Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

Advances in DNA methylation and demethylation in medicinal plants: a review.

DNA methylation and demethylation are widely acknowledged epigenetic phenomena which can cause heritable and phenotypic changes in functional genes without changing the DNA sequence. They can thus affect phenotype formation in medicinal plants. However, a comprehensive review of the literature summarizing current research trends in this field is lacking. Thus, this review aims to provide an up-to-date summary of current methods for the detection of 5-mC DNA methylation, identification and analysis of DNA methyltransferases and demethyltransferases, and regulation of DNA methylation in medicinal plants. The data showed that polyploidy and environmental changes can affect DNA methylation levels in medicinal plants. Changes in DNA methylation can thus regulate plant morphogenesis, growth and development, and formation of secondary metabolites. Future research is required to explore the mechanisms by which DNA methylation regulates the accumulation of secondary metabolites in medicinal plants.

The evaluation of chitosan hydrogel based curcumin effect on DNMT1, DNMT3A, DNMT3B, MEG3, HOTAIR gene expression in glioblastoma cell line.

BACKGROUND: Cancer is one of the most important causes of death worldwide. Some types of cancer, including glioblastoma, with a high potential for growth, invasion, and resistance to general treatments, chemotherapy, and radiotherapy, have a high potential for recurrence. Many chemical drugs have been used to treat it, but herbal drugs are more effective with fewer side effects; Therefore, this research aims to investigate the effect of curcumin-chitosan nano-complex on the expression of MEG3, HOTAIR, DNMT1, DNMT3A, DNMT3B genes in the glioblastoma cell line. METHODS: In this research, glioblastoma cell line, PCR and spectrophotometry techniques, MTT test and transmission, field emission transmission, and fluorescent electron microscopes were used. RESULTS: The morphological examination of the curcumin-chitosan nano-complex was without clumping, and the fluorescent microscope examination showed the nano-complex enters the cell and affects the genes expression. In its bioavailability studies, it was found that it significantly increases the death of cancer cells in a dose- and time-dependent manner. Gene expression tests showed that this nano-complex increased MEG3 gene expression compared to the control group, which is statistically significant (p < 0.05). It also decreased HOTAIR gene expression compared to the control group, which was not statistically significant (p > 0.05). It decreased the expression of DNMT1, DNMT3A, and DNMT3B genes compared to the control group, which is statistically significant (p < 0.05). CONCLUSION: By using active plant substances such as curcumin, the active demethylation of brain cells can be directed to the path of inhibiting the growth of brain cancer cells and eliminating them.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy.

BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells.

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

Banxia xiexin decoction affects drug sensitivity in gastric cancer cells by regulating MGMT expression via IL­6/JAK/STAT3­mediated PD­L1 activity.

Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PD­L1), 6­O­methylguanine­DNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent non­cancer tissues of patients with GC, and in vitro experimentation was conducted using drug­resistant and ­sensitive GC cells. The expression levels of PD­L1, MGMT and STAT3 were determined using reverse transcription­quantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit­8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL­6, IFN­Î³, JAK/STAT3 pathway proteins, PD­L1 and MGMT. The association between PD­L1 and MGMT protein expression levels was subsequently assessed via co­immunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drug­resistant tumor­bearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PD­L1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatin­resistant cells. Furthermore, BXXX inhibited the proliferation of drug­resistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drug­resistant cells. BXXX also inhibited the expression levels of IL­6, IFN­Î³ and JAK/STAT3 pathway proteins, as well as the expression levels of PD­L1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drug­resistant GC cells, and significantly reversed the effect of BXXX on PD­L1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPD­L1, which was itself mediated by IL­6/JAK/STAT3 signaling.

Molecular Characteristics of Thalamic Gliomas in Adults.

The 2016 World Health Organization classification of central nervous system tumor firstly introduces molecular diagnosis to glioma, while the molecular features of adult thalamic gliomas (ATGs) in a relatively large sample have not been reported. We aimed at exploring molecular characteristics in ATGs. The data of 97 and 575 newly diagnosed ATGs and superficial gliomas (SGs) patients were collected, and we performed a comparative analysis of molecular characteristics between them. We analyzed expressions of molecules as follow: H3 K27M, isocitrate dehydrogenase1 (IDH1), Ki-67, O6-Methylguanine-DNA methyltransferase (MGMT) promoter, EGFR, p53, ATRX, GFAP, Oligo2, PTEN, MGMT, and MMP9 by immunohistochemistry. Direct gene sequencing was performed to test the H3 K27M, IDH1, and TERT promoter mutation. The median age at diagnosis of ATGs was 36.0 years, and majority of them were high-grade glioma. We found a significant difference in H3 K27M mutation (P = 0.003), IDH1 mutation (P < 0.001), MGMT promoter methylation (P = 0.005), and Ki67 > 0.1 (P < 0.001) between ATGs and SGs. The statuses of IDH1 (P < 0.001), MGMT promoter (P < 0.001), and Ki67 (P < 0.001) were significantly different between these two groups in lower-grade gliomas. And statuses of IDH1 (P < 0.001), Ki67 (P < 0.001), and EGFR (P = 0.032) were different between these two groups in high-grade gliomas. Only Ki67 > 0.1 was differentially expressed between lower- and high-grade gliomas in ATGs (P = 0.014). The high occurrence of H3 K27M mutation and Ki67 > 0.1, rare occurrence of IDH1 mutation, and MGMT promoter methylation in ATGs suggested that ATGs may be a distinct type of glioma entity.

