Self-Powered Biosensing Platform Based on "Signal-On" Enzymatic Biofuel Cell for DNA Methyltransferase Activity Analysis and Inhibitor Screening.

Aberrant DNA methylation catalyzed by DNA methyltransferases (MTase) has proved to be associated with human diseases such as cancers. Thus, the development of an efficient strategy to accurately detect DNA MTase is highly desirable in medical diagnostics. Herein, we proposed a robust "signal-on" enzymatic biofuel cell (EBFC)-based self-powered biosensing platform with excellent anti-interference ability for DNA MTase activity analysis and inhibitor screening. In the presence of target MTase, the MTase-catalyzed DNA methylation occurred and hindered the HpaII endonuclease-catalyzed dsDNA dissociation, which enabled more bilirubin oxidase (BOD) to immobilize at the cathode surface via amidation. Then, BOD-catalyzed oxygen reduction took place by accepting electrons generated at the anode via glucose oxidation, thus leading to an elevated open-circuit voltage value, the amplitude of which was directly related to MTase concentration. The direct detection limit of the M.SssI assay was down to 0.005 U/mL, which was lower than that of those reported results. Notably, the as-proposed protocol was competent to detect DNA MTase activity directly in human serum samples without enrichment and separation, and applicable to the screening of M.SssI inhibitors. Considering the virtues of the excellent anti-interference ability, no requirement of external power, simplicity, and high accuracy, the biosensing platform would hold great potential in DNA MTase bioassay and clinical diagnosis of cancers.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

Epigenetic targeting DNMT1 of pancreatic ductal adenocarcinoma using interstitial control release biodegrading polymer reduced tumor growth through hedgehog pathway inhibition.

Annually, 48,000 people die from pancreatic ductal adenocarcinoma (PDAC), ranking it the fourth among cancer-related deaths in the United States. Currently, anti-cancer drugs are not effective against PDAC, and only extends survival by 3 months. Aberrant DNA methylation has been shown to play an important role during carcinogenesis in PDAC, with approximately 80% of tumor overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP), which is identified from a Chinese herbal drug. n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells and inducing cell cycle arrest at G0/G1 phase leading apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray studies, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.

Fluorescent copper nanoclusters as a nano-dye for DNA methyltransferase activity analysis and inhibitor screening.

Fluorescent copper nanoslusters (CuNCs) as a new class of fluorophores have attracted more and more attention due to their ease of synthesis, excellent optical properties, and low cost. In this study, a novel label-free fluorescent method was developed for the detection of DNA methyltransferases based on template length-dependent of dsDNA-CuNCs. In the absence of DNA adenine methylation methyltransferase (Dam MTase), the dsDNA containing the methylation-responsive sequence could effectively template the formation of fluorescent CuNCs with bright fluorescence. When the dsDNA substrate is methylated by Dam MTase, the methylation-sensitive restriction endonuclease Dpn I cleaves the methylated dsDNA and produces shorter dsDNA product, which fails to template fluorescent CuNCs. So, the Dam MTase activity could be identified by the changes of CuNCs' fluorescence. Based on this method, a linear range of 0.5-10 U/mL was achieved with high sensitivity and selectivity. Moreover, we also demonstrate the proposed method can be applied to evaluation and screening of inhibitors for Dam MTase.

Tanshinone IIA induces apoptosis via inhibition of Wnt/ß­catenin/MGMT signaling in AtT­20 cells.

A strategy to suppress the expression of the DNA repair enzyme O6­methylguanine­DNA methyltransferase (MGMT) by inhibition of Wnt/ß­catenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/ß­catenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT­20 cells in a concentration­dependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC­1 staining, which were associated with activation of caspase­3 and DNA fragmentation detected by TUNEL in AtT­20 cells. T­cell factor (TCF)­lymphoid­enhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between ß­catenin and TCF­4 were detected using a co­immunoprecipitation kit. The results indicated TSA treatment increased ß­catenin phosphorylation, inhibited ß­catenin nuclear translocation, reduced ß­catenin/TCF­4 complex formation and TCF­LEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in ß­catenin knock down AtT­20 cells abrogated the TSA­mediated effects in AtT­20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/ß­catenin­dependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza.

