In vitro and in silico insights into moniliformin induced global DNA methylation in liver cells

Deoxynivalenol induces global DNA hypomethylation by modulating the expression of miR-29b and DNA methylation regulators in HepG2 cells.

Obesity is defined by increased adipose tissue volume and has become a major risk factor for reproduction. Recent studies have revealed a substantial link between obesity and epigenetics. The epigenome is dynamically regulated mainly by DNA methylation. DNA methylation, which is controlled by DNA methyltransferases (Dnmts), has been widely studied because it is essential for imprinting and regulation of gene expression. In our previous study, we showed that the levels of Dnmt1, Dnmt3a and global DNA methylation was dramatically altered in the testis and ovary of high-fat diet (HFD)-induced obese mice. However, the effect of HFD on Dnmts and global DNA methylation in mouse uterus has not yet been demonstrated. Therefore, in the present study, we aimed to evaluate the effect of HFD on the level of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l and global DNA methylation in uterus. Our results showed that HFD significantly altered the levels of Dnmts and global DNA methylation in the uterus. The total expression of Dnmt1, Dnmt3a and Dnmt3b was significantly upregulated, while level of Dnmt3l and global DNA methylation were dramatically decreased (p < 0.05). Furthermore, we observed that the expression of Dnmt3b and Dnmt3l was significantly increased in endometrium including gland and epithelium (p < 0.05). Although Dnmt3b was the only protein whose expression significantly increased, the level of global DNA methylation and Dnmt3l significantly decreased in stroma and myometrium (p < 0.05). In conclusion, our results show for the first time that obesity dramatically alters global DNA methylation and expression of Dnmts, and decreased DNA methylation and Dnmt expression may cause abnormal gene expression, especially in the endometrium.

ABSTRACTFusaric acid (FA), a mycotoxin contaminant of maize, displays toxicity in plants and animals; however, its epigenetic mechanism is unknown. DNA methylation, an epigenetic modification that regulates gene expression, is mediated by DNA methyltransferases (DNMTs; DNMT1, DNMT3A, and DNMT3B) and demethylases (MBD2). The expression of DNMTs and demethylases are regulated by promoter methylation, microRNAs (miR-29b) and post-translational modifications (ubiquitination). Alterations in these DNA methylation modifying enzymes affect DNA methylation patterns and offer novel mechanisms of FA toxicity. We determined the effect of FA on global DNA methylation as well as a mechanism of FA-induced changes in DNA methylation by transcriptional (promoter methylation), post-transcriptional (miR-29b) and post-translational (ubiquitination) regulation of DNMTs and MBD2 in the human hepatocellular carcinoma (HepG2) cell line. FA induced global DNA hypomethylation (p < 0.0001) in HepG2 cells. FA decreased the mRNA and protein expression of DNMT1 (p < 0.0001), DNMT3A (p < 0.0001), and DNMT3B (p < 0.0001) by upregulating miR-29b (p < 0.0001) and inducing promoter hypermethylation of DNMT1 (p < 0.0001) and DNMT3B (p < 0.0001). FA decreased the ubiquitination of DNMT1 (p = 0.0753), DNMT3A (p = 0.0008), and DNMT3B (p < 0.0001) by decreasing UHRF1 (p < 0.0001) and USP7 (p < 0.0001). FA also induced MBD2 promoter hypomethylation (p < 0.0001) and increased MBD2 expression (p < 0.0001). Together these results indicate that FA induces global DNA hypomethylation by altering DNMT promoter methylation, upregulating miR-29b, and increasing MBD2 in HepG2 cells.

Event Abstract Back to Event Antiproliferative effects of NDC2 and its analogues against human hepatocellular carcinoma (HepG2) cell line Sau Har Lee1*, Cheng Foh Le2 and Chu Xin Ng1 1 School of Biosciences, Taylor's University, Malaysia 2 University of Nottingham Malaysia Campus, Malaysia Background Liver cancer is the second most common cause of mortality from cancer worldwide, with a mortality to incidence ratio as high as 0.95. This is mainly attributed to multidrug resistance developed by liver cancer cells after exposure to conventional chemotherapies. Hence, development of therapeutic peptides as a new chemotherapeutic drug might be a promising approach. NDC2 is an Aurein 1.2-like antibacterial and antitumour peptide that was shown to display minimal cell toxicity. We selected this peptide as the template to design five synthetic analogues (ND1–ND5) to enhance its anticancer potential. Therefore, the purpose of this study is to evaluate the in vitro antiproliferative properties of NDC2 and its analogues against human hepatocellular carcinoma (HepG2) cell line. Methods Each ND analogue was introduced with multiple residual alterations at specific positions at the C-terminal arm. Its anticancer potentials were predicted using the AntiCP anticancer peptide prediction tool. Subsequently, MTT assay was carried out to investigate the potential cytotoxic properties of NDs against HepG2 cells, up to a concentration of 256μg/ml for 24, 48 and 72 hours. Results Based on AntiCP algorithm, an anticancer peptide should exhibit SVM score greater than 1.0. From our analysis, only ND1–ND4 showed SVM scores ranging from 1.43–1.78, indicating their higher potential as anticancer peptides. These findings were supported by MTT results that showed both ND5 and the template NDC2 do not possess antiproliferative activity against HepG2 cells. Contrarily, ND1–ND4 exhibited antiproliferative activity against HepG2 cells with IC50 values ranging from 54–104µg/mL after 24 hours treatment. Among these analogues, ND4 was found to be most potent against HepG2 cells, with IC50 value of 54.811±3.403µg/mL. Our findings also showed that there was no significant time-dependent effects for each ND on HepG2 cells. Conclusion The four ND1–ND4 analogues exhibited antiproliferative activity against HepG2 cells, thus could represent as promising candidates for treatment against liver cancer. Hence, the anticancer mechanisms of the ND analogues should be further studied. Keywords: Anticancer peptides (ACPs), Drug Discovery, liver cancer, HepG2, cancer therapy Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Lee S, Le C and Ng C (2019). Antiproliferative effects of NDC2 and its analogues against human hepatocellular carcinoma (HepG2) cell line. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00148 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Sau Har Lee, School of Biosciences, Taylor's University, Subang Jaya, Selangor Darul Ehsan, 47500, Malaysia, sauhar.lee@taylors.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sau Har Lee Cheng Foh Le Chu Xin Ng Google Sau Har Lee Cheng Foh Le Chu Xin Ng Google Scholar Sau Har Lee Cheng Foh Le Chu Xin Ng PubMed Sau Har Lee Cheng Foh Le Chu Xin Ng Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Changes in gene expression and assessment of DNA methylation in primary human hepatocytes and HepG2 cells exposed to the environmental contaminants—Hexabromocyclododecane and 17-β oestradiol