Pulsed and Discontinuous Electromagnetic Field Exposure Decreases Temozolomide Resistance in Glioblastoma by Modulating the Expression of O6-Methylguanine-DNA Methyltransferase, Cyclin-D1, and p53.

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.

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.

Vitamin D deficiency during pregnancy affects the function of Th1/Th2 cells and methylation of IFN-γ gene in offspring rats.

The effects of maternal vitamin D status on offspring's Th1/Th2 cell function and the related mechanisms have not been reported. In this study, we established the rat model of vitamin D deficiency during pregnancy. 48 female Sprague-Dawley rats (8 weeks old) were randomly assigned to three groups (n = 16/group): control group (fed with standard AIN-93 G diet until parturition), vitamin D deficiency group (VDD group, fed with vitamin D deficient diet until parturition) and vitamin D supplementation group (VDS group, fed with vitamin D deficient diet prior to mating and with standard AIN-93 G diet during pregnancy). At 4 weeks of age, the ratio of T helper type 1/ T helper type 2 (Th1/Th2) cells and the levels of Th1/Th2 cytokines (IFN-γ, IL-4, IL-5, IL-6, IL-10 and IL-13) in offspring rats were determined by Flow Cytometry and Meso Scale Discovery, respectively. Furthermore, DNA methyltransferase (DNMT) activity as well as the methylation levels of IFN-γ and IL-4 genes were measured. As a result, rats in the VDD group showed a significant decrease in Th1/Th2 ratio and IFN-γ level and an increase in IL-4 level. Additionally, up-regulated DNMT activity and increased methylation rate of IFN-γ gene was shown in VDD offspring rats. Supplementation with vitamin D during pregnancy reversed the above abnormalities. In conclusion, maternal vitamin D deficiency affected the function of Th1/Th2 cells and methylation of IFN-γ gene in offspring rats. Meanwhile, maternal vitamin D deficiency had the potential to regulate DNMT activity, which may determine the status of methylation.

High frequency of H3K27M immunopositivity in adult thalamic glioblastoma.

Adult thalamic glioblastomas (GBM) are uncommon tumors with limited available molecular data. One of the reported molecular alterations in these tumors is the H3K27M mutation. It has been documented that H3K27M mutation is found in a high proportion of pediatric thalamic gliomas. In this study, we have analyzed the molecular alterations exclusive to adult thalamic GBM. This is a 6 years retrospective study of adult thalamic GBM patients who underwent surgical decompression of the tumor. Clinical data were obtained from the case records. Immunohistochemistry (IHC) was performed on the tumors using antibodies directed against the gene products of R132H mutant isocitrate dehydrogenase 1 (IDH1), alpha-thalassemia/mental retardation X-linked (ATRX), p53, H3K27M, H3K27me3, and V600E mutant BRAF. Molecular analyses were carried out to detect other IDH1 and IDH2 mutations, O6 -methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation, and epidermal growth factor gene (EGFR) and telomerase reverse transcriptase gene (TERT) promoter mutations. A total of 42 cases of adult thalamic GBM were studied. The mean age of presentation was 42 years with age range of 19-58 years. Male predominance was noted. All the tumors were IDH wild-type, BRAF (V600E)-immunonegative and unmethylated for MGMT promoter. H3K27M immunopositivity was noted in 60% of tumors. Of these 33.3% were from older adults above the age of 50 years. Of the H3K27M-immunopositive cases, ATRX loss of expression was seen in 32%, p53 immunopositivity in 24% and EGFR amplification in 12%. Higher frequency of TERT promoter mutations was noted in H3K27M-immunonegative cases (58.8%) compared to immunopositive cases (20%). Ours is one of the few studies elucidating the molecular alterations exclusive to adult thalamic GBM. We show a high frequency of H3K27M immunopositivity, suggestive of its mutational status in these tumors, including in older adults.

