Abstract
Lysine-specific demethylase 1 (LSD1) is abnormally overexpressed in various tumour tissues of patients and has been an attractive anticancer target. In this work, a potent LSD1 inhibitor (compound 14) was designed and synthesised by the molecular hybridisation strategy. It displays the potent antiproliferative activity against HepG2, HEP3B, HUH6, and HUH7 cells with IC50 values of 0.93, 2.09, 1.43, and 4.37 μM, respectively. Furthermore, compound 14 is a selective and reversible LSD1 inhibitor with an IC50 value of 0.18 μM and increases the methylation levels of H3K4me1/2. Molecular docking studies showed that it formed hydrogen bonds, hydrophilic interactions and hydrophobic interactions with residues of LSD1. Anticancer mechanisms demonstrated that it suppresses migration and epithelial–mesenchymal transition process in HepG2 cells. Importantly, it exhibits potent anti-liver cancer effects in vivo without obvious toxic effects. These interesting findings suggested that compound 14, a novel LSD1 inhibitor, may be a promising therapeutic agent to treat liver cancer.
Keywords: LSD1, anti-liver cancer effects, H3K4me1/2, migration, epithelial–mesenchymal transition
Graphical abstract
Introduction
Cancer is a global disease with a high mortality rate, and it imposes an immense threat on the health of human beings around the world1. Epigenetics is heritable and dynamic changes in gene functions that occur independently of the DNA sequence variations2. Epigenetic modifications can generally be divided into three categories: non-coding RNAs, histone modifications, and DNA or RNA methylations, which are considered to be the main regulatory mechanisms in the progression of cancer3. Lysine-specific demethylase 1 ([LSD1], also named KDM1A) as a member of the monoamine oxidase family is the first identified histone demethylase in epigenetics4. LSD1 could specifically remove methyl groups from Lys9me1/2 or Lys4me1/2 of histone H3, and it is abnormally overexpressed in various tumour tissues of patients5. LSD1 has been an attractive anticancer target, and pharmacological inhibition of LSD1 could significantly slow tumour progression in acute myeloid leukaemia and solid tumours6.
Recently, numerous LSD1 inhibitors with potent anticancer effcts are designed and reported7. Seclidemstat (compound 1, Figure 1) reversibly inhibits LSD1 with an IC50 value of 13 nM and has completed the clinical trial to treat patients with advanced solid tumours (NCT03895684)8. Quinazoline derivative 2 displays the potent antiproliferative activity with an IC50 value of 0.3 μM against THP-1 cells and increases the expression levels of H3K4me29. Tetrazole analogue 3 as a selective LSD1 inhibitor displays the synergistic effect against NB cells with bortezomib10. Raloxifene (compound 4) as a LSD1 inhibitor with an IC50 value of 2.08 μM inhibits migration against ACHN cells11. Styrenylcyclopropylamine LSD1 inhibitor 5 covalently inhibits LSD1 and shows the potent anticancer effects in a Kasumi-1 xenograft model of acute myeloid leukaemia12. N-benzylarylamide analogue 6 inhibits LSD1 and induces the accumulation of H3K4me1/2 with potent anticancer effects in vitro and in vivo13. Compared with covalent inhibitors, only a few reversible inhibitors have entered the clinic trials. Therefore, the development of novel and potent reversible LSD1 inhibitors is very significant for the clinical treatment of cancer.
Figure 1.
Reported LSD1 inhibitors.
Piperidine-2,6-dione scaffold belongs to the privileged fragment in drug design and piperidine-2,6-dione is widely used in the development of anticancer agents. Piperidine-2,6-dione analogue 7 (Figure 2) induces cell cycle arrest and reduces clonogenic capacity in glioblastoma cells14. Piperidine-2,6-dione derivative 8 displays the potent antiproliferative activity against HepG2, PC3, and MCF-7 cell lines with IC50 values of 9.81, 15.49, and 10.09 μM, respectively15. Piperidine-2,6-dione analogue 9 induces apoptosis and inhibits proliferation in diffuse large B-cell lymphoma cells16. Piperidine-2,6-dione derivative 10 exhibits the antiproliferative activity with IC50 values of 5.52 and 5.17 μM against myeloma MOLP-8 and KMS-12-PE cells17. In addition, propanamide scaffold is a promising linker in anticancer agents. Compound 11 with the propanamide linker as a carbonic anhydrase inhibitor displays the antiproliferative activity against HCT-116 and MCF-7 cells with IC50 values of 0.57 and 0.67 μM18. Compound 12 significantly inhibits colony formation and induces apoptosis against ovarian cancer cells19. 4(3H)-quinazolinone derivative with the propanamide linker 13 exhibits extensive-spectrum anticancer effects against various cell lines20. Thus, piperidine-2,6-dione scaffold and propanamide linker are both promising fragments to design anticancer agents.
