Overexpression Of DNA Methyltransferase Research Articles

Nerolidol reduces depression-like behavior in mice and suppresses microglia activation by down-regulating DNA methyltransferase 1.

Modern medicine has unveiled that essential oil made from Aquilaria possesses sedative and hypnotic effects. Among the chemical components in Aquilaria, nerolidol, a natural sesquiterpene alcohol, has shown promising effects. This study aimed to unravel the potential of nerolidol in treating depression. Chronic unpredictable mild stress (CUMS) was utilized to induce depression-like behavior in mice, and open field test, sucrose preference, and tail suspension test was conducted. The impacts of nerolidol on the inflammatory response, microglial activation, and DNA methyltransferase 1 (DNMT1) were assessed. To study the regulatory role of DNMT1, lipopolysaccharide (LPS) was used to treat BV2 cells, followed by the evaluation of cell viability and DNMT1 level. Additionally, the influence of DNMT1 overexpression on BV2 cell activation was determined. Behavioral analysis revealed that nerolidol reduced depression-like behavior in mice. Nerolidol reduced the levels of inflammatory factors and microglial activation caused by CUMS. Nerolidol treatment was found to reduce DNMT1 levels in mouse brain tissue and it also decrease the LPS-induced increase in DNMT1 levels in BV2 cells. DNMT1 overexpression reversed the impacts of nerolidol on the inflammation response and cell activation. This study underscores the potential of nerolidol in reducing CUMS-induced depressive-like behavior and inhibiting microglial activation by downregulating DNMT1. These findings offer valuable insights into the potential of nerolidol as a therapeutic option for depression.

Cracking a cancer code DNA methylation in epigenetic modification: an in-silico approach on efficacy assessment of Sri Lanka-oriented nutraceuticals

DNA methyltransferase (DNMTs) are essential epigenetic modifiers that play a critical role in gene regulation. These enzymes add a methyl group to cytosine’s 5'-carbon, specifically within CpG dinucleotides, using S-adenosyl-L-methionine. Abnormal overexpression of DNMTs can alter the gene expression patterns and contribute to cancer development in the human body. Therefore, the inhibition of DNMT is a promising therapeutic approach to cancer treatment. This study was aimed to identify potential nutraceutical inhibitors from the Sri Lanka Flora database using computational methods, which provided an atomic-level description of the drug binding site and examined the interactions between nutraceuticals and amino acids of the DNMT enzyme. A series of nutraceuticals from Sri Lanka-oriented plants were selected and evaluated to assess their inhibitory effects on DNMT using absorption, distribution, metabolism, excretion and toxicity analysis, virtual screening, molecular docking, molecular dynamics simulation and trajectory analysis. Azacitidine, a DNMT inhibitor approved by the US Food and Drug Administration, was selected as a reference inhibitor. The complexes with more negative binding energies were selected and further assessed for their potency. Seven molecules were identified from 200 nutraceuticals, demonstrating significantly negative binding energies against the DNMT enzyme. Various trajectory analyses were conducted to investigate the stability of the DNMT enzyme. The results indicated that petchicine (NP#0003), ouregidione (NP#0011) and azacitidine increased the stability of the DNMT enzyme. Consequently, these two nutraceuticals showed inhibitory efficacies similar to azacitidine, making them potential candidates for therapeutic interventions targeting DNMT enzyme-related cancers. Additional bioassay testing is recommended to confirm the efficacies of these nutraceuticals and explore their applicability in clinical treatments. Communicated by Ramaswamy H. Sarma

MiR-216a reduces apoptosis of pulmonary microvascular endothelial cells in COPD by targeting DNMT1.

