Targeting histone demethylases JMJD3 and UTX: selenium as a potential therapeutic agent for cervical cancer.

BACKGROUND: The intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease. RESULT: Selenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets. CONCLUSIONS: Our study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

Targeting the PHF8/YY1 axis suppresses cancer cell growth through modulation of ROS.

High levels of mitochondrial reactive oxygen species (mROS) are linked to cancer development, which is tightly controlled by the electron transport chain (ETC). However, the epigenetic mechanisms governing ETC gene transcription to drive mROS production and cancer cell growth remain to be fully characterized. Here, we report that protein demethylase PHF8 is overexpressed in many types of cancers, including colon and lung cancer, and is negatively correlated with ETC gene expression. While it is well known to demethylate histones to activate transcription, PHF8 demethylates transcription factor YY1, functioning as a co-repressor for a large set of nuclear-coded ETC genes to drive mROS production and cancer development. In addition to genetically ablating PHF8, pharmacologically targeting PHF8 with a specific chemical inhibitor, iPHF8, is potent in regulating YY1 methylation, ETC gene transcription, mROS production, and cell growth in colon and lung cancer cells. iPHF8 exhibits potency and safety in suppressing tumor growth in cell-line- and patient-derived xenografts in vivo. Our data uncover a key epigenetic mechanism underlying ETC gene transcriptional regulation, demonstrating that targeting the PHF8/YY1 axis has great potential to treat cancers.

Combination Treatment of a Phytochemical and a Histone Demethylase Inhibitor-A Novel Approach towards Targeting TGFß-Induced EMT, Invasion, and Migration in Prostate Cancer.

Minimizing side effects, overcoming cancer drug resistance, and preventing metastasis of cancer cells are of growing interest in current cancer therapeutics. Phytochemicals are being researched in depth as they are protective to normal cells and have fewer side effects. Hesperetin is a citrus bioflavonoid known to inhibit TGFß-induced epithelial-to-mesenchymal transition (EMT), migration, and invasion of prostate cancer cells. Targeting epigenetic modifications that cause cancer is another class of upcoming therapeutics, as these changes are reversible. Global H3K27me3 levels have been found to be reduced in invasive prostate adenocarcinomas. Combining a demethylase inhibitor and a known anti-cancer phytochemical is a unique approach to targeting cancer to attain the aforementioned objectives. In the current study, we used an H3K27 demethylase (JMJD3/KDM6B) inhibitor to study its effects on TGFß-induced EMT in prostate cancer cells. We then gave a combined hesperetin and GSK-J4 treatment to the PC-3 and LNCaP cells. There was a dose-dependent increase in cytotoxicity and inhibition of TGFß-induced migration and invasion of prostate cancer cells after GSK-J4 treatment. GSK-J4 not only induced trimethylation of H3K27 but also induced the trimethylation of H3K4. Surprisingly, there was a reduction in the H3K9me3 levels. GSK-J4 alone and a combination of hesperetin and GSK-J4 treatment effectively inhibit the important hallmarks of cancer, such as cell proliferation, migration, and invasion, by altering the epigenetic landscape of cancer cells.

[Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1].

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Promising natural lysine specific demethylase 1 inhibitors for cancer treatment: advances and outlooks.

Lysine specific demethylase 1 (LSD1), a transcriptional corepressor or coactivator that serves as a demethylase of histone 3 lysine 4 and 9, has become a potential therapeutic target for cancer therapy. LSD1 mediates many cellular signaling pathways and regulates cancer cell proliferation, invasion, migration, and differentiation. Recent research has focused on the exploration of its pharmacological inhibitors. Natural products are a major source of compounds with abundant scaffold diversity and structural complexity, which have made a major contribution to drug discovery, particularly anticancer agents. In this review, we briefly highlight recent advances in natural LSD1 inhibitors over the past decade. We present a comprehensive review on their discovery and identification process, natural plant sources, chemical structures, anticancer effects, and structure-activity relationships, and finally provide our perspective on the development of novel natural LSD1 inhibitors for cancer therapy.

