Grape seed extract prevents chlorpyrifos-induced toxicity in rat liver through the modulation of phase I detoxification pathway.

Chlorpyrifos (CPF) poisoning is a public health problem for which there is not currently any effective prophylaxis. In this study, we investigated the protective effect of grape seed extract (GSE) against CPF-induced hepatotoxicity. Rats were daily treated either with CPF (2 mg/kg) or CPF and GSE (20 mg/kg) for 1 week, sacrificed, and their livers dissected for biochemical, molecular, and histopathological analyses. CPF generated liver dysfunction by altering carbohydrate, lipid, amino acid, ammonia and urea metabolism, and provoked mitochondrial impairment through disturbing tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and mitochondrial viability. CPF also induced cholinergic excitotoxicity along with oxidative stress and histopathological alterations. Interestingly, treatment with GSE prevented all the detrimental effects of CPF through the regulation of cytochrome P450 (CYP450) gene expression. Molecular docking analysis indicated that GSE-containing polyphenols acted as epigenetic modulators through inhibiting DNA (cytosine-5)-methyltransferase 1 (DNMT1), thus favoring the CYP2C6 detoxification pathway. Thereby, GSE might be a promising strategy in the protection of the liver against CPF toxicity.

Annonaceous acetogenins as promising DNA methylation inhibitors to prevent and treat leukemogenesis - an in silico approach.

Leukemia is a haematological malignancy affecting blood and bone marrow, ranking 10th among the other common cancers. DNA methylation is an epigenetic dysregulation that plays a critical role in leukemogenesis. DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B are the key enzymes catalysing DNA methylation. Inhibition of DNMT1 with secondary metabolites from medicinal plants helps reverse DNA methylation. The present study focuses on inhibiting DNMT1 protein (PDB ID: 3PTA) with annonaceous acetogenins through in-silico studies. The docking and molecular dynamic (MD) simulation study was carried out using Schrödinger Maestro and Desmond, respectively. These compounds' drug likeliness, ADMET properties and bioactivity scores were analysed. About 76 different acetogenins were chosen for this study, among which 17 showed the highest binding energy in the range of -8.312 to -10.266 kcal/mol. The compounds with the highest negative binding energy were found to be annohexocin (-10.266 kcal/mol), isoannonacinone (-10.209 kcal/mol) and annonacin (-9.839 kcal/mol). MD simulation results reveal that annonacin remains stable throughout the simulation time of 100 ns and also binds to the catalytic domain of DNMT1 protein. From the above results, it can be concluded that annonacin has the potential to inhibit the DNA methylation process and prevent leukemogenesis.Communicated by Ramaswamy H. Sarma.

Jieduquyuziyin prescription enhances CD11a and CD70 DNA methylation of CD4+ T cells via miR-29b-sp1/DNMT1 pathway in MRL/lpr mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Jieduquyuziyin prescription (JP) is a traditional Chinese medicine utilized to treat systemic lupus erythematosus (SLE). Its efficacy has been confirmed through clinical trials and empirical evidence, leading to its authorized use in Chinese hospitals. The development of JP exemplifies the integration of traditional wisdom and scientific approaches, demonstrating the interdisciplinary essence of ethnopharmacology. These results emphasize the potential value of traditional medicine in addressing autoimmune disorders. AIM OF THE STUDY: This study aims to address the effect of JP in MRL/lpr mice and elucidate the pharmacological mechanism by which JP targets CD11a and CD70 DNA methylation via the miR-29b-sp1/DNMT1 pathway. MATERIALS AND METHODS: MRL/lpr mice were divided into three groups: the model group (received distilled water), the positive group (administered AAV/miR-29b-3p inhibitor), and the JP group (treated with JP decoction). C57BL/6 mice were constituted as a control group. Through ELISA assay, serum and urine samples were assessed for anti-dsDNA, TNF-α, TGF-ß, IL-2, and UP. HE and Masson staining were conducted to reveal renal pathology. Genome DNA was extracted from CD4+ T cells of mice spleens to evaluate methylation level. The methylation of CD11a, CD70, and CD40L promoter regions was analyzed by targeted bisulfate sequencing. Their expression at the mRNA and protein levels was examined using quantitative real-time PCR, western blot analysis, immunohistochemistry, and immunofluorescence staining of kidney tissues. Furthermore, the molecular mechanisms underlying the regulation of the miR-29b-sp1/DNMT1 pathway by JP were explored with Jurkat cells transfected with miR-inhibitors or miR-mimics. RESULTS: Mice treated with JP exhibited a significant decrease in anti-dsDNA, TNF-α, TGF-ß, and UP, accompanied by a significant increase in IL-2. HE staining revealed JP effectively mitigated renal inflammatory response, while Masson staining indicated a reduction in collagen fiber content. In addition, JP exhibited a significant impact on the global hypomethylation of SLE, as evidenced by the induction of high methylation levels of CD11a and CD70 promoter regions, mediated through the miR-29b-sp1/DNMT1 pathway. CONCLUSION: Our findings demonstrate JP exerts a protective effect against spontaneous SLE development, attenuates renal pathological changes, and functions as a miRNA inhibitor to enhance CD11a and CD70 DNA methylation through the modulation of the miR-29b-sp1/DNMT1 pathway.