Figure 2.
Anticancer agents containing a piperidine-2,6-dione scaffold or a propanamide linker.
Molecular hybridisation as a well-exploited drug design strategy could combine different chemical fragments in a novel and bioactive compound21. For medicinal chemists, molecular hybridisation has been a powerful tool to discover potent anticancer agents22. In this work, targeted compound 14 is designed by the molecular hybridisation strategy based on structures of a N-benzylarylamide derivative 6, a piperidine-2,6-dione analogue 7 and a propanamide derivative 13 (Figure 3). We assumed that linking a piperidine-2,6-dione scaffold and a propanamide linker to the N-benzylarylamide moiety could generate compound 14, which might be a novel LSD1 inhibitor with the potential antiproliferative activity. Luckily, compound 14 was identified as a selective and reversible LSD1 inhibitor with potent anti-liver cancer effects in vitro and in vivo. To the best of our knowledge, it has not been reported in references regarding a piperidine-2,6-dione-propanamide hybrid as the reversible LSD1 inhibitor with anti-liver cancer effects so far.
Figure 3.
Discovery of a novel LSD1 inhibitor 14 with potent anti-liver cancer effects by the molecular hybridisation strategy.
Results and discussion
Synthesis of compound 14
Following synthetic procedures outlined in Scheme 1, 3,4,5-trimethoxyaniline I was reacted with 1-(chloromethyl)-4-methoxybenzene and 3-bromopropionyl chloride in the presence of potassium carbonate at room temperature to obtain the crude intermediate II in 83.6% yield. Piperidine-2,6-dione was reacted with intermediate II in the presence of triethylamine at 85 °C to obtain the targeted compound 14 in 37.6% yield. Chemical structure of compound 14 was characterised by 1H NMR and 13C NMR. The detailed data of NMR and HRMS are provided in the Supporting information.
Scheme 1.
Synthesis of compound 14. Reagents and conditions: (a) 1-(Chloromethyl)-4-methoxybenzene, potassium carbonate, dichloromethane, 3-bromopropionyl chloride, rt. (b) Piperidine-2,6-dione, triethylamine, acetonitrile, 85 °C.
Compound 14 has a broad-spectrum antiproliferative activity
Human prostate cancer cells (C42B and PC3), colon cancer cells (Caco-2), liver cancer cells (HepG2), breast cancer cells (MCF7), leukaemia cells (HL-60), thyroid cancer cells (8505 C), ovarian cancer cells (OVCAR-3), and lung cancer cells (A549) were cultured to evaluate the viability of compound 14. 5-Fluorouracil, a broad-spectrum anticancer drug, was selected as the control. In our CCK8 assays, 5-fluorouracil exhibited antiproliferative activities with IC50 values of 16.72, 12.28, >20, 9.87, >20, 7.25, 12.37, >20, and >20 μM against C42B, PC3, Caco-2, HepG2, MCF7, HL-60, 8505 C, OVCAR-3, and A549 cell lines, respectively. From results of cell viability in Figure 4, compound 14 could inhibit the proliferation in a concentration-dependent manner against all cell lines. When cancer cells were treated with compound 14 at 10 μM for 48 h, cell viability rates were 87.99%, 52.03%, 72.27%, 14.18%, 49.72%, 35.66%, 49.68%, 80.70%, and 41.07% against C42B, PC3, Caco-2, HepG2, MCF7, HL-60, 8505 C, OVCAR-3, and A549 cells, respectively. All these results indicated that compound 14 has a broad-spectrum antiproliferative activity.
Figure 4.
Cell viability of various cancer cell lines treated with compound 14. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Compound 14 displays potent anticancer effects in vitro against liver cancer cells
Among antiproliferative results of above nine cancer cell lines, compound 14 displays the most potent inhibitory activity against liver cancer HepG2 cells. Therefore, four human liver cancer cell lines were selected to evaluate its anticancer effects in vitro. Based on the inhibitory results of Figure 5, its IC50 values of HepG2, HEP3B, HUH6, and HUH7 cells were 0.93, 2.09, 1.43, and 4.37 μM, respectively. All these results in Figure 5 showed that compound 14 displays potent anticancer effects in vitro against liver cancer cells.
Figure 5.