[Expression pattern of the histone lysine demethylase family and its potential role in bladder cancer: a multi-omics analysis].

OBJECTIVE: To investigate the expression patterns of 19 histone lysine demethylases (KDMs) and their role in bladder cancer. METHODS: In this study, UALCAN and GSCALite were used to analyze the transcriptional expression, methylation level and somatic variation of KDMs in bladder cancer samples from TCGA. Kaplan Meier-Plotter and Assistant for clinical bioinformatics were used to investigate the effect of KDMs expression on the prognosis of BLCA samples. The immune infiltration and drug sensitivity of KDMs in bladder cancer were analyzed by Timer and GSCALite. RESULTS: The KDMs did not show consistent expressions patterns in bladder cancer, where the expressions of KDM1A/1B/2B/4A/4B/5B/5C were significantly upregulated while those of KDM3B/6B/7C were significantly downregulated. Methylation data analysis showed that methylation levels of KDM1A/3B/4A/4B/4C/5A/5B/5C/7B were significantly downregulated and that of KDM7C was upregulated. The transcription levels of 14 KDMs had significant negative correlations with their methylation levels, and among them KDM1A showed the strongest correlation. Mutation analysis revealed that KDM6A had the highest frequency of nonsynonymous mutations with the largest variety, and these mutations were complementary to nonsynonymous mutations of the other KDMs. Survival analysis showed that KDM3A/4C/5D/6A/7B were protective for OS while KDM3B/5B/5C adversely affected RFS of BLCA patients. Further comprehensive prognostic modeling confirmed that KDM4C/6A/7B were potential prognostic biomarkers of bladder cancer, and their expressions were positively correlated with immune infiltration in BLCA patients. KDM2B/3B/4B/4C/5A were negatively correlated with the sensitivity to most anticancer drugs, while KDM2B/4B were positively correlated with the sensitivity to 4 anticancer drugs. CONCLUSION: The expression patterns of the KDMs in bladder cancer highlight a high mutation complementarity and a negative correlation between over-expression and hypomethylation level closely related with the prognosis, immune infiltration and drug sensitivity.

Machine-Learning-Enabled Virtual Screening for Inhibitors of Lysine-Specific Histone Demethylase 1.

A machine learning approach has been applied to virtual screening for lysine specific demethylase 1 (LSD1) inhibitors. LSD1 is an important anti-cancer target. Machine learning models to predict activity were constructed using Morgan molecular fingerprints. The dataset, consisting of 931 molecules with LSD1 inhibition activity, was obtained from the ChEMBL database. An evaluation of several candidate algorithms on the main dataset revealed that the support vector regressor gave the best model, with a coefficient of determination (R2) of 0.703. Virtual screening, using this model, identified five predicted potent inhibitors from the ZINC database comprising more than 300,000 molecules. The virtual screening recovered a known inhibitor, RN1, as well as four compounds where activity against LSD1 had not previously been suggested. Thus, we performed a machine-learning-enabled virtual screening of LSD1 inhibitors using only the structural information of the molecules.

X-linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of hypothalamic neurons.

Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17ß-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization.

A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay.

Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity.

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

IOX1 activity as sepsis therapy and an antibiotic against multidrug-resistant bacteria.

Sepsis is caused by organ dysfunction initiated by an unrestrained host immune response to infection. The emergence of antibiotic-resistant bacteria has rapidly increased in the last decades and has stimulated a firm research platform to combat infections caused by antibiotic-resistant bacteria that cannot be eradicated with conventional antibiotics. Strategies like epigenetic regulators such as lysine demethylase (Kdm) has received attention as a new target. Thus, we sought to investigate the epigenetic mechanisms in sepsis pathophysiology with the aim of discovering new concepts for treatment. A transcriptome analysis of dendritic cells during their inflammatory state identified Kdm as a critical molecule in sepsis regulation. Next, 8-hydroxyquinoline-5-carboxylic acid (IOX1) ability to control endotoxemia induced by Lipopolysaccharide and bacterial sepsis was demonstrated. IOX1 has been shown to regulate endotoxemia and sepsis caused by Escherichia coli and carbapenem-resistant Acinetobacter baumannii and has also contributed to the suppression of multidrug-resistant bacterial growth through the inhibition of DNA Gyrase. These findings show that IOX1 could be a component agent against bacterial sepsis by functioning as a broad-spectrum antibiotic with dual effects.