Deciphering the active constituents of Dabushen decoction of ameliorating osteoarthritis via PPARγ preservation by targeting DNMT1.

Osteoarthritis (OA) is a multifactorial and chronic degenerative joint disease. Due to the adverse effects of currently used drugs, a safer and more effective therapy for treating OA is needed. Peroxisome proliferator-activated receptor-γ (PPARγ) is a key protein protecting cartilage. DNMT1-mediated hypermethylation of PPARγ promoter leads to its suppression. Therefore, DNMT1 might be an effective target for exerting cartilage protective effects by regulating the epigenetic expression of PPARγ. Dabushen decoction (DD) is a representative prescription of Dunhuang ancient medical prescription, which has a potential therapeutic effect on OA. So far, the research of the efficacy and material basis of DD in the treatment of OA remains unclear. In this study, Micro-CT, HE staining, S-O staining, and immunohistochemistry analysis were used to demonstrate that DD increased the expression of PPARγ and collagen synthesis in an OA rat model. Next, the structure of DNMT1 was used to screen the active constituents of DD by molecular docking method for treatment OA. Seven potential active constituents, including isoliquiritigenin, emodin, taxifolin, catalpol, alisol A, zingerone, and schisandrin C were hited. The protective effect of the potential active constituents to chondrocytes were evaluated by protein capillary electrophoresis, immunofluorescence assays, and ex vivo culture of rat knee cartilage. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C could promote the expression of PPARγ and ameliorate IL-1ß-induced downregulation of collagen II and the production of MMP-13. Alisol A and Emodin could effectively mitigate cartilage damage. At last, molecular dynamics simulations with MM-GBSA method was applied to investigate the interaction pattern of the active constituents and DNMT1 complexes. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C achieved a stable binding pattern with DNMT1, in which alisol A has a relatively high binding free energy. In conclusion, this study elucidates that the active constituents of DD (alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C) could ameliorate osteoarthritis via PPARγ preservation by targeting DNMT1.These findings facilitated clinical use of DD and provided a valuable strategy for developing natural epigenetic modulators from Chinese herbal formula.

Shema Oral Liquid Ameliorates the Severity of LPS-Induced COPD via Regulating DNMT1.

Background: Chronic obstructive pulmonary disease (COPD) is the most common respiratory disease with high morbidity and mortality. Shema oral liquid (Shema) is a traditional Chinese medicine (TCM) approved for the treatment of respiratory diseases. Clinical applications have shown that Shema has antitussive, expectorant, and anti-asthmatic effects, but its definite efficacy to COPD is still unclear. This study aimed to explore the therapeutic capacity and potential mechanism of Shema in treatment of COPD. Methods: Network pharmacology was used to investigated the possible pharmacological mechanism of Shema against COPD. A rat model of lipopolysaccharide (LPS)-induced COPD was established to determine pulmonary ventilatory function, serum inflammatory cytokines, and pulmonary pathological change. Subsequently, tandem mass tag (TMT)-based quantitative proteomics was used to further reveal the therapeutic targets related with Shema against COPD. Western blot was finally performed to validate the expression of targeted proteins screened by proteomics research. Results: Network pharmacology analysis indicated that Shema against COPD mainly inhibited the inflammation and affected the immune system. The animal experiment demonstrated that Shema treatment protected the lung tissue from LPS induced injury, inhibited the levels of serum inflammatory cytokines such as interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α, and improved the respiratory ventilatory function by upregulating forced expiratory volume in 0.1 s (FEV0.1), FEV0.3, forced vital capacity (FVC), and the ratios of FEV0.1 (0.3)/FVC. Proteomic analysis and western blot both proved that Shema inhibited the expression of DNA methyltransferase 1 (DNMT1) in the lung tissue. Conclusion: The therapeutic mechanism of Shema in treatment of COPD may involve inhibiting inflammatory response, improving pulmonary ventilatory function, and alleviating LPS-induced lung injury through regulating the expression of DNMT1. This study also shed light on the development of therapeutic strategies in treating COPD by intervening DNMT-related pathways.