Dietary fatty acids modulate adipocyte TNFa production via regulation of its DNA promoter methylation levels.

The factors regulating TNF alpha (TNFa) levels could be considered therapeutic targets against metabolic syndrome development. DNA methylation is a potent regulator of gene expression and may be associated with protein levels. In this study we investigate whether the effect of dietary fatty acids on TNFa released from adipocytes might be associated with modifications of the TNFa promoter DNA methylation status. A group of rats was assigned to three diets with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of visceral adipose tissues were taken for adipocyte isolation, in which released TNFa levels were measured, and for methylation and expression studies. In addition, 3 T3-L1 cells were treated with palmitic, oleic and linoleic acids, with and without 5-Azacitydine (5-AZA). After treatments, cells and supernatants were included in the same analyses as rat samples. TNFa promoter methylation levels, gene expression and secretion were different according to the diets and fatty acid treatments associated with them. Cells treated with 5-AZA displayed higher TNFa levels than in the absence of 5-AZA, without differences between fatty acids. According to our results, dietary fatty acid regulation of adipocyte TNFa levels may be mediated by epigenetic modifications of the TNFa promoter DNA methylation levels.

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

Comparison of the Effects of Curcumin and RG108 on NGF-Induced PC-12 Adh Cell Differentiation and Neurite Outgrowth.

DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel drugs, especially for neurodegenerative disorders. In recent years, there has been increased interest in small molecules that can cross the blood-brain barrier for the treatment of neurodegenerative diseases. Therefore, comparing the neuronal differentiative effects of a natural compound curcumin and a synthetic small molecule RG108 was the aim of this study. The effects of curcumin and RG108 on neuronal differentiation and neurite outgrowth were investigated in the PC-12 Adh cell line. First, a nontoxic concentration was determined to be 100 nM with WST-1 assay. Subsequently, cells were treated with 100 nM curcumin and RG108 alone or in combination with 50 nM nerve growth factor (NGF). Cell differentiations were evaluated by a real-time cell analyzer system. Neurite outgrowth was determined and morphologically shown by immunofluorescence staining with anti-beta III tubulin antibody on PC-12 Adh cells. Also, growth-associated protein-43 (GAP-43) and ß-tubulin III mRNA expression levels, associated with neurite outgrowth promotion, were determined with real-time polymerase chain reaction (RT-PCR). According to our results, 100 nM curcumin and RG108 significantly induced neurite outgrowth of PC-12 Adh cells with 50 nM NGF. Curcumin + NGF combination further increased cell differentiations and total neurite lengths more than curcumin alone and RG108 + NGF combination groups. Strikingly, curcumin and NGF combination upregulated GAP-43 and ß-tubulin mRNA expression levels excessively. In conclusion, curcumin was found to be more effective than RG108 on neuronal differentiation and neurite outgrowth of PC-12 Adh cells in a combination with NGF. Therefore, natural DNMT1 inhibitors, such as curcumin, can be a novel approach for the neurodegenerative disorders treatment.

Aberrant DNA methylations in chondrosarcoma.

Chondrosarcoma (CS) is the second most common primary malignant bone tumor. Unlike other bone tumors, CS is highly resistant to conventional chemotherapy and radiotherapy, thus resulting in poor patient outcomes. There is an urgent need to establish alternative therapies for CS. However, the etiology and pathogenesis of CS still remain elusive. Recently, DNA methylation-associated epigenetic changes have been found to play a pivotal role in the initiation and development of human cancers, including CS, by regulating target gene expression in different cellular pathways. Elucidating the mechanisms of DNA methylation alteration may provide biomarkers for diagnosis and prognosis, as well as novel treatment options for CS. We have conducted a critical review to summarize the evidence regarding aberrant DNA methylation patterns as diagnostic biomarkers, predictors of progression and potential treatment strategies in CS.