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.

Lower levels of global DNA methylation in white blood cell (WBC) DNA have been associated with adult cancers. It is unknown whether individuals with a family history of cancer also have lower levels of global DNA methylation early in life. We examined global DNA methylation in WBC (measured in three repetitive elements, LINE1, Sat2 and Alu, by MethyLight and in LINE1 by pyrosequencing) in 51 girls ages 6-17. Compared to girls without a family history of breast cancer, methylation levels were lower for all assays in girls with a family history of breast cancer, and statistically significantly lower for Alu and LINE1 pyrosequencing. After adjusting for age, body mass index (BMI), and Tanner stage, only methylation in Alu was associated with family history of breast cancer. If these findings are replicated in larger studies, they suggest that lower levels of global WBC DNA methylation observed later in life in adults with cancer may also be present early in life in children with a family history of cancer.

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Differential crosstalk between global DNA methylation and metabolomics associated with cell type specific stress response by pristine and functionalized MWCNT.

Identification of NTCP as an HBV Receptor: The Beginning of the End or the End of the Beginning?

Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.

Cholesterol 7alpha-hydroxylase is the rate-limiting enzyme in the degradation of cholesterol to bile salts and plays a central role in regulating cholesterol homeostasis. The mechanisms involved in the transcriptional control of the human gene are largely unknown. HepG2 cells represent an appropriate model system for the study of the regulation of the gene. To identify liver-specific DNA sequences in the promoter of the human CYP7 gene, we first examined the DNase I hypersensitivity in the 5'-region of the gene. An area of hypersensitivity was observed in the region from -50 to -200 of the human gene in nuclei from transcriptionally active HepG2 cells, but was absent in transcriptionally inactive HeLa cell nuclei or in free DNA. Various 5'-promoter deletion constructs were made and transfected into HepG2 cells. About 300 base pairs of upstream sequence are required for high level promoter activity of the human CYP7 gene in HepG2 cells. DNase I footprinting of the hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from -80 to -70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to the segment from -148 to -127 of the human CYP7 promoter. Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from -313 to -285 is bound by members of the HNF-3 family and acts as an enhancer. Additionally, the segment from -197 to -173 binds a negative regulatory protein that is present in Chinese hamster ovary cell extracts and in HepG2 cell extracts. These experiments define the key control elements responsible for basal transcription of the human CYP7 gene in HepG2 cells.

10-Hydroxycamptothecin (HCPT), a DNA topoisomerase I inhibitor, is an antitumor alkaloid isolated from a Chinese tree, Camptotheca acuminata, and exhibits a remarkable antihepatoma effect. We studied HCPT to determine whether or not its anti-hepatoma activity occurs through apoptosis induction and cell cycle disturbance using the MTT method, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. The results showed that HCPT inhibited proliferation of human hepatoma Hep G2, Bel-7402 and Bel-7404 cells at an optimal concentration of 0.1 microg/ml. This growth inhibition was dose and time dependent, and was accompanied by evidence of apoptotic changes and cell cycle perturbation in Hep G2 cells. Chromatin condensation and nuclear fragmentation were observed in Hep G2 cells by fluorescence microscopy. Agarose gel electrophoresis showed internucleosomal DNA fragmentation ('ladder pattern') of Hep G2 cells following treatment with HCPT, in a concentration- and time-dependent manner. Flow cytometry showed that HCPT induced a massive hypodiploid cell population and arrested cells in G2/M phase (at low dose) or in S phase (at high dose) in Hep G2 cells. The results of this study suggest that the anti-hepatoma effect of HCPT may result from apoptosis induction and cell cycle disturbance.

Triclosan reduces the levels of global DNA methylation in HepG2 cells