Antiproliferative activity of compound 14 against human liver cancer cell lines. **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Compound 14 is a selective and reversible LSD1 inhibitor
LSD1 has about 70% sequence similarity with monoamine oxidases MAO-A/B23. In order to evaluate the selectivity against LSD1, we also tested the inhibitory activity of compound 14 against MAO-A and MAO-B. As shown in Figure 6(A), compound 14 has weak inhibition against MAO-A and MAO-B with IC50 values of >1 μM. Seclidemstat as a potent LSD1 inhibitor has entered the clinical trial to treat advanced solid tumours. Thus, seclidemstat was selected as a control drug in the enzymatic activity experiments and animal experiments. Based on the results in Figure 6(B), compound 14 is a potent LSD1 inhibitor with an IC50 value of 0.18 μM. An irreversible inhibitor GSK-LSD1 was used in the dialysis assay to evaluate the reversibility of compound 14. As shown in Figure 6(C), compound 14 inhibits LSD1 in a reversible manner. In addition, compound 14 could increase the methylation levels of H3K4me1 and H3K4me2 as LSD1 substrates in a concentration-dependent manner (Figure 6(D)). All these results indicated that compound 14 is a potent, selective and reversible LSD1 inhibitor.
Figure 6.
Identification of compound 14 as a potent, selective and reversible LSD1 inhibitor. (A) Inhibitory rates of compound 14 against LSD1, MAO-A, and MAO-B. (B) The inhibitory activity of compound 14 and seclidemstat against LSD1. (C) Dialysis assay to evaluate the reversibility. (D) Western blotting of LSD1 substrates H3K4me1/2.
Molecular docking studies
3D structure of LSD1 was used as the template for docking studies in this work (PDB: 5LHI). All water molecules, inhibitors and ligands were removed to prepare the PDB file. AutoDock 4.2.6 and PyMOL softwares were used to perform these docking studies. As shown in Figure 7(A,B), compound 14 is located into the active pocket of LSD1. Based on the docking results in Figure 7(C), oxygen atom of 4-methoxybenzyl unit formed two hydrogen bonds with residues T189 (2.1 Å) and Y255 (3.0 Å). Piperidine-2,6-dione scaffold of compound 14 formed two hydrogen bonds with the residue Q191 (3.0 Å, 3.3 Å). 3,4,5-Trimethoxyphenyl unit of compound 14 also formed a hydrogen bond with the residue R805 (2.2 Å). Furthermore, compound 14 formed hydrophilic interactions with residues S190, S817, and G823. It also formed hydrophobic interactions with residues L261, I804, L816, L819, and F264, respectively. These docking results might explain the potent inhibitory activity of compound 14 against LSD1.
Figure 7.
Docking studies of compound 14 targeting LSD1. (A) Surface map of compound 14 in LSD1. (B) Hydrogen bonds between compound 14 and surrounding residues in LSD1. (C) Detailed 2D binding models to show hydrogen bonds, hydrophilic interactions, and hydrophobic interactions.
Compound 14 inhibits migration and epithelial–mesenchymal transition process in liver cancer HepG2 cells
Recent reference reported that the interruption of LSD1 with siRNA or small molecule inhibitors could inhibit proliferation and migration of cancer cells24. Wound healing and transwell assays were performed to investigate migration effects of compound 14 against HepG2 cells. Compared to the migration effect of control group, compound 14 significantly suppresses the migration of HepG2 cells (Figure 8(A,B)). Epithelial–mesenchymal transition plays a crucial role in the occurrence and migration of various tumours. In addition, LSD1 could promote the epithelial–mesenchymal transition in human cancer cells25. Western blotting was used to determine expression levels of epithelial–mesenchymal transition-related proteins (E-cadherin, N-cadherin, and Vimentin) in HepG2 cells treated with compound 14. As shown in Figure 8(C), compound 14 increases the expression level of E-cadherin and decreases the expression levels of N-cadherin and Vimentin in HepG2 cells. All these results investigated that compound 14 inhibits migration and epithelial–mesenchymal transition process in liver cancer HepG2 cells.
Figure 8.
Effects of migration and epithelial–mesenchymal transition process in liver cancer HepG2 cells treated with compound 14. (A) Wound healing assay. (B) Transwell assay. (C) Western blotting analysis of epithelial–mesenchymal transition related proteins.