Abnormal apoptosis of pulmonary microvascular endothelial cells (PMVECs) participates in the pathogenesis of COPD. Studies have shown that microRNAs (miRNAs) contribute to the pathogenesis of pulmonary diseases by regulating cell apoptosis. The present study aimed to investigate the effects of miR-216a in cigarette smoke extract (CSE)-induced apoptosis of PMVECs in COPD and explore the potential mechanisms. The emphysema model mice were treated with CSE and CS exposure. The expression of miR-216a and DNA methyltransferase 1 (DNMT1) was assessed in emphysema mice and COPD patients. The miR-216a mimic and Lenti-DNMT1 were transfected into PMVECs to identify the underlying mechanisms. The expression levels of miR-216a and DNMT1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) or Western blot. Moreover, cell apoptosis was examined by flow cytometry assays. The results show that the expression of miR-216a was decreased, whereas the expression of DNMT1 was increased in the lung tissue of emphysema mice and COPD patients. In addition, the expression of miR-216a was significantly reduced in CSE-treated PMVECs, and the overexpression of miR-216a attenuated CSE-induced PMVEC apoptosis. Furthermore, the expression of DNMT1 was increased in the CSE-induced PMVECs and then was reduced after the overexpression of miR-216a in the CSE-stimulated PMVECs. Luciferase reporter assays confirmed the target reaction between miR-216a and DNMT1. Also, the overexpression of DNMT1 was able to reverse the anti-apoptotic effect of miR-216a in CSE-induced PMVECs. The results indicate that miR-216a may play a crucial role in CSE-induced apoptosis by directly regulating its target gene DNMT1 in COPD. It provides insights into the function of MiR-216a/DNMT1 as a potential molecule in COPD.

DNMT1-mediated DNA methylation in toll-like receptor 4 (TLR4) inactivates NF-κB signal pathway-triggered pyroptotic cell death and cellular inflammation to ameliorate lipopolysaccharides (LPS)-induced osteomyelitis

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

Simvastatin prevents BMP-2 driven cell migration and invasion by suppressing oncogenic DNMT1 expression in breast cancer cells

Both epigenetic and genetic changes in the cancer genome act simultaneously to promote tumor development and metastasis. Aberrant DNA methylation, a prime epigenetic event, is often observed in various cancer types. The elevated DNA methyltransferase 1 (DNMT1) enzyme creates DNA hypermethylation at CpG islands to drive oncogenic potential. This study emphasized to decipher the molecular mechanism of endogenous regulation of DNMT1 expression for finding upstream signaling molecules. Cancer database analyses found an upregulated DNMT1 expression in most cancer types including breast cancer. Overexpression of DNMT1 showed an increased cell migration, invasion, and stemness potential whereas 5-azacytidine (DNMT1 inhibitor) and siRNA mediated knockdown of DNMT1 exhibited inhibition of such cancer activities in breast cancer MDA-MB-231 and MCF-7 cells. Infact, cancer database analyses further found a positive correlation of DNMT1 transcript with both cholesterol pathway regulatory genes and BMP signaling molecules. Experimental observations documented that the cholesterol-lowering drug, simvastatin decreased DNMT1 transcript as well as protein, whereas BMP-2 treatment increased DNMT1 expression in breast cancer cells. In addition, expression of various key cholesterol regulatory genes was found to be upregulated in response to BMP-2 treatment. Moreover, simvastatin inhibited BMP-2 induced DNMT1 expression in breast cancer cells. Thus, this study for the first time reveals that both BMP-2 signaling and cholesterol pathways could regulate endogenous DNMT1 expression in cancer cells.

DNMT1 regulates polarization of macrophage-induced intervertebral disc degeneration by modulating SIRT6 expression and promoting pyroptosis in vivo.