The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO's therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.

The phytochemical brazilin suppress DNMT1 expression by recruiting p53 to its promoter resulting in the epigenetic restoration of p21 in MCF7cells.

BACKGROUND: Cancer is an outcome of uncontrolled cell division eventually associated with dysregulated epigenetic mechanisms, including DNA methylation. DNA methyltransferase 1 is ubiquitously expressed in the proliferating cells and is essential for the maintenance of DNA methylation. It causes the abnormal silencing of tumor suppressor genes in human cancer which is necessary for proliferation, cell cycle progression, and survival. DNMT1 is involved in tumorigenesis of several cancers, its upregulation potentially upscale the promoter level inactivation of transcription of a tumor inhibitory gene by introducing repressive methylation marks on the CpG islands. This epigenetic perturbation caused by DNMT is targeted for cancer therapeutics. PURPOSE: To demonstrate the proliferative inhibitory potential of brazilin in human breast cancer cell line (MCF-7) with concurrent mitigation of DNMT1 functional expression and to understand its effect on downstream targets like cell cycle inhibitor p21. STUDY DESIGN/ METHODS: The impact of brazilin on the growth and proliferation of the MCF-7 cells was determined using the XTT assay. The global DNA 5-methyl cytosine methylation pattern was analyzed upon brazilin treatment. The gene and protein expression of DNMTs were determined with quantitative RTPCR and western blots respectively. The potential binding sites of transcription factors in the human DNMT1 promoter were predicted using the MatInspector tool on the Genomatix software. The chromatin immunoprecipitation (ChIP) assay was performed to demonstrate the transcription factors occupancy at the promoter. Methylation of promoter CpG islands was determined by the methylation-specific PCR (MSP) upon brazilin treatment. The molecular docking of the human DNMT1 with brazilin (ligand) was performed using the Schrödinger suite. RESULTS: The heterotetracyclic compound brazilin, present in the wood of Caesalpinia sappan, inhibited the proliferation of the human breast cancer cell line (MCF-7) and reduced the DNMT1 expression with a decrease in global DNA methylation. Brazilin, by activating p38 MAPK and elevating p53 levels within the exposed cells. The elevated level of p53 enriched the occupancy at binding sites within 200 bp upstream to the transcription start site in the DNMT1 promoter, resulting in reduced DNMT1 gene expression. Furthermore, the brazilin restored the p21 levels in the exposed cells as the CpGs in the p21 promoter (-128 bp/+17 bp) were significantly demethylated as observed in the methylation-specific PCR (MSP). CONCLUSION: Highly potential anti-proliferative molecule brazilin can modulate the DNMT1 functional expression and restore the cell cycle inhibitor p21expression. We propose that brazilin can be used in therapeutic interventions to restore the deregulated epigenetic mechanisms in cancer.

Sennoside A alleviates inflammatory responses by inhibiting the hypermethylation of SOCS1 in CCl4-induced liver fibrosis.

Liver fibrosis is the consequence of chronic liver injury and is a major challenge to global health. However, successful therapy for liver fibrosis is still lacking. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve hepatic disease, but the underlying mechanisms remain largely elusive. Here, we show for the first time that SA enhanced suppressor of cytokine signaling 1 (SOCS1) expression in a DNA methyltransferase 1 (DNMT1)-dependent manner and thereby attenuated liver fibrosis. Consistently, SA inhibited the expression of the liver fibrogenesis markers α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) and suppressed inflammatory responses in vivo and in vitro. Coculture experiments with macrophages/hepatic stellate cells (HSCs) revealed that SA suppressed HSC proliferation by downregulating proinflammatory cytokines in macrophages. Mechanically, SA promoted the aberrant expression of SOCS1 in liver fibrosis. However, blocking SOCS1 expression weakened the inhibitory effect of SA on HSC proliferation, indicating that SOCS1 may play an important role in mediating the antifibrotic effect of SA. Furthermore, SA inhibited DNMT1-mediated SOCS1 and reduced HSC proliferation by inhibiting inflammatory responses in carbon tetrachloride (CCl4) -induced liver fibrosis.