Compound 14 exhibits potent anti-liver cancer effects in vivo
Finally, we subcutaneously inoculated human liver cancer HepG2 cells in nude mice to evaluate anti-liver cancer effects in vivo of compound 14. As shown in Figure 9(A), nude mice treated with compound 14 did not show significant changes in body weight compared to the control group. Furthermore, compound 14 and seclidemstat inhibited the growth of subcutaneous tumours in nude mice (Figure 9(B)). The tumour volume of nude mice treated with compound 14 was significantly lower than that in control group. After the intragastric administration for 21 d, tumours were isolated from nude mice. Tumour weight of nude mice treated with compound 14 was lower than that of control group, and the tumour growth inhibition rate of compound 14 was 50.3% (Figure 9(C)). Haematoxylin and eosin staining of liver, spleen, lung, and kidney in nude mice showed no morphological differences in nude mice treated with compound 14 compared to the control group (Figure 9(D)). These results in animal studies indicated that compound 14 potently inhibits tumour growth in nude mice bearing HepG2 cells without obvious toxic effects.
Figure 9.
Xenograft studies of compound 14. (A) Weight of mice. (B) Tumour volume. (C) Tumour weight. (D) The images of tumours. (E) Haematoxylin and eosin staining of liver, spleen, lung, and kidney. **p < 0.01.
Conclusions
In summary, we designed and synthesised a piperidine-2,6-dione-propanamide-trimethoxyphenyl hybrid 14 using the molecular hybridisation strategy. Its potential inhibitory effects against C42B, PC3, Caco-2, HepG2, MCF7, HL-60, 8505 C, OVCAR-3, and A549 cells indicated that compound 14 has a broad-spectrum antiproliferative activity. Compound 14 exhibits potent antiproliferative activity against human liver cancer HepG2, HEP3B, HUH6, and HUH7 cells with IC50 values of 0.93, 2.09, 1.43, and 4.37 μM, respectively. Furthermore, compound 14 is a potent, selective and reversible LSD1 inhibitor with an IC50 value of 0.18 μM and increases methylation levels of H3K4me1 and H3K4me2. Molecular docking studies showed that compound 14 formed five hydrogen bonds with residues T189, Y255, Q191, and R805 of LSD1. Anticancer mechanisms in liver cancer HepG2 cells investigated that compound 14 inhibits migration and epithelial–mesenchymal transition process. Importantly, compound 14 exhibits potent anti-liver cancer effects in vivo. Therefore, compound 14 could be a promising lead compound to discover more potent anticancer agents, and the development of LSD1 inhibitors might be an effective strategy to treat liver cancer.
Experimental section
General
PC3 prostate cancer cells, Caco-2 colon cancer cells, HepG2, HEP3B, HUH6, and HUH7 liver cancer cells, MCF7 breast cancer cells, HL-60 leukaemia cells, OVCAR-3 ovarian cancer cells, and A549 lung cancer cells were obtained from Wuhan Pricella Biotechnology Co., Ltd (Wuhan, China). The catalogue numbers of PC3, Caco-2, HepG2, MCF7, HL-60, OVCAR-3, A549, HEP3B, HUH6, and HUH7 cell lines in Wuhan Pricella Biotechnology Co., Ltd are CL-0185, CL-0050, CL-0103, CL-0149, CL-0110, CL-0178, CL-0016, CL-0102, CL-0119, and CL-0120, respectively. C42B prostate cancer cells were obtained from Wuhan SUNNCELL Biotech Co., Ltd (Wuhan, China). The catalogue number of C42B cells in Wuhan SUNNCELL Biotech Co., Ltd is SNL-161. 8505 C thyroid cancer cells were obtained from Shanghai Xuanya Biotechnology Co., Ltd (Shanghai, China). The catalogue number of 8505 C cells in Shanghai Xuanya Biotechnology Co., Ltd is XY-XB-2153. Cell lines were maintained in different medium with 10% foetal bovine serum and 1% penicillin/streptomycin (Servicebio, Wuhan, China). All cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Chemical agents 3,4,5-trimethoxyaniline (I), 1-(chloromethyl)-4-methoxybenzene, potassium carbonate, dichloromethane, 3-bromopropionyl chloride, piperidine-2,6-dione, triethylamine and acetonitrile to synthesise compound 14 were obtained from Innochem (Beijing, China).
Procedure for the synthesis of compound 14
A mixture of 3,4,5-trimethoxyaniline I (3 mmol), 1-(chloromethyl)-4-methoxybenzene (3 mmol), potassium carbonate (3 mmol), and dichloromethane (20 ml) was stirred at room temperature for 6 h. Then, 3-bromopropionyl chloride (4 mmol) dissolved in dichloromethane (6 ml) was added. The system was stirred at room temperature for 3 h. The reaction system was filtered and concentrated to obtain the crude intermediate II. Yield, 83.6%.
