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.

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.

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.

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.

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.

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.

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.

Mechanisms of anti-cancer effects of ascorbate: Cytotoxic activity and epigenetic modulation.

Vitamin C (Vit C or Ascorbate) is essential for many fundamental biochemical processes. Vit C is an essential nutrient with redox functions at normal physiologic concentrations. The main physiologic function of this vitamin is related to its capacity to act as a co-factor for a large family of enzymes, collectively known as Fe and 2-oxoglutarate-dependent dioxygenases. It also modulates epigenetic gene expression through the control of TET enzymes activity. Vit C also has several biological properties allowing to restore the deregulated epigenetic response observed in many tumors. High-dose Vit C has been investigated as a treatment for cancer patients since the 1969. Pharmacologic ascorbate acts as a pro-drug for hydrogen peroxide formation (H2O2) and, through this mechanism, kills cancer cells. To achieve high in vivo concentrations, Ascorbate must be injected by i.v. route. Initial clinical studies of Ascorbate cancer treatment have provided encouraging results, not confirmed in subsequent studies. Recent clinical studies using i.v. injection of high-dose Ascorbate have renewed the interest in the field, showing that significant anti-tumor activity. Pre-clinical studies have led to identify tumors sensitive to Ascorbate that could potentially benefit from this treatment either through an epigenetic modulator effect or through tumor killing by oxidative stress.

Iodine excess did not affect the global DNA methylation status and DNA methyltransferase expression in T and B lymphocytes from NOD.H-2h4 and Kunming mice.

Dysregulated DNA methylation in lymphocytes has been linked to various autoimmune disorders. Excessive iodine intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT) flares in humans and animals. However, whether excessive iodine modifies the DNA methylation status in lymphocytes is unknown. Twenty NOD.H-2h4 mice and 20 Kunming mice were randomly divided into high iodine and control groups. We scored lymphatic infiltration in the thyroid by hematoxylin and eosin (H&E) staining and assayed serum thyroglobulin antibody (TgAb) levels by an indirect enzyme-linked immunosorbent assay. CD3+ T cells and CD19+ B cells were separated by flow cytometry. Global DNA methylation levels were examined by absorptiometry. Methylation of long interspersed nucleotide element-1 (LINE-1) repeats was detected with bisulfite sequencing PCR. Expression of DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b mRNA and protein were determined by real-time PCR and Western blot, respectively. We observed evident thyroiditis in the high­iodine-treated NOD.H-2h4 mice, while mice in the other three groups did not develop thyroiditis. No differences were found in the global methylation levels and methylation status of LINE-1 repeats in T and B lymphocytes from high­iodine-treated NOD.H-2h4 mice and Kunming mice compared with those from normal­iodine-supplemented controls. We did not find obvious changes in DNMT mRNA and protein expression levels in T and B lymphocytes among the studied groups. In conclusion, we showed for the first time that excess iodine did not affect the global methylation status or DNMT expression in T and B lymphocytes in NOD.H-2h4 and Kunming mice.

Selenium and Epigenetics in Cancer: Focus on DNA Methylation.

Chemopreventive activity of selenium (Se) may influence epigenome. In this review, we have discussed two aspects of Se and epigenetics in cancer, related to (1) the association between Se and epigenetic regulation in cancer development and prevention; (2) epigenetic modification of selenoprotein-encoding genes in different cancers. In both issues, we focused on DNA methylation as the most investigated epigenetic mechanism. The existing evidence from experimental data in human cancer cell lines, rodents, and human studies in cancer-free subjects indicates that: high Se exposure leads to the inhibition of DNA methyltransferase expression/activity; the association between Se and global methylation remains unclear and requires further investigation with respect to the underlying mechanisms and possible nonlinear character of this relationship; Se affects methylation of specific tumor suppressor genes, possibly in a sex-dependent manner; and cancer phenotype is often characterized by altered methylation of selenoprotein-encoding genes, mainly glutathione peroxidase 3.

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.

Olea europaea Leaf Extract Improves the Efficacy of Temozolomide Therapy by Inducing MGMT Methylation and Reducing P53 Expression in Glioblastoma.

Unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter leads to Temozolomide (TMZ) resistance in most of the glioblastoma multiforme (GBM) patients. We previously investigated the synergistic effect of Olea europaea leaf extract (OLE) on TMZ cytotoxicity through modulating microRNA expression. To date, knowledge about the effect of OLE on MGMT methylation is insufficient. The aim of the current study was to evaluate the potential modulating effect of OLE on the TMZ response of GBM tumors through MGMT methylation. Exposure to 1 mg/mL OLE caused a significant induction of CpG island methylation in the MGMT gene using Methyl quantitative PCR assay (P < 0.001). In WST-1 analysis, the use of 350 µM TMZ plus 1 mg/mL OLE significantly increased the TMZ response of MGMT unmethylated cells (P = 0.003). Using the comet assay, the impact of 1 mg/mL OLE plus 350 µM TMZ on the formation of DNA strand breaks was significantly higher than that of 450 µM TMZ alone (P < 0.001) and Western blot analysis revealed that, when cells are treated with 1-mg/mL OLE, the total p53 protein levels tended to decrease. The results presented in this study uniquely demonstrated that OLE synergistically increased the TMZ response of GBM tumors by regulating MGMT gene methylation and p53 expression. However, further studies to validate our findings are required.

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.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk.

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear ß-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.

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.