Intervertebral disc degeneration (IDD) is a complex phenomenon and a multifactorial degenerative disease that creates a heavy economic burden on health systems globally. Currently, there is no specific treatment proven to be effective in reversing and delaying the progression of IDD. This study consisted of animal and cell culture experiments. The role of DNA methyltransferase 1 (DNMT1) on regulating the M1/M2 macrophages polarization and pyroptosis, as well as its effect on Sirtuin 6 (SIRT6) expression in an IDD rat model and in tert-butyl hydroperoxide (TBHP)-treated nucleus pulposus cells (NPCs) were explored. Rat models were constructed, followed by transfection with lentiviral vector to inhibit DNMT1 or overexpress SIRT6. The NPCs were treated with THP-1-cells conditioned medium, and their pyroptosis, apoptosis, and viability were evaluated. Western blot, histological and immunohistochemistry staining, ELISA, PCR, and flow cytometry were all used to evaluate the role of DNMT1/ SIRT6 on macrophage polarization. Silencing DNMT1 inhibited apoptosis, the expression of related inflammatory mediators (e.g., iNOS) and inflammatory cytokines (e.g., IL6 and TNF-α). Moreover, silencing DNMT1 significantly inhibited the expression of pyroptosis markers IL- 1β, IL-6, and IL-18 and decreased the NLRP3, ASC, and caspase-1 expression. On the other hand, M2 macrophage specific markers CD163, Arg-1, and MR were overexpressed upon silencing DNMT1 or SIRT6 overexpression. At the same time, silencing DNMT1 exerted a regulatory effect on increasing the SIRT6 expression. DNMT1 may be a promising potential target for IDD treatment due to its ability to ameliorate the progression of the disease.

IL-27 induces autophagy through regulation of the DNMT1/lncRNA MEG3/ERK/p38 axis to reduce pulmonary fibrosis

PurposePrevious studies have shown that interleukin-27 (IL-27) can reduce bleomycin (BLM)-induced pulmonary fibrosis (PF). However, the underlying mechanism by which IL-27 attenuates PF is not fully clear.MethodsIn this research, we used BLM to construct a PF mouse model, and MRC-5 cells stimulated by transforming growth factor-β1 (TGF-β1) were used to construct a PF model in vitro. The lung tissue status was observed by Masson and hematoxylin and eosin (HE) staining. To detect gene expression, RT‒qPCR was used. The protein levels were detected by western blotting and immunofluorescence staining. EdU and ELISA were used to detect cell proliferation viability and hydroxyproline (HYP) content, respectively.ResultsAberrant IL-27 expression was observed in BLM-induced mouse lung tissues, and the use of IL-27 attenuated mouse lung tissue fibrosis. TGF-β1 induced autophagy inhibition in MRC-5 cells, and IL-27 alleviated MRC-5 cell fibrosis by activating autophagy. The mechanism is inhibition of DNA methyltransferase 1 (DNMT1)-mediated lncRNA MEG3 methylation and ERK/p38 signaling pathway activation. Overexpression of DNMT1, knockdown of lncRNA MEG3, autophagy inhibitor or ERK/p38 signaling pathway inhibitors reversed the positive effect of IL-27 in a lung fibrosis model in vitro.ConclusionIn conclusion, our study shows that IL-27 upregulates MEG3 expression through inhibition of DNMT1-mediated lncRNA MEG3 promoter methylation, which in turn inhibits ERK/p38 signaling pathway-induced autophagy and attenuates BLM-induced PF, providing a contribution to the elucidation of the potential mechanisms by which IL-27 attenuates PF.

DNMT1 involved in the analgesic effect of folic acid on gastric hypersensitivity through downregulating ASIC1 in adult offspring rats with prenatal maternal stress.

Gastric hypersensitivity (GHS) is a characteristic pathogenesis of functional dyspepsia (FD). DNA methyltransferase 1 (DNMT1) and acid-sensing ion channel 1 (ASIC1) are associated with GHS induced by prenatal maternal stress (PMS). The aim of this study was to investigate the mechanism of DNMT1 mediating the analgesic effect of folic acid (FA) on PMS-induced GHS. GHS was quantified by electromyogram recordings. The expression of DNMT1, DNMT3a, DNMT3b, and ASIC1 were detected by western blot, RT-PCR, and double-immunofluorescence. Neuronal excitability and proton-elicited currents of dorsal root ganglion (DRG) neurons were determined by whole-cell patch clamp recordings. The expression of DNMT1, but not DNMT3a or DNMT3b, was decreased in DRGs of PMS rats. FA alleviated PMS-induced GHS and hyperexcitability of DRG neurons. FA also increased DNMT1 and decreased ASIC1 expression and sensitivity. Intrathecal injection of DNMT1 inhibitor DC-517 attenuated the effect of FA on GHS alleviation and ASIC1 downregulation. Overexpression of DNMT1 with lentivirus not only rescued ASIC1 upregulation and hypersensitivity, but also alleviated GHS and hyperexcitability of DRG neurons induced by PMS. These results indicate that increased DNMT1 contributes to the analgesic effect of FA on PMS-induced GHS by reducing ASIC1 expression and sensitivity.