To a stirred solution of piperidine-2,6-dione (4 mmol) and triethylamine (4 mmol) in acetonitrile (10 ml), a solution of the crude intermediate II (3 mmol) in acetonitrile (15 ml) was added. The reaction mixture was refluxed at 85 °C for 6 h to synthesise compound 14. Then, it was purified by column chromatography (petroleum:ethyl acetate = 10:1).
3-(2,6-dioxopiperidin-1-yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)propanamide (14):
Yield: 37.6%, yellow liquid.1H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 6.14 (s, 2H), 4.65 (s, 2H), 4.04 − 3.87 (m, 2H), 3.78 (s, 3H), 3.71 (s, 3H), 3.65 (s, 6H), 2.55 (t, J = 6.5 Hz, 4H), 2.30 − 2.22 (m, 2H), 1.90 − 1.82 (m, 2H).13C NMR (100 MHz, CDCl3) δ 171.48, 169.10, 157.96, 152.45, 136.50, 129.52, 128.75, 112.60, 104.81, 59.91, 55.14, 54.26, 51.12, 35.27, 31.78, 30.96, 15.97. HRMS (ESI): [M-H]- calcd. for C25H30N2O7: 469.19693, found: 469.19836.
CCK8 assay
C42B, PC3, Caco-2, HepG2, MCF7, HL-60, 8505 C, OVCAR-3, and A549 were cultured to screen the broad-spectrum antiproliferative activity of compound 14. For above cell lines, three thousand cells per well were seeded in the 96-well plate and treated by compound 14 for 48 or 72 h at different concentrations (0, 5, 10, and 20 μM). In addition, HepG2, HEP3B, HUH6, and HUH7 were cultured to screen its antiproliferative activity against liver cancer cell lines. For these liver cancer cell lines, two thousand cells per well were seeded in the 96-well plate and treated by compound 14 for 48 h at different concentrations (0, 0.625, 1.25, 2.5, and 5 μM). Then, 10 μl of CCK8 was added into each well and cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C for 4 h. The 96-well plate was measured with a microplate reader (Bio-Tek, San Diego, CA) at a wavelength of 450 nm to calculate IC50 values.
Inhibitory activity against LSD1, MAO-A and MAO-B
LSD1 screening assay kit was purchased from Cayman Chemical (Ann Arbor, MI) to evaluate the enzymatic activity of compound 14. An aliquot of 20 μl of LSD1 assay reagent, 20 μl LSD1 assay peptide, 120 μl of LSD1 buffer solution and 10 μl of compound 14 at different concentrations (6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1000 nM) were added to each well in the 96-well plate. After the incubation for 30 min at 37 °C, a mixture of horseradish peroxidase solution (20 μl) and fluorometric substrate solution (10 μl) was added to each well. Then, the 96-well plate was incubated for 15 min at 37 °C. Finally, the fluorescence of each well in the 96-well plate was measured at an emission wavelength of 590 nm and an excitation wavelength of 530 nm by a fluorimeter (Agilent Technologies Inc., Palo Alto, CA). Seclidemstat, a famous LSD1 inhibitor, was used as the control drug in this assay.
In order to analyse the enzymatic selectivity of compound 14, MAO-A/B was purchased from Servicebio (Wuhan, China). MAO-A was dissolved in phosphate buffer (0.1 M, pH = 7.4) to get MAO-A enzymatic buffer with a concentration of 0.006 mg/ml. MAO-B was also dissolved in phosphate buffer (0.1 M, pH = 7.3) to get MAO-B enzymatic buffer with a concentration of 0.015 mg/mL. Kynuramine dihydrochloride (Servicebio, Wuhan, China) was used to get the assay buffer with a concentration of 40 μM for MAO-A and 20 μM for MAO-B. An aliquot of 97 μl of assay buffer and 3 μl of compound 14 at different concentrations (250, 500, and 1000 nM) were added into each well on the 96-well black plate. The plate was incubated at 37 °C for 20 min, and 80 μl of 2 N sodium hydroxide was added into each well to stop the reaction. The fluorescence of each well was measured using a microplate reader (Agilent Technologies Inc., CA) with an excitation at 310 nm and an emission at 380 nm.
Dialysis assay
A mixture of compound 14 and the recombinant LSD1 was incubated for 60 min at 37 °C. Then, the system was dialysed against 50 mmol/l HEPES buffer at 37 °C for 12 h. Finally, the HEPES buffer was replaced and the system was incubated for 12 h at 37 °C. In the dialysis experiment, an irreversible inhibitor GSK-LSD1 was purchased as the reference drug (Innochem, Beijing, China).
