in [17]), SGI-1027 (synthetized as previously described in [20]), DAC (Sigma-Aldrich, Milan, Italy), and MC3353 were resuspended in DMSO (Sigma-Aldrich, Milan, Italy) at 100 mM, 50 mM, 10 mM, and 1 mM, respectively.
DNA methyltransferase assays
DNMT1 assay
His-DNMT1 (182 kDa, human) was cloned, expressed, and purified as described by Lee et al. [15]. The DNMT1 assay was performed according to Gros et al. [25]. Briefly, the DNMT1 enzymatic assay is based on the use of radiolabeled SAM, and the methylation occurs in homogeneous phase in 384-well microplates. The reaction is performed with DNMT1 at the final concentration of 90 nM in a total volume of 10 μL including also the chemical compound to be tested at the desired concentration, 1.25 μM of SAM//[methyl-3H] SAM (3TBq/mmol) mix in a ratio of 3:1 and 0.3 μM of biotinylated DNA duplex. DNMT1, as “maintenance” methyltransferase, requires hemimethylated DNA for the reaction. After 2 h incubation at 37 °C, 8 μL of the reaction’s solution is transferred into a streptavidin 96-well scintillant coated FlashplateTM containing 190 μL of 20 mM SAH in 50 mM Tris-HCl (pH 7.4). The FlashplateTM is then agitated at room temperature for 1 h and washed. The plate is read with TopCount (PerkinElmer, Villebon-sur-Yvette, France). The greater the DNMT1 inhibition, the lower will be the scintillation signal correlated to the incorporation of the tritiated methyl groups. In this assay, the negative and positive controls were defined as wells without enzyme and wells without any compound but just DMSO, respectively.
DNMT3A assay
DNMT3A enzyme inhibition was adapted from the restriction-based fluorescence assay protocol described in Ceccaldi et al. [18]. Briefly, it is performed in a 384-well microplate in a total volume of 50 μL. An oligonucleotide labeled at the 5′ end with biotin is hybridized to his complementary strand labeled at the 3′ end with 6-carboxyfluorescein. The DNA duplex thus formed contains only one single CpG site overlapping with a restriction site of a methylation sensitive restriction enzyme. The duplex is transferred in the wells coated with avidin. Thus, the duplex is fixed to the plate. The methylation reaction is performed by adding human C-terminal DNMT3A at the final concentration of 4 ng/μL in a total volume of 50 μL in the presence of the chemical compound to be tested at the desired concentration and the cofactor SAM at the final concentration of 20 μM. After incubation (1 h at 37 °C), the plate is washed and the methylation sensitive restriction enzyme HpyCH4IV is added. After 1-h incubation at 37 °C, the plate is washed and the fluorescence measured with a PerkinElmer Envision Multilabel Plate Reader (PerkinElmer, Villebon-sur-Yvette, France). The data are expressed as a percentage of inhibition vs. log concentration (M). Data are normalized referring to the “restriction control” (wells coated with labeled duplex not treated nor exposed to DNMT3A, but only cleaved by HpyCH4IV) as the maximum of inhibition (100%) and to the “DMSO control” (wells coated with labeled duplex but treated just with 0.1% DMSO exposed to DNMT3A and then to HpyCH4IV) as the minimum of inhibition (0%; total methylation).
CMV-luc assay in KG-1 cells
The assay was carried out as described by Rilova et al. [26]. The KG-1 cell line was stably transfected with the luciferase firefly (Luc+ from pGL3 by Promega, Madison, WI, USA) reporter gene under the control of a methylated CMV promoter (from pEGFP-N1 by Clontech) and selected for the maintenance of the methylation of CMV and luciferase expression silencing (KG-1 CMV-luc). The stably integrated KG-1 CMV-luc cells are cultivated in RPMI-1640 medium (Lonza, Strasbourg, France), supplemented with 10% FCS (Lonza, Strasbourg, France) and 0.5 mg/mL of geneticin (Sigma-Aldrich, Saint-Quentin Fallavier, France), under 5% CO2, at 37 °C and seeded at 20,000 cells per well in 96-well plates. After 5 or 24 h of incubation in the presence of compounds or the solvent DMSO, the induction of the promoter is measured by quantification of luciferase with the Brite-lite assay system (PerkinElmer, Villebon-sur-Yvette, France) according to the manufacturer’s protocol. The luminescence is measured on an EnVision™ Multilabel Plate Reader (PerkinElmer, Villebon-sur-Yvette, France), and the data are expressed as fold induction compared to DMSO control. The mean of 2–4 experiments and its standard error are reported.