A Comparative Study on the In Vitro Effects of the DNA Methyltransferase Inhibitor 5-Azacytidine (5-AzaC) in Breast/Mammary Cancer of Different Mammalian Species.

Murine models are indispensible for the study of human breast cancer, but they have limitations: tumors arising spontaneously in humans must be induced in mice, and long-term follow up is limited by the short life span of rodents. In contrast, dogs and cats develop mammary tumors spontaneously and are relatively long-lived. This study examines the effects of the DNA methyltransferase (DNMT) inhibitor 5-Azacytidine (5-AzaC) on normal and tumoral mammary cell lines derived from dogs, cats and humans, as proof of concept that small companion animals are useful models of human breast cancer. Our findings show that treatment with 5-AzaC reduces in vitro tumorigenicity in all three species based on growth and invasion assays, mitochondrial activity and susceptibility to apoptosis. Interestingly, we found that the effects of 5-AzaC on gene expression varied not only between the different species but also between different tumoral cell lines within the same species, and confirmed the correlation between loss of methylation in a specific gene promotor region and increased expression of the associated gene using bisulfite sequencing. In addition, treatment with a high dose of 5-AzaC was toxic to tumoral, but not healthy, mammary cell lines from all species, indicating this drug has therapeutic potential. Importantly, we confirmed these results in primary malignant cells isolated from canine and feline adenocarcinomas. The similarities observed between the three species suggest dogs and cats can be useful models for the study of human breast cancer and the pre-clinical evaluation of novel therapeutics.

Azacitidine-Induced Pericarditis: A Case Series.

STUDY OBJECTIVE: To describe three cases of pericarditis probably related to azacitidine administration in a span of 3 years at our center. DESIGN: Case series. SETTING: Comprehensive cancer center within a large, academic medical center. PATIENTS: Three patients with high-grade myelodysplastic syndrome or acute myeloid leukemia who received azacitidine. INTERVENTION: None. MEASUREMENTS: None. MAIN RESULTS: Patient 1 presented with pericarditis after cycle 2 of azacitidine, patient 3 presented 3 weeks after completing cycle 5, and patient 2 presented during cycle 1. All patients were treated symptomatically and responded to corticosteroids. None of the patients were re-challenged with hypomethylating agents. Use of the Naranjo adverse drug reaction probability scale indicated a probable adverse drug reaction (score of 6) for patients 1 and 3 and a possible adverse drug reaction (score of 3) for patient 2. CONCLUSION: With the exclusion of other common causes of pericarditis, we believe it is likely that azacitidine was responsible for the findings in our patients. Providers caring for patients receiving hypomethylating agents should consider this potential adverse drug reaction in the setting of unexplained chest pain or other clinical signs consistent with cardiotoxicity.

Improving cancer immunotherapy with DNA methyltransferase inhibitors.

Immunotherapy confers durable clinical benefit to melanoma, lung, and kidney cancer patients. Challengingly, most other solid tumors, including ovarian carcinoma, are not particularly responsive to immunotherapy, so combination with a complementary therapy may be beneficial. Recent findings suggest that epigenetic modifying drugs can prime antitumor immunity by increasing expression of tumor-associated antigens, chemokines, and activating ligands by cancer cells as well as cytokines by immune cells. This review, drawing from both preclinical and clinical data, describes some of the mechanisms of action that enable DNA methyltransferase inhibitors to facilitate the establishment of antitumor immunity.

Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.

5-aza-2'-Deoxycytidine, a DNA methyltransferase inhibitor, facilitates the inorganic phosphorus-induced mineralization of vascular smooth muscle cells.