Western blotting
Compound 14 at different concentrations (0, 0.5, 1, and 2 μM) was added into cancer cells solution and incubated for 48 h. Total protein was isolated from cells using RIPA buffer (Servicebio, Wuhan, China). An amount of 30 mg of total protein were separated by sodium dodecyl sulphate polycrylamide gel electrophoresis and transferred to PVDF membranes. 5% non-fat milk (Servicebio, Wuhan, China) was used to block PVDF membranes and incubated with primary antibody for 12 h at 4 °C. Then, PVDF membranes were washed by TBST buffer (Servicebio, Wuhan, China) and incubated with secondary antibody at room temperature for 2 h. The enhanced chemiluminescence kit (Servicebio, Wuhan, China) was used to visualise immunoreactive bands. H3, GAPDH, Vimentin, N-cadherin, and E-cadherin as antibodies were purchased from Servicebio (Wuhan, China). Catalogue numbers of H3, GAPDH, Vimentin, N-cadherin, and E-cadherin as antibodies in Servicebio are GB11102, GB15004, GB11192, GB12135, and GB12868, respectively. H3K4me1 and H3K4me2 were obtained from Proteintech Group, Inc (Wuhan, China). Catalogue numbers of H3K4me1 and H3K4me2 as antibodies in Proteintech Group are 91289 and 39679, respectively.
Molecular docking
Protein structure of LSD1 was obtained from protein data bank (https://www.rcsb.org/). The resolution of LSD1 in molecular docking studies was 3.40 Å (PDB code: 5LHI). Water and ligand molecules (FAD, 6 × 5 and GOL) of the protein were deleted. AutoDock 4.2.6 was used to add hydrogen and partial charges. In the docking process, number of points in x-dimension, y-dimension and z-dimension were 70, 50, and 86, respectively. Docking results between compound 14 and LSD1 were reserved as a PBD file. Hydrogen–bond interaction, hydrophobic interaction and hydrophilic interaction were studied by PyMOL.
Wound healing
Cancer cells were seeded into a 6-well plate for 12 h and each well was scratched using a sterile micropipette tip. Cellular debris was removed by PBS solution (Servicebio, Wuhan, China), and compound 14 at different concentrations (0, 0.5 μM, 1 μM, and 2 μM) was added. The 6-well plate was incubated for 48 h and photographed on a microscope.
Migration
Cancer cells were seeded into a Transwell 24-well plate (Corning, NY) and compound 14 was added. About 20% foetal bovine serum and 1% heat-inactivated foetal bovine serum were respectively added into the lower and upper chamber. The 24-well plate was incubated for 48 h at 37 °C, and it was washed by water. Cancer cells were fixed with cold ethanol for 20 min and stained with haematoxylin solution (Servicebio, Wuhan, China).
Xenograft study
Tumorigenesis in nude mice could stimulate the growth process of human tumours in the body, and could be used to develop and evaluate the effectiveness and safety of anti-cancer candidates. Therefore, xenograft studies in nude mice were performed to evaluate anticancer effects of compound 14. Animals were treated according to the protocols established by the ethics committee of The Fourth Clinical College of Xinxiang Medical University. The in vivo experiments were carried out in accordance with the approved guidelines and approved by the ethics committee of The Fourth Clinical College of Xinxiang Medical University. The number of ethics approvals is XMU-2024–01-03. All authors have adhered to the ARRIVE guidelines (https://arriveguidelines.org/). BALB/c nude mice were purchased from Beijing Huafukang Biotechnology Co., LTD (Beijing, China), and fifteen mice were used in this study (aged 6–7 weeks and weighing 20–30 g). Before the animal experiments, all nude mice were given a week to adjust to the clean, hygienic and specific-pathogen-free environment. All nude mice were taken care of instainless steel cages with a constant temperature of 60% humidity, 25 °C and a 12/12 light/dark cycle. Nude mice were fed with refreshing water and nourishing food everyday.
Then, nude mice were anaesthetised with 2% isoflurane and 0.3 l/min of oxygen by the small animal anaesthesia machine (Beijing Yi Zejia Technology Co., LTD, Beijing, China). 3 × 105 human liver cancer HepG2 cells were subcutaneously injected in the right armpit of mice. Until the volume of tumours reached 60–100 mm3, all mice were randomly divided into three groups and each group had five nude mice. An amount of 60 mg/kg of compound 14, 10 mg/kg of seclidemstat and water as the control group were given by gavage every day for 21 days. Weight of mice and tumour volume were measured every 3 d. All nude mice in this xenograft studies were performed euthanizing procedures using a carbon dioxide euthanasia chamber (Shanghai Yuyan Instruments Co., Ltd, Shanghai, China). The flow rate of carbon dioxide was adjusted at 20% of the volume of the euthanasia chamber per minute. Once nude mice were unconscious, the flow rate was increased to 100% of the euthanasia chamber volume per minute. Death was confirmed by immobility, non-breathing and pupil enlargement. Tumours form BALB/c nude mice were isolated and weighed.