To determine the antiproliferative activity of MC3353 in KG-1 cells, the cells were seeded at day 0 in a 96-well plate and treated with test compound solutions at a dose range spanning from 3.2 × 10−9 to 1 × 10−5 M at days 1, 2, and 3. On day 4, cell viability was assessed using the ATPLite kit (PerkinElmer, Villebon-sur-Yvette, France).
UCHL1 promoter demethylation assay (luciferase activity induction) in HCT116 colon cancer cells
Cell culture and treatments
Human colon cancer HCT116 (ATCC, VA, USA) cells were propagated in McCoy’s 5A medium (Euroclone, Milan, Italy) with 10% fetal bovine serum (FBS; Euroclone), 2 mM l-glutamine (Euroclone), and antibiotics (100 U/mL penicillin, 100 g/mL streptomycin) (Euroclone). For the treatments with MC3353, 5-AZA, and DAC as positive controls, 2 × 104 cells were seeded in 6-well plates in triplicate. Cells were treated every 2 days, and the medium was changed. On day 5, cells were observed under a fluorescent microscope. The nuclei were counterstained with DAPI (10 μg/mL, Invitrogen, Tournai, Belgium).
Fluorescence detection
Fluorescence of EGFP and DAPI in living cells was analyzed by Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTeK, Milan, Italy).
Transfection
Before the transfection, the pUMLIEP vector was linearized by with ClaI restriction enzyme (New England Biolabs, Milan, Italy) and then it was methylated by SssI-methylase (New England Biolabs, Milan, Italy) for 60 min. The methylated vector was transfected into HCT116 cells using Lipofectamine™2000 (Invitrogen, Monza, Italy) and Opti-MEM® (Gibco, Monza, Italy), and stably introduced clones were selected with puromycin (1.0 μg/mL, Sigma-Aldrich, Milan, Italy). The detailed protocol for the generation of cells highly responsive to DNA demethylating agents is described in [27].
Cell lines and culture conditions
U-937 (acute myeloid leukemia), RAJI (Burkitt’s lymphoma), PC-3 (prostate cancer), and MDA-MB-231 (breast cancer) cell lines were obtained from the Deutsche Sammlung für Mikroorganismen and Zellkulturen (DSZM, Braunschweig, Germany) and were cultured in RPMI 1640 (Lonza, Verviers, Belgium) complemented with 10% FBS (Lonza, Verviers, Belgium) and 1% antibiotic-antimycotic (Lonza). RPMI1788 (normal B lymphocytes) and KG-1 (acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC) and were maintained in RPMI 1640 complemented with 20% FBS and IMDM complemented with 10% FBS, respectively. Cell lines were treated with compounds at the indicated concentrations in the exponential growth phase.
Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated as previously described [28]. Freshly isolated PBMCs were either cultured [28] and treated at 1.106 cells/mL (non-proliferating PBMCs) or stimulated with phytohaemoagglutinin (PHA) and interleukin (IL)-2 (called PHA_PBMCs) to induce blastogenesis in T lymphocytes before treatments as previously described [29]. Briefly, freshly isolated PBMCs were seeded at a concentration of 2.106 cells/mL in RMPI 1640 supplemented with 1% antibiotic-antimycotic (Lonza, Verviers, Belgium) and 10% human AB serum (Corning, Fisher scientific, Merelbeke, Belgium). After overnight incubation, floating lymphoid cells were collected and seeded at 1.106 cells/mL in RMPI 1640 supplemented with 1% antibiotic-antimycotic, 5% FBS, 10% human AB serum, 1 μg/mL PHA (Gentaur, Kampenhout, Belgium), 50 U/mL IL-2 (Roche, Prophac, Luxembourg City, Luxembourg), 1% non-essential amino acids (Invitrogen, Luxembourg), 1% HEPES (Invitrogen, Luxembourg), 1% sodium pyruvate (Invitrogen, Luxembourg), and 0.1% β-mercaptoethanol (Invitrogen, Luxembourg). After 72 h, the cells were seeded at 1.106 cells/mL in the same mitogenic growth medium described just above and treated with test compounds.
The patient-derived human OS cell lines Saos-2, U-2OS, and MG-63 were obtained from ATCC (1992). The patient-derived OS cell lines IOR/OS9, IOR/OS20, and IOR/SARG were established and previously characterized at the Laboratory of Experimental Oncology of Rizzoli Orthopedic Institute [30]. Cell lines were profiled for DNA copy number changes [31] and DNA methylation status at approximately 27,000 CpG sites [32]. The cell line PDX-OS#1-C4 was obtained from OS patient-derived xenograft (PDX) after 4 passages in the animal. All cell lines were tested for mycoplasma contamination (Mycoalert Mycoplasma Detection Kit, Lonza, Verviers, Belgium) before use and authenticated by short-tandem repeat polymerase chain reaction (STR PCR) analysis (last control December 2017) using PowerPlex ESX fast System kit (Promega, Madison, WI, USA). The following loci were verified: AMEL, D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1054, VWA, D8S1179, FGA, D2S441, D12S391, D19S433, and SE33. All cell lines were immediately amplified to constitute liquid nitrogen stocks and were never passaged for more than 1 month upon thawing. Cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated FBS (Lonza, Verviers, Belgium), penicillin (20 U/mL), and streptomycin (100 μg/mL) (Sigma-Aldrich, Milan, Italy) in 37 °C humidified at 5% CO2.
Cell proliferation experiments
DKO HCT116 (ATCC, VA, USA) cells were propagated in Dulbecco’s modified Eagle medium (DMEM) (Euroclone, Milan, Italy) with 10% fetal bovine serum (FBS; Euroclone, Milan, Italy), 2 mM l-glutamine (Euroclone, Milan, Italy) and antibiotics (100 U/mL penicillin, 100 g/mL streptomycin) (Euroclone, Milan, Italy).
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich, Milan, Italy) assay was used to determine the proliferation of HCT116 clone and DKO HCT116 cells after treatment with MC3353, 5-AZA, and DAC. 5 × 104 cells/well for both cell lines were plated in 24-well plate and treated, in duplicate, with compounds at several concentrations for 24 and 48 h of induction. After treatments, MTT solution was added for 3 h at 0.5 mg/mL, the purple formazan crystals were dissolved in DMSO (Sigma-Aldrich, Milan, Italy), and the absorbance was read at a wavelength of 570 nm with reader TECAN M-200.
Proliferation and viability of U-937, KG-1, RAJI, PC-3, MDA-MB-231, RPMI1788, PBMCs, and PHA_PBMCs were assessed by trypan blue exclusion assays at the indicated time points. Nuclear morphology changes associated with apoptosis and necrosis were evaluated by fluorescence microscopy after Hoechst and propidium iodide (PI) staining as previously described [33].
To perform osteosarcoma cell growth inhibition experiments, 2 × 105 cells/well were plated, and MC3353 (0.1–30 μM) or DAC (0.5 μM) was added after 24 h. Cells were exposed for up to 96 h before being counted by Trypan blue vital cell count (Sigma-Aldrich, Milan, Italy). In parallel, the cells were treated with DMSO-containing medium as a control. The highest final concentration of DMSO in the medium was < 0.005%, and DMSO had no effect on cell growth.