AIM: Vascular calcification, an independent risk factor for cardiovascular disease in patients with chronic kidney disease(CKD), refers to the mineralization of vascular smooth muscle cells(VSMCs) caused by phenotypic changes toward osteoblast-like cells. DNA methylation, mediated by DNA methyltransferases(DNMTs), plays an important role in the differentiation of osteoblasts. We herein assessed the effects of a DNMT inhibitor on phenotypic changes in VSMCs and the development of vascular calcification. METHODS: The effects of 5-aza-2'-deoxycytidine(5-aza-dC), a DNMT inhibitor, on human aortic smooth muscle cells(HASMCs) were evaluated. The expression and DNA methylation status of osteogenic genes were determined using RT-qPCR and bisulfite sequencing, respectively. Mineralization of HASMCs was induced by high concentrations of inorganic phosphate(Pi), as confirmed by quantitation of the calcium levels and von Kossa staining. Moreover, we examined the effects of the suppression of DNMT1 and/or alkaline phosphatase(ALP) on the mineralization of HASMCs. RESULTS: 5-aza-dC increased the expression and activity of ALP and reduced the DNA methylation levels of the ALP promoter region in the HASMCs. In addition, both treatment with 5-aza-dC and downregulation of the DNMT1 expression promoted the Pi-induced mineralization of HASMCs. Moreover, both treatment with phosphonoformic acid(PFA), a sodium-dependent phosphate transporter inhibitor, and suppression of the ALP expression inhibited the 5-aza-dC-promoted mineralization of HASMCs. CONCLUSIONS: The present study showed that DNMT inhibitors facilitate the Pi-induced development of vascular calcification via the upregulation of the ALP expression along with a reduction in the DNA methylation level of the ALP promoter region.

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.

Molecular modeling and virtual screening of DNA methyltransferase inhibitors.

Inhibition of DNA methyltransferases (DNMTs) is a promising approach for the therapeutic treatment of cancer and other diseases. In this work, we review the recent progress on the molecular modeling and virtual screening toward the identification of key structural features associated with the enzyme inhibitory action of active compounds and to identify DNMT inhibitors with novel molecular scaffolds. We discuss the molecular modeling with the co-factor binding site using a recent crystallographic structure of the methyltransferase domain of human DNMT1. We also review the emerging synergy of molecular modeling and chemoinformatic approaches applied to epigenetic therapies targeting DNMTs.

Myelodysplastic syndromes: who and when in the course of disease to transplant.

The myelodysplastic syndromes are clonal hematopoietic disorders for which hematopoietic stem cell transplantation remains the only curative therapy. The timing of transplantation, methods of disease risk stratification, patient selection, pretransplantation therapies, and preparative regimens have evolved over the years, resulting in increasing disease-free survival. In recent years, alternative donor sources have been demonstrated to be a viable alternative to traditional sibling and matched unrelated donor stem cell sources. Efforts at transplantation regimen development continue with the aim of maximizing the chances of cure with minimal toxicity and improved quality of life. Integrating new knowledge regarding disease biology will be critical to continue to improve the success of hematopoietic stem cell transplantation. Exciting areas of ongoing research that may lead to reductions in posttransplantation relapse rate include posttransplantation therapies such as DNA methyltransferase inhibitors, vaccine strategies, and donor lymphocyte infusions to enhance the GVL effect.

Neonatal isolation decreases cued fear conditioning and frontal cortical histone 3 lysine 9 methylation in adult female rats.