Haematoxylin and eosin staining
Liver, spleen, lung, and kidney were isolated from BALB/c nude mice and stained by haematoxylin and eosin solution. The detailed methods were performed according to a reported reference26. First, liver, spleen, lung, and kidney were added into tubes of 4% paraformaldehyde fixative (Servicebio, Wuhan, China). Then, tissues were dehydrated in the alcohol solution, and xylene was added into the solution for 2 h. Finally, sectioning and staining were performed to finish this experiments.
Statistical analyses
All biological data were obtained at least three independent experiments. Statistical differences of two groups were determined with analysis of Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant.
Supplementary Material
Funding Statement
The authors gratefully acknowledge the financial support of Henan Province Medical Science and Technology Tackling Program Joint Co-Construction Project (No. LHGJ20221004).
Author contributions
Xiao-Song Zhang: Writing, Methodology, Investigation, Data curation, Formal analysis. Jin-Zhan Liu: Methodology, Data curation, Formal analysis. Ying-Ying Mei: Methodology, Formal analysis. Meng Zhang: Methodology, Formal analysis. Li-Wei Sun: Methodology, Data curation.
Disclosure statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.
Data availability statement
The data in this study are available from the corresponding author upon the reasonable request.
References
- 1.Siegel RL, Giaquinto AN, Jemal A.. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12–49. [DOI] [PubMed] [Google Scholar]
- 2.Lu Y, Chan YT, Tan HY, Li S, Wang N, Feng Y.. Epigenetic regulation in human cancer: the potential role of epi-drug in cancer therapy. Mol Cancer. 2020;19(1):79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bennett RL, Licht JD.. Targeting epigenetics in cancer. Annu Rev Pharmacol Toxicol. 2018;58(1):187–207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Pallavi R, Gatti E, Durfort T, Stendardo M, Ravasio R, Leonardi T, Falvo P, Duso BA, Punzi S, Xieraili A, et al. Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion. Nat Commun. 2024;15(1):828. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Lynch JT, Harris WJ, Somervaille TCP.. LSD1 inhibition: a therapeutic strategy in cancer? Expert Opin Ther Targets. 2012;16(12):1239–1249. [DOI] [PubMed] [Google Scholar]
- 6.Perillo B, Tramontano A, Pezone A, Migliaccio A.. LSD1: more than demethylation of histone lysine residues. Exp Mol Med. 2020;52(12):1936–1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Yang GJ, Lei PM, Wong SY, Ma DL, Leung CH.. Pharmacological inhibition of LSD1 for cancer treatment. Molecules. 2018;23(12):3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Noce B, Di Bello E, Fioravanti R, Mai A.. LSD1 inhibitors for cancer treatment: Focus on multi-target agents and compounds in clinical trials. Front Pharmacol. 2023;14:1120911. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Menna M, Fiorentino F, Marrocco B, Lucidi A, Tomassi S, Cilli D, Romanenghi M, Cassandri M, Pomella S, Pezzella M, et al. Novel non-covalent LSD1 inhibitors endowed with anticancer effects in leukemia and solid tumor cellular models. Eur J Med Chem. 2022;237:114410. [DOI] [PubMed] [Google Scholar]
- 10.Mills CM, Turner J, Piña IC, Garrabrant KA, Geerts D, Bachmann AS, Peterson YK, Woster PM.. Synthesis and evaluation of small molecule inhibitors of LSD1 for use against MYCN-expressing neuroblastoma. Eur J Med Chem. 2022;244:114818. [DOI] [PubMed] [Google Scholar]
- 11.Ma Y, Zheng Y, Ji Y, Wang X, Ye B.. Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma. Future Med Chem. 2021;13(6):533–542. [DOI] [PubMed] [Google Scholar]