RNA extraction, reverse transcription, and quantitative PCR in PC-3 and HCT116 cells for EMT pathway evaluation
RNAs were extracted by ReliaPrep™ RNA Tissue Miniprep (Promega, Madison, WI, USA) and reverse transcribed with PrimeScript RT Master Mix (Takara, Kusatsu, Shiga, Japan). cDNAs were amplified by a qPCR reaction using GoTaq qPCR Master Mix (Promega) and analyzed with the oligonucleotide pairs specific for the target genes. Relative amounts, determined with the 2(−ΔCt) method, were normalized with respect to the human housekeeping gene L32. The primers used are as follows: L32 (forward: 5′-GGAGCGACTGCTACGGAAG-3′, reverse: 5′-GATACTGTCCAAAAGGCTGGAA-3′), E-cadherin (forward: 5′-TACGCCTGGGACTCCACCTA-3′, reverse: 5′-CCAGAAACGGAGGCCTGAT-3′), and MMP2 (forward: 5′-ATGCCGCCTTTAACTGGAG-3′, reverse: 5′-GGAAAGCCAGGATCCATTTT-3′).
Western blots
Cells were lysed in Laemmli buffer; subsequently, the proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.45-μm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The following primary antibodies were used for immunoblotting: α-E-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ), α-MMP2 (Abcam), α-DNMT3a (Santa Cruz Biotechnologies, Dallas, TX), and α-GAPDH (Millipore Corp., Bedford, MA), used as a loading control. The immune complexes were detected with horseradish peroxidase-conjugated species-specific secondary antiserum (Bio-Rad Laboratories, Milan, Italy) then by enhanced chemiluminescence reaction (Bio-Rad Laboratories, Milan, Italy). Densitometric analysis of protein expression was performed by using the Fiji ImageJ image processing package.
Osteosarcoma (Saos-2) differentiation towards osteoblasts
Four days after seeding, Saos-2 cells were exposed to specific osteogenic medium (IMDM supplemented with 2% FBS, 5 mM β-glycerophosphate, and 50 μg/mL ascorbic acid, Sigma-Aldrich, Milan, Italy) without (control) or with MC3353 (0.5–1 μM) or DAC (0.1–0.5 μM) and maintained in differentiating conditions for up to 21 days. The medium was renewed every 4 days, and the cells were harvested at various time points to collect total RNA and verify bone mineralization. Specific osteoblastic markers were evaluated by quantitative real-time PCR (RT-qPCR) as previously described [34]. The primers or assays (Applied Biosystems, Foster City, CA, USA) used are as follows: GAPDH (forward: 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse: 5′-GAAGATGGTGATGGGATTTC-3′ and probe: 5′-CAAGCTTCCCGTTCTCAGCC-3′), OCN (forward: 5′-GGGCTCCCAGCCATTGAT-3′, reverse: 5′-CAAAGCCTTTGTGTCCAAGCA-3′), ALP (Hs01029144_m1), and COL1A2 (Hs01028970_m1). Amplification reactions were performed using a ViiA7™ Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). To visualize bone mineralization (osteoblastic differentiation) after 7–21 days of MC3353 or DAC treatment, the plates were stained with 40 mM Alizarin red stain (ARS, Sigma-Aldrich, Milan, Italy). ARS staining was visualized with an ECLIPSE 90i microscope (Nikon, Minato, Tokyo, Japan) equipped with Plan fluor 10× NA 0.3. Images were acquired with an autocapture setting using a digital color camera (Nikon DS5 MC) and NIS-Elements AR3.10 software (Nikon, Minato, Tokyo, Japan).
IC50 values determination and statistical analyses
IC50 values of compounds against cellular viability were determined by using nonlinear regression fitting curves with GraphPad Prism 8 or CalcuSyn software The t test used for statistical analyses was performed with GraphPad Prism 8. All the tests were one-tailed, and a p value < 0.05 was considered statistically significant.