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out at pH 7.4 in the presence of 50 µM thionine and 0.5 mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 µA·µM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use of glutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

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.

Polyamine Metabolism and Gene Methylation in Conjunction with One-Carbon Metabolism.

Recent investigations have revealed that changes in DNA methylation status play an important role in aging-associated pathologies and lifespan. The methylation of DNA is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in the presence of S-adenosylmethionine (SAM), which serves as a methyl group donor. Increased availability of SAM enhances DNMT activity, while its metabolites, S-adenosyl-l-homocysteine (SAH) and decarboxylated S-adenosylmethionine (dcSAM), act to inhibit DNMT activity. SAH, which is converted from SAM by adding a methyl group to cytosine residues in DNA, is an intermediate precursor of homocysteine. dcSAM, converted from SAM by the enzymatic activity of adenosylmethionine decarboxylase, provides an aminopropyl group to synthesize the polyamines spermine and spermidine. Increased homocysteine levels are a significant risk factor for the development of a wide range of conditions, including cardiovascular diseases. However, successful homocysteine-lowering treatment by vitamins (B6, B12, and folate) failed to improve these conditions. Long-term increased polyamine intake elevated blood spermine levels and inhibited aging-associated pathologies in mice and humans. Spermine reversed changes (increased dcSAM, decreased DNMT activity, aberrant DNA methylation, and proinflammatory status) induced by the inhibition of ornithine decarboxylase. The relation between polyamine metabolism, one-carbon metabolism, DNA methylation, and the biological mechanism of spermine-induced lifespan extension is discussed.

Achievable Central Nervous System Concentrations of the Green Tea Catechin EGCG Induce Stress in Glioblastoma Cells in Vitro.

The polyphenolic compounds present in green tea are preventative against cancer in several animal tumor models. However, direct cytotoxic effects on cancer cells have also been reported. In order to determine whether drinking of green tea has chemopreventive or cytotoxic effects on brain cancer cells, we investigated the effect of the major green tea polyphenol EGCG as a pure substance and as tea extract dietary supplement on primary human glioblastoma cell cultures at the CNS-achievable concentration of 100 nM reported in the literature. We compared this with the effect of the cytotoxic concentration of 500 µM determined to be specific for the investigated primary glioblastoma cultures. After treatment with 500 µM EGCG, strong induction of autophagy and apoptosis was observed. Under treatment with 100 nM EGCG, glioblastoma cells proliferated over the entire observation period of 6 days without any detectable signs of cell death. Only within the first 12 h of treatment was increased accumulation of autophagic vacuoles and increased reactive oxygen species production as a stress response demonstrated. Mild forms of stress, such as treatment with 100 nM EGCG, activate different endogenous repair mechanisms to protect cells. Our data imply that drinking of green tea may have chemopreventive effects, but no direct cytotoxic properties.

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.

The Treatment of Gliomas in Adulthood.

BACKGROUND: Gliomas are the most common intrinsic tumors of the brain, with an incidence of 6 per 100 000 persons per year. Recent years have seen marked changes in the diagnosis and treatment of gliomas, with molecular parameters now being an integral part of the diagnostic evaluation. METHODS: This review is based on pertinent articles retrieved by a selective search in PubMed, with special attention to the new WHO glioma classification. RESULTS: The classification of gliomas on the basis of additional molecular parameters enables more accurate prognostication and serves as a basis for therapeutic decision-making and treatment according to precisely specified algorithms. PET scanning with 18F-fluoroethyl tyrosine and 11C-methionine for the measurement of metabolic activity in gliomas has further refined the diagnostic evaluation. The median overall survival of patients with glioblastoma who have undergone resection of all tumor tissue with a disrupted blood-brain barrier (i.e., all contrast-enhancing tumor tissue) has been prolonged to up to 20 months. The 5-year survival of patients with WHO grade II gliomas is now as high as 97% after near-total resection. The surgical resection of all contrast-enhancing tumor tissue and subsequent radiotherapy and chemotherapy remain the key elements of treatment. New surgical strategies and new methods of planning radiotherapy have made these techniques safer and more effective. The percutaneous application of tumor-treating fields is a new therapeutic option that has gained a degree of acceptance. Accompanying measures such as psycho-oncology and palliative care are very important for patients and should be considered mandatory. CONCLUSION: The consistent application of the existing multimodal treatment options for glioma has led in recent years to improved survival. Areas of important current and future scientific activity include immunotherapy and targeted and combined chemotherapy, as well as altered neurocognition, modern approaches to palliative care, and complementary therapies.