DNMT1-induced miR-133b suppression via methylation promotes myocardial fibrosis after myocardial infarction.

Myocardial fibrosis is an underlying cause of many cardiovascular diseases. Novel insights into the epigenetic control of myocardial fibrosis are now emerging. The current work is focused on investigating the biological role of DNA methyltransferase 1 (DNMT1) in myocardial fibrosis as well as the underlying mechanism. Our findings revealed that DNMT1 expression levels were upregulated, whereas miR-133b expression levels were decreased in a rat model of myocardial fibrosis following myocardial infarction. In vitro, the expression levels of DNMT1 increased and those of miR-133b decreased after Ang-II treatment in cardiac fibroblasts. DNMT1 knockdown inhibited Ang-II-induced cardiac myofibroblast activation, and DNMT1 overexpression increased the proliferation and collagen generation of cardiac myofibroblasts. Furthermore, DNMT1 expression levels decreased, while miR-133b expression levels increased after treatment with 5-Aza (5-Azacytidine, a known inhibitor of DNA methylation) in Ang-II-induced cardiac fibroblasts. BSP (Bisulfite sequencing PCR) results showed a marked decrease in methylation levels in the miR-133b promoter region upon overexpression of DNMT1, whereas knockdown of DNMT1 blocked increased methylation levels in the miR-133b promoter region in Ang-II-induced cardiac fibroblasts. Finally, 5-Aza treatment reduced the progression of myocardial fibrosis after myocardial infarction in rats in vivo. Collectively, our results suggest that DNMT1 mediates CTGF expression in cardiac fibroblast activation by regulating the methylation of miR-133b. The present work reveals the unique role of the DNMT1/miR-133b/CTGF axis in myocardial fibrosis, thus suggesting its great therapeutic potential in the treatment of cardiac diseases.

Ginsenoside Rg1 Epigenetically Modulates Smad7 Expression in Liver Fibrosis via MicroRNA-152

BackgroundGinsenoside Rg1, a bioactive component of Ginseng, has demonstrated anti-inflammatory, anti-cancer, and hepatoprotective effects. It is known that the epithelial–mesenchymal transition (EMT) plays a key role in the activation of hepatic stellate cells (HSCs). Recently, Rg1 has been shown to reverse liver fibrosis by suppressing EMT, although the mechanism of Rg1-mediated anti-fibrosis effects is still largely unclear. Interestingly, Smad7, a negative regulator of the transforming growth factor β (TGF-β) pathway, is often methylated during liver fibrosis. Whether Smad7 methylation plays a vital role in the effects of Rg1 on liver fibrosis remains unclear. MethodsAnti-fibrosis effects were examined after Rg1 processing in vivo and in vitro. Smad7 expression, Smad7 methylation, and microRNA-152 (miR-152) levels were also analyzed. ResultsRg1 significantly reduced the liver fibrosis caused by carbon tetrachloride, and reduced collagen deposition was also observed. Rg1 also contributed to the suppression of collagenation and HSC reproduction in vitro. Rg1 caused EMT inactivation, reducing Desmin and increasing E-cadherin levels. Notably, the effect of Rg1 on HSC activation was mediated by the TGF-β pathway. Rg1 induced Smad7 expression and demethylation. The over-expression of DNA methyltransferase 1 (DNMT1) blocked the Rg1-mediated inhibition of Smad7 methylation, and miR-152 targeted DNMT1. Further experiments suggested that Rg1 repressed Smad7 methylation via miR-152-mediated DNMT1 inhibition. MiR-152 inhibition reversed the Rg1-induced promotion of Smad7 expression and demethylation. In addition, miR-152 silencing led to the inhibition of the Rg1-induced EMT inactivation. ConclusionRg1 inhibits HSC activation by epigenetically modulating Smad7 expression and at least by partly inhibiting EMT.