Protective effect of plastrum testudinis extract on dopaminergic neurons in a Parkinson's disease model through DNMT1 nuclear translocation and SNCA's methylation.

The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.

Cissus quadrangularis extract attenuates diabetic nephropathy by altering SIRT1/DNMT1 axis.

OBJECTIVES: Hyperglycemia-induced SIRT1, DNMT1, SODs, as well as oxidative stress, play a pivotal role in the progression of diabetic nephropathy. Cissus quadrangularis, holds antioxidant and hypoglycemic activity; however, a direct link between its activity and prevention of diabetic nephropathy has not been ascertained yet. Accordingly, we aimed to delineate the protective effect of ethanolic extract of Cissus quadrangularis (EECQ) against high-fat diet/streptozotocin (HFD/STZ) induced diabetic nephropathy rats. METHODS: The control group was fed with a normal chow diet. Rats kept on an HFD for 12 weeks with a single low dose of STZ manifested the features of diabetic nephropathy. The treatment was done by the oral administration of EECQ (200 mg/kg) for six weeks (six rats in each group). KEY FINDINGS: Treatment with EECQ demonstrated substantial attenuation of elevated insulin resistance, lipid profile and creatinine level. Additionally, EECQ restored albuminuria, glomerular filtration rate and creatinine clearance in diabetic nephropathy rats. Furthermore, HFD consumption in rats culminated in reduced SIRT1 and enhanced DNMT1 expression, nonetheless, rescued by EECQ. Moreover, EECQ augmented the SOD 1 and 3 levels, thereby safeguarded from oxidative damage and renal inflammation. Besides, treatment protected from renal fibrosis by downregulating TGFß, Smad2/3 and col1/3 expression in diseased rats. CONCLUSIONS: Thus, based on the above findings, we conclude that EECQ shows a protective effect against diabetic nephropathy.

A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A.

Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme's active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.

Alteration of CYP2E1, DBN1, DNMT1, miRNA-335, miRNA-21, c-Fos and Cox-2 gene expression in prefrontal cortex of rats' offspring submitted to prenatal ethanol exposure during their neurodevelopment and the preventive role of nancocurcumin administration: A histological, ultrastructural and molecular study.

Ethanol (EtOH) has been linked to neurotoxic effects on the fetus and prenatal alcohol exposure (PAE) has a negative impact on brain neurodevelopment. Therefore, the present study was aimed to focus on the underlying mechanisms of alcohol-induced oxidative stress and apoptotic cell death in addition to shedding the light on the modulatory effect of nanocurcumin in rats' offspring prefrontal cortices. The current study investigated the effects of prenatal maternal exposure to EtOH intragastric (i.g.) administration of 0.015 mL/g of a 10 % v/v ethanol solution throughout gestation and the concomitant use of nanocurcumin, on 21-day-old offspring Wistar rat prefrontal cortex parameters. CYP2E1, DBN1, DNMT1, miRNA-335, miRNA-21, c-Fos and Cox-2 gene expression as well as the accompanying histological and ultrastructural alterations were assessed. The implemented experimental setting has revealed that ethanol exposure caused significant alterations in the above mentioned parameters. Changes observed in nanocurcumin-treated animals were significantly different to the ethanol-treated group when nanocurcumin was concomitantly administered.

Hairpin-bisulfite sequencing of cells exposed to decitabine documents the process of DNA demethylation.

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

Sennoside A prevents liver fibrosis by binding DNMT1 and suppressing DNMT1-mediated PTEN hypermethylation in HSC activation and proliferation.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.

Caffeic acid phenethyl ester, a coffee polyphenol, inhibits DNA methylation in vitro and in vivo.

DNA methylation represents an important epigenetic regulation of the genome. Earlier studies have suggested that dietary phenolic compounds including those contained in coffee, tea and soy products may modulate the level of DNA methylation. In this study, we first characterize the effect of caffeic acid phenethyl ester (CAPE) and other dietary phenolic compounds on DNA methylation in vitro. The IC50 values of CAPE, daidzein, isorhamnetin and genistein are 7.6, 6.9, 6.2, and 4.3 µM, respectively, in an in-vitro enzymatic assay system. Computational analysis indicates that CAPE, daidzein, isorhamnetin and genistein can bind inside the DNA substrate-binding site in human DNMT1 with a favorable binding energy. In an animal study, we find that maternal CAPE treatment shifts the coat color distribution of the 21-day-old Avy/a offspring towards the yellow phenotype, indicating that CAPE inhibits the methylation of the agouti gene promoter sequence in vivo. The results from this study may shed light on the potential epigenetic effect in the offspring resulting from maternal intake of certain coffee phenolics during pregnancy.