- 12.Gehling VS, McGrath JP, Duplessis M, Khanna A, Brucelle F, Vaswani RG, Côté A, Stuckey J, Watson V, Cummings RT, et al. Design and synthesis of styrenylcyclopropylamine LSD1 inhibitors. ACS Med Chem Lett. 2020;11(6):1213–1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Yuan XY, Song CH, Liu XJ, Wang X, Jia MQ, Wang W, Liu WB, Fu XJ, Jin CY, Song J, et al. Discovery of novel N-benzylarylamide-dithiocarbamate based derivatives as dual inhibitors of tubulin polymerization and LSD1 that inhibit gastric cancers. Eur J Med Chem. 2023;252:115281. [DOI] [PubMed] [Google Scholar]
- 14.Roberto GM, Paiva HH, Botelho de Souza LE, Pezuk JA, Vieira GM, de Oliveira HF, Umezawa K, Tone LG, Brassesco MS.. DTCM-glutarimide Delays Growth and Radiosensitizes Glioblastoma. Anticancer Agents Med Chem. 2018;18(9):1323–1329. [DOI] [PubMed] [Google Scholar]
- 15.Ward MMSA, Abdallah AE, Zayed MF, Ayyad RR, El-Zahabi MA.. Design, synthesis and biological evaluation of newly triazolo-quinoxaline based potential immunomodulatory anticancer molecules. J Mol Struct. 2024;1298:137041. [Google Scholar]
- 16.Hagner PR, Man HW, Fontanillo C, Wang M, Couto S, Breider M, Bjorklund C, Havens CG, Lu G, Rychak E, et al. CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL. Blood. 2015;126(6):779–789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Krasavin M, Adamchik M, Bubyrev A, Heim C, Maiwald S, Zhukovsky D, Zhmurov P, Bunev A, Hartmann MD.. Synthesis of novel glutarimide ligands for the E3 ligase substrate receptor Cereblon (CRBN): Investigation of their binding mode and antiproliferative effects against myeloma cell lines. Eur J Med Chem. 2023;246:114990. [DOI] [PubMed] [Google Scholar]
- 18.Elbadawi MM, Eldehna WM, Nocentini A, Abo-Ashour MF, Elkaeed EB, Abdelgawad MA, Alharbi KS, Abdel-Aziz HA, Supuran CT, Gratteri P, et al. Identification of N-phenyl-2-(phenylsulfonyl)acetamides/propanamides as new SLC-0111 analogues: Synthesis and evaluation of the carbonic anhydrase inhibitory activities. Eur J Med Chem. 2021;218:113360. [DOI] [PubMed] [Google Scholar]
- 19.Zhang J, Zhang J, Hao G, Xin W, Yang F, Zhu M, Zhou H.. Design, synthesis, and structure–activity relationship of 7-propanamide benzoxaboroles as potent anticancer agents. J Med Chem. 2019;62(14):6765–6784. [DOI] [PubMed] [Google Scholar]
- 20.Mohamed MA, Ayyad RR, Shawer TZ, Abdel-Aziz AAM, El-Azab AS.. Synthesis and antitumor evaluation of trimethoxyanilides based on 4(3H)-quinazolinone scaffolds. Eur J Med Chem. 2016;112:106–113. [DOI] [PubMed] [Google Scholar]
- 21.Konidala SK, Kotra V, Danduga RCSR, Kola PK, Bhandare RR, Shaik AB.. Design, multistep synthesis and in-vitro antimicrobial and antioxidant screening of coumarin clubbed chalcone hybrids through molecular hybridization approach. Arab J Chem. 2021;14(6):103154. [Google Scholar]
- 22.Farag AK, Hassan AHE, Chung KS, Lee JH, Gil HS, Lee KT, Roh EJ.. Diarylurea derivatives comprising 2,4-diarylpyrimidines: discovery of novel potential anticancer agents via combined failed-ligands repurposing and molecular hybridization approaches. Bioorg Chem. 2020;103:104121. [DOI] [PubMed] [Google Scholar]
- 23.Carradori S, Petzer JP.. Novel monoamine oxidase inhibitors: a patent review (2012-2014). Expert Opin Ther Pat. 2015;25(1):91–110. [DOI] [PubMed] [Google Scholar]
- 24.Lv T, Yuan D, Miao X, Lv Y, Zhan P, Shen X, Song Y.. Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer. PLoS One. 2012;7(4):e35065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Zhao ZK, Dong P, Gu J, Chen L, Zhuang M, Lu WJ, Wang DR, Liu YB.. Overexpression of LSD1 in hepatocellular carcinoma: a latent target for the diagnosis and therapy of hepatoma. Tumour Biol. 2013;34(1):173–180. [DOI] [PubMed] [Google Scholar]
- 26.Takahashi M, Fujii T, Nakano M.. Visualization of features in digital slides of HE-stained hepatic histological sections. Pathology. 2014;46:S78. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data in this study are available from the corresponding author upon the reasonable request.