Geniposide restricts angiogenesis in experimentary arthritis via inhibiting Dnmt1-mediated PTEN hypermethylation

Neovascularization in rheumatoid arthritis (RA) is a key bridge between malignant proliferative synovial tissue and pannus. In view of previous studies on the efficacy of Geniposide (GE) in experimentary arthritis, the purpose of this study was to investigate the possible mechanism of GE inhibiting angiogenesis by regulating the gene of phosphate and tension homology deleted on chromosome ten (PTEN). In this study, human umbilical vein endothelial cells (HUVEC) and adjuvant arthritis (AA) rat models were performed to research in vitro and in vivo. The results showed that GE treatment significantly reduced synovitis and angiogenesis in AA rats, which may be associated with the increased expression of PTEN with GE treatment. Meanwhile, the hypermethylation of PTEN accompanied by the over-expression of DNA methyltransferases (Dnmts) was demonstrated in TNF-α-induced HUVEC and AA rats. Knockdown of Dnmt1 by Dnmt1- siRNA significantly inhibited the tube formation of HUVEC in vitro. GE significantly restricted the angiogenesis of HUVEC by inhibiting DNA methylation, which was attributed to the down-regulation of Dnmt1 rather than Dnmt3a and Dnmt3b. The anti-angiogenesis effect of GE was further verified in AA model by the inhibition of Dnmt1. These results indicate that GE exhibited anti-angiogenesis effects in experimentary arthritis by inhibiting Dnmt1-mediated PTEN gene hypermethylation, which may brings new insights for the prevention and research of RA.

Extracellular-superoxide dismutase DNA methylation promotes oxidative stress in homocysteine-induced atherosclerosis.

In the present study, we investigate the effect of homocysteine (Hcy) on extracellular-superoxide dismutase (EC-SOD) DNA methylation in the aorta of mice, and explore the underlying mechanism in macrophages, trying to identify the key targets of Hcy-induced EC-SOD methylation changes. ApoE -/- mice are fed different diets for 15 weeks, EC-SOD and DNA methyltransferase 1 (DNMT1) expression levels are detected by RT-PCR and western blot analysis. EC-SOD methylation levels are assessed by ntMS-PCR. After EC-SOD overexpression or knockdown in macrophages, following the transfection of macrophages with pEGFP-N1-DNMT1, the methylation levels of EC-SOD are detected. Our data show that the concentrations of Hcy and the area of atherogenic lesions are significantly increased in ApoE -/- mice fed with a high-methionine diet, and have a positive correlation with the levels of superoxide anions, which indicates that Hcy-activated superoxide anions enhance the development of atherogenic lesions. EC-SOD expression is suppressed by Hcy, and the content of superoxide anion is increased when EC-SOD is silenced by RNAi in macrophages, suggesting that EC-SOD plays a major part in oxidative stress induced by Hcy. Furthermore, the promoter activity of EC-SOD is increased following transfection with the -1/-1100 fragment, and EC-SOD methylation level is significantly suppressed by Hcy, and more significantly decreased upon DNMT1 overexpression. In conclusion, Hcy may alter the DNA methylation status and DNMT1 acts as the essential enzyme in the methyl transfer process to disturb the status of EC-SOD DNA methylation, leading to decreased expression of EC-SOD and increased oxidative stress and atherosclerosis.

Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1.