GPx1-mediated DNMT1 expression is involved in the blocking effects of selenium on OTA-induced cytotoxicity and DNA damage.

Ochratoxin A (OTA) is a potent nephrotoxin. Selenium (Se) is an essential micronutrient for humans and animals, and plays a key role in antioxidant defense. To date, little is known about the effect of Se on OTA-induced DNA damage. In this study, the protective effects of Se (from selenomethionine) against OTA-induced cytotoxicity and DNA damage were investigated by using PK15 cells as a model. The results showed that OTA at 4.0 µg/mL induced cytotoxicity and DNA damage. Se at 0.5, 1, 2 and 4 µM significantly blocked OTA-induced cytotoxicity and DNA damage. Furthermore, Se blocked the increases of DNMT1, DNMT3a and HDAC1 mRNA and protein expression, reversed the decreases of glutathione peroxidase 1 (GPx1) mRNA and protein expression, and promoted the increases of SOCS3 mRNA and protein expression induced by OTA. Overexpression of GPx1 by pcDNA3.1-GPx1 inhibited the OTA-induced DNMT1 expression, promoted OTA-induced SOCS3 expression, and prevented the OTA-induced cytotoxicity and DNA damage. In contrast, knock-down of GPx1 by using a GPx1-specific siRNA had the opposite effects. The results suggest that GPx1-mediated DNMT1 expression is involved in the blocking effects of selenium on OTA-induced cytotoxicity and DNA damage.

NALP3 orchestrates cellular bioenergetics to facilitate non-small cell lung cancer cell growth.

AIMS: Previous work has reported the closely correlation between inflammation and carcinogenesis, while the role of NALP3, the key component of inflammasome activation in NSCLC remains elusive. This study was to unravel the mechanism of NALP3 on modulating NSCLC cancer cell growth. METHODS: IHC and immuno-blot were performed to analyze expression of NALP3 and indicated molecules. CCK-8 and xenograft nude mice assay were used to evaluate cell growth in vitro and in vivo. Bioenergetics assay was performed to measure OXPHOS and aerobic glycolysis. siRNA and shRNA were constructed to knockdown endogenous NALP3 and DNMT1. Co-immunoprecipitation was applied to confirm the interaction between NALP3 and DMAP1. BioProfile FLEX analyzer and Lactate Reagent Kit were used to measure relative level glucose uptake and lactate production. KEY FINDINGS: We reported NALP3 were up-regulated in NSCLC tumor tissues. NALP3 depletion suppressed cancer cell growth in vitro and in vivo. Moreover, data showed depletion of NALP3 promoted cell bioenergetics switch from aerobic glycolysis to OXPHOS. Additionally, we found NALP3 interacted with DMAP1 and alteration of NALP3 increased DNMT1 level. Subsequently, we clarified depletion of DNMT1 significantly suppressed NSCLC cell growth and orchestrated cellular metabolism which was similar to the effects of NALP3 knockdown. Finally, our data showed high NALP3 was associated with poor outcomes, and correlated with TNM stage and differentiation. SIGNIFICANCE: Current study elucidated NALP3 could promote metabolic reprogramming to regulate NSCLC cell growth and suggested that NALP3 may be considered as a novel biomarker and therapeutic target for NSCLC patients.

Crucifera sulforaphane (SFN) inhibits the growth of nasopharyngeal carcinoma through DNA methyltransferase 1 (DNMT1)/Wnt inhibitory factor 1 (WIF1) axis.

BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.

Programmed Epigenetic DNA Methylation-Mediated Reduced Neuroprogenitor Cell Proliferation and Differentiation in Small-for-Gestational-Age Offspring.