Target separation and antitumor metastasis activity of sesquiterpene-based lysine-specific demethylase 1 inhibitors from zedoary turmeric oil.

Lysine-specific histone demethylase 1 (LSD1) was the first histone demethylase identified in epigenetics and has recently emerged as an attractive therapeutic target for treating tumors. To date, almost all reported LSD1 inhibitors have been chemosynthesized; however, natural products possess pharmacological and biological activity and can be sources for drug development. Here, we established a target separation countercurrent chromatography technique to isolate LSD1 inhibitors from zedoary turmeric oil. Four sesquiterpene-based LSD1 inhibitors were efficiently obtained with an inhibition ratio equal to or less than that of the positive control drug. Compound 2 showed the most potent inhibitory activity, with a half-maximal inhibitory concentration of 3.97 µM, and was further tested to determine its ability to inhibit LSD1 and its antitumor metastatic effects in MDA-MB-231 cells. These four compounds are the first sesquiterpene-based natural LSD1 inhibitors to be characterized. Our findings provide a new molecular framework for studying LSD1 inhibitors and offer a template for designing more sesquiterpene-based LSD1 inhibitors with potential antitumor activity.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma.

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 µM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.

KDM6A promotes imatinib resistance through YY1-mediated transcriptional upregulation of TRKA independently of its demethylase activity in chronic myelogenous leukemia.

Rationale: Despite landmark therapy of chronic myelogenous leukemia (CML) with tyrosine kinase inhibitors (TKIs), drug resistance remains problematic. Cancer pathogenesis involves epigenetic dysregulation and in particular, histone lysine demethylases (KDMs) have been implicated in TKI resistance. We sought to identify KDMs with altered expression in CML and define their contribution to imatinib resistance. Methods: Bioinformatics screening compared KDM expression in CML versus normal bone marrow with shRNA knockdown and flow cytometry used to measure effects on imatinib-induced apoptosis in K562 cells. Transcriptomic analyses were performed against KDM6A CRISPR knockout/shRNA knockdown K562 cells along with gene rescue experiments using wildtype and mutant demethylase-dead KDM6A constructs. Co-immunoprecipitation, luciferase reporter and ChIP were employed to elucidate mechanisms of KDM6A-dependent resistance. Results: Amongst five KDMs upregulated in CML, only KDM6A depletion sensitized CML cells to imatinib-induced apoptosis. Re-introduction of demethylase-dead KDM6A as well as wild-type KDM6A restored imatinib resistance. RNA-seq identified NTRK1 gene downregulation after depletion of KDM6A. Moreover, NTRK1 expression positively correlated with KDM6A in a subset of clinical CML samples and KDM6A knockdown in fresh CML isolates decreased NTRK1 encoded protein (TRKA) expression. Mechanistically, KDM6A was recruited to the NTRK1 promoter by the transcription factor YY1 with subsequent TRKA upregulation activating down-stream survival pathways to invoke imatinib resistance. Conclusion: Contrary to its reported role as a tumor suppressor and independent of its demethylase function, KDM6A promotes imatinib-resistance in CML cells. The identification of the KDM6A/YY1/TRKA axis as a novel imatinib-resistance mechanism represents an unexplored avenue to overcome TKI resistance in CML.

The role of PHF8 and TLR4 in osteogenic differentiation of periodontal ligament cells in inflammatory environment.