To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism. Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L). DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells. CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

20(S)- Protopanaxadiol suppresses hepatic stellate cell activation via WIF1 demethylation-mediated inactivation of the Wnt/β-catenin pathway

Background20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. MethodsThe anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. ResultsPPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl4)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. ConclusionPPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the down-regulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

Inhibition of DNA Methyltransferases By Volasertib in HMA Resistant Cell Lines

Introduction: Resistance to hypomethylating agents (HMA) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is a concerning problem. We established HMA resistant cell lines with overexpression of DNA methyltransferases (DNMTs), which were not suppressed by azacitidine or decitabine. Polo-like kinase 1 (PLK1) is a key cell cycle modulator and is associated with regulation of the PIK3/Akt pathway, which was reported to stabilize DNMTs. We hypothesized that volasertib, a PLK inhibitor, might overcome HMA resistance through regulation of PIK3/Akt pathway and DNMTs.Methods: Cell viability was determined in HMA resistant cell lines (MOLM/AZA-1 and MOLM/DEC-5) and MOLM-13. Specific antibodies including phosphorylated-histone H3 (PHH3), caspase 3, PARP, and XIAP were used to evaluate apoptosis or DNA damage. We also analyzed phosphorylated-Akt (p-Akt), Akt, and DNMTs (DNMT1, 3A, and 3B) by immunoblot assay.Results: Volasertib effectively inhibit proliferation of MOLM/AZA-1 and MOLM/DEC-5 cells as well as MOLM-13 cells in a dose dependent manner. It also showed superior anti-leukemic effects compared to cytarabine, azicitidine or decitiabine. Volasertib showed a dose dependent increased expression of PHH3, caspase 3, and PARP, and decrease of XIAP. Treatment of volasertib caused decreased expression of p-AKT and all DNMTs (DNMT1, 3A, and 3B). GDC-0941, a PI3K inhibitor, suppressed DNMT1 and DNMT3A as well as p-Akt, but it could not decrease DNMT3B. A combination of volasertib and decitabine showed synergistic effects in MOLM/DEC-5. Anti-proliferative effects of volasertib were sustained in ex vivo AML or MDS samples.Conclusion: In our HMA resistant models, volasertib effectively inhibited all DNMTs. DNMT1 and 3A seemed to be suppressed via regulation of PI3K/Akt, but suppression of DNMT3B by volasertib might have separate mechanisms. Our data suggest that volasertib with or without decitabine has potential role in overcoming the HMA resistance in patients with AML and MDS. DisclosuresKo:Boehringer Ingelheim: Research Funding. Hur:Boehringer Ingelheim: Research Funding. Goo:Boehringer Ingelheim: Research Funding. Moon:Boehringer Ingelheim: Research Funding. Choi:Boehringer Ingelheim: Research Funding. Park:Boehringer Ingelheim: Research Funding. Lee:Boehringer Ingelheim: Research Funding. Lee:Boehringer Ingelheim: Research Funding.

3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of breast cancer, but drug resistance seriously limits its clinical use. The aim of the present work was to explore the effect of 3,3’-diindolylmethane (DIM) on PTX sensitivity and its possible mechanism in breast cancer. The expression of Krüppel-like factor 4 (KLF4) and DNA-methyltransferase 1 (DNMT1) in breast cancer tissues were assessed by immunohistochemistry and Western blotting. The methylation of KLF4 was evaluated by the MassARRAY platform. The lentivirus carrying KLF4 and DNMT1 gene or shRNA targeting DNMT1 were used to overexpress KLF4 or knockdown DNMT1 in MCF-7 and T47D breast cancer cells and the role of KLF4 and DNMT1 in regulation of PTX sensitivity was investigated. The effect of PTX on inhibiting the proliferation of MCF-7 and T47D cells was measured by CCK-8 assay. Flow cytometry was used to examine cell apoptosis. The expression of mRNA and protein was evaluated by qRT-PCR and Western blotting analysis, respectively. Our data showed that the expression of DNMT1 was increased, and the methylation level of CpG sites (−148 bp) in the KLF4 promoter was increased while the KLF4 expression was significantly decreased in breast cancer tissues. Overexpression of KLF4 increased the sensitivity of MCF-7 and T47D cells to PTX. DNMT1 increased the methylation of the KLF4 promoter and decrease the expression of KLF4. Knockdown of DNMT1 increased the sensitivity of MCF-7 and T47D cells to PTX. DIM enhanced the PTX sensitivity of MCF-7 and T47D cells, decreased the expression of DNMT1 and the methylation level of KLF4 promoter, thus increasing the level of KLF4. Furthermore, overexpression of DNMT1 attenuated the effect of DIM on the regulation of PTX sensitivity. Collectively, our data indicated that DNMT1-mediated hypermethylation of KLF4 promoter leads to downregulation of KLF4 in breast cancer. The level of KLF4 is correlated with the sensitivity of MCF-7 and T47D cells to PTX. DIM could enhance the antitumor efficacy of PTX on MCF-7 and T47D cells by regulating DNMT1 and KLF4.