Early life stress is thought to enhance adult susceptibility to stress and stress-related mood disorders. In this study, fear-potentiated startle was used to model the acquisition of a traumatic event-related memory in female rats experiencing early life stress. Daily 1-hr maternal and sibling separation throughout day 2-9 postpartum (D2-9 PP) caused a decrease in the fear-potentiated startle, but not acoustic startle baseline, in adult female rats. The separation procedure did not affect corticosterone secretion but produced an increase in serum estradiol concentration. Moreover, the separation procedure did not affect histone 3 lysine 9 (H3K9) acetylation but decreased H3K9 mono- and tri-methylation in frontal cortices. Treatment with 5-aza-2'-deoxycytidine (AZA) (5mg/kg at alternative days from D2PP to D9PP or 10mg/kg at D5PP and D9PP), a DNA methylation inhibitor, did not affect the separation-decreased fear-potentiated startle. Treatment with valproic acid (VPA), a histone deacetylase inhibitor, at 3 dosing regimens (300mg/kg at D2-9PP; 100mg/kg at D2-4PP, 200mg/kg at D5-7PP, 300mg/kg at D8-9PP; 100mg/kg at D2-5PP, 200mg/kg at D6-9PP) prior to daily separation reversed such a decrease in fear-potentiated startle. The lowest effective VPA dosing regimen used (100mg/kg at D2-5PP, 200mg/kg at D6-9PP) reversed the separation-decreased H3K9 mono- and tri-methylation in frontal cortices. Eight-day VPA (300mg/kg/day) and AZA (5mg/kg/day) administrations starting at D28PP were ineffective in altering the separation-decreased fear-potentiated startle. We, hereby, suggest that decreased frontal cortical H3K9 mono- and tri-methylation may be involved in early life separation-decreased fear memory of adult rats.

Induced production of mycotoxins in an endophytic fungus from the medicinal plant Datura stramonium L.

Epigenetic modifiers, including DNA methyltransferase (DNMT) or histone deacetylase (HDAC) inhibitors, are useful to induce the expression of otherwise dormant biosynthetic genes under standard laboratory conditions. We isolated several endophytic fungi from the medicinal plant Datura stramonium L., which produces pharmaceutically important tropane alkaloids, including scopolamine and hyoscyamine. Although none of the endophytic fungi produced the tropane alkaloids, supplementation of a DNMT inhibitor, 5-azacytidine, and/or a HDAC inhibitor, suberoylanilide hydroxamic acid, to the culture medium induced the production of mycotoxins, including alternariol, alternariol-5-O-methyl ether, 3'-hydroxyalternariol-5-O-methyl ether, altenusin, tenuazonic acid, and altertoxin II, by the endophytic fungus Alternaria sp. This is the first report of a mycotoxin-producing endophytic fungus from the medicinal plant D. stramonium L. This work demonstrates that treatments with epigenetic modifiers induce the production of mycotoxins, thus providing a useful tool to explore the biosynthetic potential of the microorganisms.

Arsenic trioxide inhibits DNA methyltransferase and restores expression of methylation-silenced CDKN2B/CDKN2A genes in human hematologic malignant cells.

Cyclin-dependent kinase inhibitors CDKN2B and CDKN2A are tumor suppressor genes that are frequently dysregulated in a variety of cancers. Aberrant regulation via DNA hypermethylation causes gene silencing. Arsenic trioxide has been successfully used to treat malignant, hematopoietic diseases and is known to act by induction of apoptosis and inhibition of cellular proliferation. However, arsenic trioxide has been recently reported to act via inhibition of DNA hypermethylation in some solid tumors. The goal of this study was to explore the mechanism of arsenic trioxide induced demethylation of the CDKN2B and CDKN2A promoters in the hematologic malignant cell lines Molt4, MUTZ-1, U937, U266 and CA46. We used bisulphate modification and nested-methylation specific PCR to determine the levels of methylated and unmethylated promoter sequences in untreated and As2O3-treated cells. We used semi-quantitative RT-PCR and immunoblotting to quantify CDKN2B and CDKN2A mRNA and protein levels, respectively. We measured DNMT activity in nuclear extracts of untreated and treated cells using radiolabeled SAM as a methyl donor. The CDKN2B promoter was hypermethylated in Molt4 and MUTZ-1 cells, while the CDKN2A promoter was hypermethylated in U937, U266 and CA46 cells. As2O3 treatment caused demethylation associated with an increase in mRNA levels of the CDKN2B and CDKN2A genes. We also demonstrated a concomitant inhibition in DNMT activity and DNMT mRNA levels in As2O3-treated cells. In summary, As2O3 restored expression levels of tumor suppressor genes in hematologic malignant cells by causing promoter demethylation along with an inhibition of DNMTs 1, 3a and 3b.