BACKGROUND: Histone methylation is considered to play an important role in the occurrence and development of periodontitis. Plant homeodomain finger protein 8 (PHF8), a histone demethylase, has been shown to regulate inflammation and osteogenic differentiation of bone marrow stromal cells (BMSCs). This study aimed to detect the functions of PHF8 and TLR4 in osteogenic differentiation in an inflammatory environment induced by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) METHODS: A periodontitis mouse model was established, and the mice were treated with TAK-242. Immunohistochemical staining was used to detect the expression of PHF8 in periodontal tissue. Periodontal ligament cells (PDLCs) were treated with mineralization induction medium supplemented with Pg-LPS and/or TAK-242, and a Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of PDLCs. Real-time PCR and western blotting were used to detect the mRNA and protein expression levels, respectively, of PHF8, toll-like receptor 4 (TLR4) and the other osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), Special AT-rich sequence-binding protein 2 (Satb2) and Runt-related transcription factor 2 (Runx2) RESULTS: Periodontitis reduced PHF8 expression in periodontal tissue, and TAK-242 partially reversed this downregulation. An in vitro experiment revealed that the mRNA and protein expression levels of PHF8 were significantly upregulated during the osteogenic differentiation of PDLCs. Alizarin red staining showed that the mineralized nodules of PDLCs in osteogenic induction group were more than those in control group. Real-time PCR and western blot results indicated that Pg-LPS inhibited PHF8 expression and upregulated TLR4 expression in PDLCs. TAK-242 inhibited TLR4 and partially reversed the inhibition of PHF8 expression and osteogenic differentiation induced by Pg-LPS in PDLCs CONCLUSION: PHF8 and TLR4 play important roles in periodontitis. Pg-LPS inhibits the expression of PHF8 via upregulation of TLR4 and might further inhibit the osteogenic differentiation of PDLCs. However, the specific mechanisms involved remain to be explored.

Capsaicin: A "hot" KDM1A/LSD1 inhibitor from peppers.

Capsaicinoids are plant secondary metabolites, and capsaicin is the main principal that responsible to the pungency of chili peppers, with widely application as food additive. In our study, capsaicin was characterized as lysine specific demethylase 1A (KDM1A/LSD1) inhibitor with IC50 of 0.6 ± 0.0421 µM in biochemical level, and can bind KDM1A recombinant directly and reversibly. Further cellular study confirmed that capsaicin can bind and inhibit KDM1A in gastric cancer cell line BGC-823 and further inhibit cell invasion and migration by reversing epithelial-mesenchymal transition (EMT). In sum, our findings identified KDM1A as a target of capsaicin and reveals capsaicin as a modifier of histone methylation for the first time, which may provide a new skeleton for further optimization of KDM1A inhibitor.

Precise separation of lysine-specific demethylase 1 inhibitors from Corydalis yanhusuo using multi-mode counter-current chromatography guided by virtual screening.

It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 µM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.

Selective lysine-specific demethylase 1 inhibitor, NCL1, could cause testicular toxicity via the regulation of apoptosis.