DNA methyltransferase 1 inhibits O6-methylguanine-DNAmethyl-transferase-mediated cell growth and metastasis of hypopharyngeal squamous carcinoma

ObjectiveTo explore the role of DNA methyltransferase 1 (DNMT1) in development and progression of hypopharyngeal squamous carcinoma. DesignA total of 32 hypopharyngeal squamous carcinoma biopsy samples and 20 normal tissue specimens were collected. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot were performed for expression analysis. The mRNA and protein expression in the specimens and subcellular localization were analyzed. hypopharyngeal squamous carcinoma cells (FaDu) were used for small interfering RNA of DNMT1, and proliferation, cell cycle, and apoptosis were determined in the transfected cells. Furthermore, metastatic ability and methylation status of O6-methylguanine-DNAmethyl-transferase (MGMT) promoter was assessed. ResultsOur results showed that DNMT1 was overexpressed, while MGMT was down expressed in hypopharyngeal squamous carcinoma. DNMT1 overexpression and MGMT down expression were significantly associated with poorly differentiated tumors, lymph node metastasis, and clinical stage. DNMT1 and MGMT were majorly distributed in the nucleus. Furthermore, knockdown of DNMT1 inhibited proliferation and metastasis, induced apoptosis and G1 phase arrest in FaDu cells, and upregulated MGMT expression to reverse methylation status of MGMT promoter. ConclusionsThis study for the first time demonstrated the clinical value and the role of DNMT1 and MGMT in the biological function of hypopharyngeal squamous carcinoma. This work suggested that DNMT1 might serve as a potential therapeutic target for patients with hypopharyngeal squamous carcinoma.

Inspired Epigenetic Modulation Synergy with Adenosine Inhibition Elicits Pyroptosis and Potentiates Cancer Immunotherapy

AbstractOvercoming innate or adaptive resistance to immune checkpoint inhibitor therapy in solid tumors with limited T‐cell responses remains challenging. Increasing evidence has indicated that epigenetic alterations, especially overexpression of DNA methyltransferase and immunosuppressive adenosine, are major obstacles to T cell activation. Here, a tumor microenvironment (TME) inspired prodrug nanomicelle (AOZN) composed of the epigenetic modulator γ‐oryzanol (Orz), the adenosine inhibitor α, β‐methylene adenosine 5′ diphosphate (AMPCP), and GSH‐activable crosslinker, is rationally designed. High glutathione redox triggers Orz and AMPCP release in the TME. The released Orz act as a DNA methyltransferases inhibitor to upregulate gasdermin D (GSDMD) expression and AMPCP converted procaspase‐1 into active caspase‐1 by increasing ATP levels. Active caspase‐1 elicited GSDMD cleavage and induced pyroptosis in tumor cells. Furthermore, it is demonstrated that Orz and AMPCP likely have a synergistic effect in combating the immunosuppressive TME. Moreover, Orz enhances programmed death‐ligand 1 (PD‐L1) expression and sensitize tumors to anti‐PD‐L1 therapy. Thus, the AOZNs nano‐formulation drastically improves the hydrophobic properties of Orz with advantages of safe, affordable, readily available, and efficiency in regressing tumor growth, enhancing PD‐L1 responsive rate and prolonging survival of the B16F10 melanoma‐bearing mouse model. As a result, AOZNs provides a promising strategy for enhancing cancer immunotherapy.