Small-for-gestational age (SGA) human newborns have an increased risk of hyperphagia and obesity, as well as a spectrum of neurologic and neurobehavioral abnormalities. We have shown that the SGA hypothalamic (appetite regulatory site) neuroprogenitor cells (NPCs) exhibit reduced proliferation and neuronal differentiation. DNA methylation (DNA methyltransferase; DNMT1) regulates neurogenesis by maintaining NPC proliferation and suppressing premature differentiation. Once differentiation ensues, DNMT1 preferentially promotes neuronal and inhibits astroglial fate. We hypothesized that the programmed dysfunction of NPC proliferation and differentiation in SGA offspring is epigenetically mediated via DNMT1. Pregnant rats received either ad libitum food (Control) or were 50% food-restricted to create SGA offspring. Primary hypothalamic NPCs from 1 day old SGA and Controls newborns were cultured and transfected with nonspecific or DNMT1-specific siRNA. NPC proliferation and protein expression of specific markers of NPC (nestin), neuroproliferative transcription factor (Hes1), neurons (Tuj1) and astrocytes (GFAP) were determined. Under basal conditions, SGA NPCs exhibited decreased DNMT1 and reduced proliferation and differentiation, as compared to Controls. In both SGA and Controls, DNMT1 siRNA in complete media inhibited NPC proliferation, consistent with reduced expression of nestin and Hes1. In differentiation media, DNMT1 siRNA decreased expression of Tuj1 but increased GFAP. In vivo data replicated these findings. In SGA offspring, impaired neurogenesis is epigenetically mediated, in part, via reduction in DNMT1 expression and suppression of Hes1 resulting in NPC differentiation. It is likely that the maturation of regions beyond the hypothalamus (e.g., cerebral cortex, hippocampus) may be impacted, contributing to poor cognitive and neurobehavioral competency in SGA offspring.

The repression and reciprocal interaction of DNA methyltransferase 1 and specificity protein 1 contributes to the inhibition of MET expression by the combination of Chinese herbal medicine FZKA decoction and erlotinib.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal medicine Fuzheng Kang-Ai (FZKA) decoction obtained from Guangdong Kangmei Pharmaceutical Company, which contains 12 components with different types of constituents, has been used as part of the adjuvant treatment of lung cancer for decades. We previously showed that FZKA decoction enhances the growth inhibition of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant non-small cell lung cancer (NSCLC) cells by suppressing glycoprotein mucin 1 (MUC1) expression. However, the molecular mechanism underlying the therapeutic potential, particularly in sensitizing or/and enhancing the anti-lung cancer effect of EGFR-TKIs, remains unclear. MATERIALS AND METHODS: Cell viability was measured using 3-(4, 5-diMEThylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 5-ethynyl -2'-deoxyuridine (EdU) assays. Western blot analysis was performed to examine the protein expressions of DNA methyltransferase 1 (DNMT1), specificity protein 1 (SP1), and MET, an oncogene encoding for a trans-membrane tyrosine kinase receptor activated by the hepatocyte growth factor (HGF). The expression of MET mRNA was measured by quantitative real-time PCR (qRT-PCR). Exogenous expression of DNMT1 and SP1, and MET were carried out by transient transfection assays. The promoter activity of MET was tested using Dual-luciferase reporter assays. A nude mouse xenografted tumor model further evaluated the effect of the combination of FZKA decoction and erlotinib in vivo. RESULTS: The combination of FZKA and erlotinib produced an even greater inhibition of NSCLC cell growth. FZKA decreased the expressions of DNMT1, SP1, and MET (c-MET) proteins, and the combination of FZKA and erlotinib demonstrated enhanced responses. Interestingly, there was a mutual regulation of DNMT1 and SP1. In addition, exogenously expressed DNMT1 and SP1 blocked the FZKA-inhibited c-MET expression. Moreover, excessive expressed MET neutralized FZKA-inhibited growth of NSCLC cells. FZKA decreased the mRNA and promoter activity of c-MET, which was not observed in cells with ectopic expressed DNMT1 gene. Similar findings were observed in vivo. CONCLUSION: FZKA decreases MET gene expression through the repression and mutual regulation of DNMT1 and SP1 in vitro and in vivo. This leads to inhibit the growth of human lung cancer cells. The combination of FZKA and EGFR-TKI erlotinib exhibits synergy in this process. The regulatory loops among the DNMT1, SP1 and MET converge in the overall effects of FZKA and EGFR-TKI erlotinib. This in vitro and in vivo study clarifies an additional novel molecular mechanism underlying the anti-lung cancer effects in response to the combination of FZKA and erlotinib in gefitinib-resistant NSCLC cells.