BACKGROUND: Recent studies have shown that epigenetic alterations, such as those involving lysine-specific demethylase 1 (LSD1), lead to oncogenic activation and highlight such alterations as therapeutic targets. However, studies evaluating the effect of LSD1 inhibitors on male fertility are lacking. OBJECTIVES: We analyzed the potential toxicity of a new selective LSD1 inhibitor, N-[(1S)-3-[3-(trans-2-aminocyclopropyl)phenoxy]-1-(benzylcarbamoyl)propyl] benzamide (NCL1), in testes. MATERIALS AND METHODS: Human testicular samples were immunohistochemically analyzed. Six-week-old male C57BL/6J mice were injected intraperitoneally with dimethyl sulfoxide vehicle (n = 15), or 1.0 (n = 15) or 3.0 (n = 15) mg/kg NCL1 biweekly. After five weeks, toxicity and gene expression were analyzed in testicular samples by ingenuity pathway analysis (IPA) using RNA sequence data and quantitative reverse transcriptase (qRT)-PCR; hormonal damage was analyzed in blood samples. NCL1 treated GC-1, TM3, and TM4 cell lines were analyzed by cell viability, chromatin immunoprecipitation, flow cytometry, and Western blot assays. RESULTS: LSD1 was mainly expressed in human Sertoli and germ cells, with LSD1 levels significantly decreased in a progressive meiosis-dependent manner; germ cells showed similar expression patterns in normal spermatogenesis and early/late maturation arrest. Histological examination revealed significantly increased levels of abnormal seminiferous tubules in 3.0 mg/kg NCL1-treated mice compared to control, with increased cellular detachment, sloughing, vacuolization, eosinophilic changes, and TUNEL-positive cells. IPA and qRT-PCR revealed NCL1 treatment down-regulated LSD1 activity. NCL1 also reduced total serum testosterone levels. Western blots of mouse testicular samples revealed NCL1 induced a marked elevation in cleaved caspases 3, 7, and 8, and connexin 43 proteins. NCL1 treatment significantly reduced GC-1, but not TM3 and TM4, cell viability in a dose-dependent manner. In flow cytometry analysis, NCL1 induced apoptosis in GC-1 cells. CONCLUSIONS: High-dose NCL1 treatment targeting LSD1 caused dysfunctional spermatogenesis and induced caspase-dependent apoptosis. This suggests the LSD1 inhibitor may cause testicular toxicity via the regulation of apoptosis.

Epigallocatechin-3-gallate (EGCG) Alters Histone Acetylation and Methylation and Impacts Chromatin Architecture Profile in Human Endothelial Cells.

Epigallocatechin gallate (EGCG), the main green tea polyphenol, exerts a wide variety of biological actions. Epigenetically, the catechin has been classified as a DNMTs inhibitor, however, its impact on histone modifications and chromatin structure is still poorly understood. The purpose of this study was to find the impact of EGCG on the histone posttranslational modifications machinery and chromatin remodeling in human endothelial cells of both microvascular (HMEC-1) and vein (HUVECs) origin. We analyzed the methylation and acetylation status of histones (Western blotting), as well as assessed the activity (fluorometric assay kit) and gene expression (qPCR) of the enzymes playing a prominent role in shaping the human epigenome. The performed analyses showed that EGCG increases histone acetylation (H3K9/14ac, H3ac), and methylation of both active (H3K4me3) and repressive (H3K9me3) chromatin marks. We also found that the catechin acts as an HDAC inhibitor in cellular and cell-free models. Additionally, we observed that EGCG affects chromatin architecture by reducing the expression of heterochromatin binding proteins: HP1α, HP1γ. Our results indicate that EGCG promotes chromatin relaxation in human endothelial cells and presents a broad epigenetic potential affecting expression and activity of epigenome modulators including HDAC5 and 7, p300, CREBP, LSD1 or KMT2A.

Natural protoberberine alkaloids, identified as potent selective LSD1 inhibitors, induce AML cell differentiation.

Natural protoberberine alkaloids were first identified and characterized as potent, selective and cellular active lysine specific demethylase 1 (LSD1) inhibitors. Due to our study, isoquinoline-based tetracyclic scaffold was identified as the key structural element for their anti-LSD1 activity, subtle changes of substituents attached to the core structure led to dramatic changes of the activity. Among these protoberberine alkaloids, epiberberine potently inhibited LSD1 (IC50 = 0.14 ± 0.01 µM) and was highly selective to LSD1 over MAO-A/B. Furthermore, epiberberine could induce the expression of CD86, CD11b and CD14 in THP-1 and HL-60 cells, confirming its cellular activity of inducing acute myeloid leukemia (AML) cells differentiation. Moreover, epiberberine prolonged the survival of THP-1 cells bearing mice and inhibited the growth of AML cells in vivo without obvious global toxicity. These findings give the potential application of epiberberine in AML treatment, and the isoquinoline-based tetracyclic scaffold could be used for further development of LSD1 inhibitors.