DNMT1-mediated methylation inhibits microRNA-214-3p and promotes hair follicle stem cell differentiate into adipogenic lineages

BackgroundDysfunction of the DNA methylation was associated with stem cell reprogramming. Moreover, DNA methyltransferase 1 (DNMT1) deficiency was involved in the differentiation of hair follicle stem cell (HFSc), but the molecular mechanisms remain unknown.MethodsHFSc from human scalp tissues were isolated and cultured. The oil red O staining was used to observe the adipogenesis. The interaction relationship between microRNA (miR)-214-3p and mitogen-activated protein kinase 1 (MAPK1) was accessed by dual-luciferase reporter gene assay. The methylation level of miR-214-3p promoter was detected by methylation-specific PCR and the enrichment of DNMT1 in miR-214-3p promoter by chromatin immunoprecipitation assay. A mouse model of trauma was established to observe the skin regeneration at 0, 6, and 14 days.ResultsExpression of DNMT1 and MAPK1 was increased in the HFSc, while the expression of miR-214-3p was reduced. Moreover, DNMT1 inhibited the expression of miR-214-3p by promoting the promoter methylation of miR-214-3p. Overexpression of DNMT1 could reduce the expression of miR-214-3p, but increase the expression of MAPK1 and the extent of extracellular signal regulated kinase (ERK)1/2 phosphorylation, leading to enhanced adipogenic differentiation. Importantly, DNMT1 promoted skin regeneration in vivo. Conversely, overexpression of miR-214-3p could reverse the effects of DNMT1 on adipogenesis of HFSc.ConclusionDNMT1 promotes adipogenesis of HFSc by mediating miR-214-3p/MAPK1/p-ERK1/2 axis. This study may provide novel biomarkers for the potential application in stem cell therapy.

Effect of 5-aza-2'-deoxycytidine on Estrogen Receptor Alpha/Beta and DNA Methyltransferase 1 Genes Expression, Apoptosis Induction, and Cell Growth Prevention of the Colon Cancer HT 29 Cell Line.

Background:Cellular activity such as gene expression is regulated by epigenetic mechanisms and modifications. In mammals, DNA methylation is an essential component of the epigenetic machinery of the cells. DNA hypermethylation of the several tumor suppressor genes (TSGs) is associated with transcriptional gene silencing resulting in colon tumorigenesis. Overexpression of DNA methyltransferase 1 (DNMT1) in colon cancer has been reported in several studies. The methylation of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) have been demonstrated in various cancers. Previously, we indicated that genistein can reactivate ERα in hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of 5-aza-2′-deoxycytidine (5-aza-CdR) on ERα/ERβ and DNMT1 gene expression, apoptosis induction, and cell viability inhibition of the colon carcinoma HT 29 cell line.Methods:The effect of 5-Aza-CdR on the colon carcinoma HT 29 cell viability was measured by MTT assay. To determine the apoptotic cells, the cells were assessed using the Annexin V-FITC/PI detection kit. The expression of ERα, ERβ, and DNMT1 genes was determined using real-time quantitative RT-PCR.Results:The results indicated that 5-Aza-CdR can inhibit cell growth significantly versus control groups, induce significant apoptosis, down-regulate DNMT1, and up-regulate ERα and ERβ genes expression at different time periods. The percentage of apoptotic cells was 85.83% and 86.84% after 24 and 48 h, respectively (P < 0.01). The IC50 value for 5-Aza-CdR was obtained at 2.5 μM.Conclusions:5-Aza-CdR can up-regulate ERα and ERβ genes expression through DNMT1 down-regulation resulting in apoptosis induction and cell growth prevention.