Methyltransferase Research Articles

Covalent 18F-Radiotracers for SNAPTag: A New Toolbox for Reporter Gene Imaging.

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG and [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.

Abstract MP11: Histone Methyl Transferase (suv39h1): Function In Smooth Muscle Cell Phenotypic Plasticity

Introduction: The phenotypic plasticity of vascular smooth muscle cells (VSMCs) is central to growth and remodeling processes, but also contributes to cardiovascular disease. This unique ability of VSMCs to reversibly differentiate and de-differentiate is incompletely understood. SUV39H1 is a histone methyltransferase that specifically trimethylates Lysine-9 of histone H3 (H3K9me3), resulting in transcriptional repression through epigenetic gene silencing. Hypothesis: We hypothesized that SUV39H1 may play a role in SMC phenotypic switch. Methods: Using in vitro and in vivo approaches including knockdown, qPCR, western, and chromatin immunoprecipitation (ChIP) assays to determine role of SUV39H1 in SMC plasticity. Results: A qPCR array screen of epigenetic regulators in VSMCs identified SUV39H1 mRNA as upregulated with PDGF induced dedifferentiation and downregulated with rapamycin induced differentiation. This was confirmed at the protein level. SUV39H1 knockdown significantly increased VSMC contractile protein mRNA, protein levels and decreased dedifferentiation associated gene expression, and also decreased PDGF-induced VSMC migration (n=4). Interestingly, we found that expression of KLF4, the master transcriptional regulator of dedifferentiation in SMCs, was dramatically decreased after SUV39H1 knockdown (n=5). We further noted that SUV39H1 knockdown decreased KLF4 mRNA stability (n=3). Mechanistically, SUV39H1 knockdown increased miRNA143, a well-known repressor of KLF4. ChIP assays at contractile gene promoters showed a significant decrease in H3K9me3 mark and an increase in H3K27 acetylation, an activation mark (n=4). Carotid artery ligation induced intimal hyperplastic lesions in wild type C57BL/6 mice, showed a significant increase in SUV39H1 and H3K9me3 expression compared to uninjured vessels. Conclusion: We identify SUV39H1 an epigenetic regulator of VSMC phenotype whose expression and activity increase with dedifferentiation in vitro and in vivo . Mechanistically, SUV39H1 influences VSMC chromatin marks and KLF4 expression to repress contractile phenotype. Understanding the role of SUV39H1 and its targets may have implications for developing new therapeutic strategies for treating vascular diseases.

Prenatal exposure to toxic and essential metal/metalloid mixtures is associated with placental genomic signatures

BACKGROUND AND AIM: Prenatal exposure to toxic metals (and metalloids) is linked to numerous adverse perinatal and later-in-life outcomes. During pregnancy, metals pass through the placental barrier to reach the developing fetus. In doing so, metals may alter placental functioning; a potential mechanism underlying adverse health outcomes. In this study, we evaluated placental gene expression in relation to cord tissue concentrations of metals/metalloids, individually and in mixtures. METHODS: Matched samples of placenta and cord tissue were collected in the ELGAN cohort (n=251), which enrolled infants delivered 28 weeks’ gestation between 2002-2004. Genome-wide RNA-sequencing of placentas quantified expression levels of 37,000 mRNA transcripts. Concentrations of 8 metals (barium, cadmium, copper, lead, manganese, mercury, strontium, zinc) and 3 metalloids (arsenic, antimony, selenium) were measured in cord tissue using inductively coupled plasma tandem-mass spectrometry. Negative binomial generalized linear models were fit within the DeSeq2 R package to identify differentially expressed genes (false discovery rate 0.1) with respect to each metal/metalloid. To assess metal mixtures, principal components analysis was also conducted. RESULTS:Individually, lead, mercury and manganese were associated with the strongest placental genomic responses, with 181, 64 and 55 differentially expressed genes, respectively. In contrast, arsenic, cadmium, antimony, selenium and strontium individually did not display associations with gene expression levels. However, in a mixtures-based analysis, the principal component that was loaded primarily with arsenic, mercury and manganese was associated with 569 differentially expressed genes. The gene that displayed the most significant association with the metals mixture was DNA Methyl Transferase 1 (DNMT1). CONCLUSIONS:By combining environmental mixtures and high-dimensional -OMICs approaches, we elucidated target genes and potential molecular pathways within the placenta altered by metal/metalloid-exposure. Lead, mercury and manganese were most strongly associated with distinct placental molecular signatures. A multi-metal analysis highlighted a critical gene involved in epigenetic patterning in the placenta. KEYWORDS: Mixtures, Molecular epidemiology, Heavy metals, Pregnancy outcomes, Birth outcomes

Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3

BackgroundIntervertebral disc degeneration (IVDD) is the breakdown of the discs supporting the vertebrae. It is one of the most frequent causes of back pain worldwide. Currently, the clinical interventions for IVDD are mainly focused on symptom releases. Thus, new therapeutic options are needed.MethodsNucleus pulposus (NP) samples were obtained from 20 patients experiencing IVDD and 10 healthy volunteers compared for mRNA N6-methyladenosine (m6A) mRNA modification as well as methyltransferase (METT) like METTL3, METTL14, and Wilms’ tumor 1-associated protein mRNA and protein abundance following exosomes exposure from mesenchymal stem cells. In addition, microRNA expressions were also compared. The correlation between the NLR family pyrin domain containing 3 (NLRP3) and METTL14 was measured by luciferase reporter assay. Cytokines were evaluated using an enzyme-linked immunosorbent assay. METTL14, NLRP3, and insulin-like growth factor 2 mRNA-binding protein 2 mRNAs were measured via a quantitative reverse transcription-polymerase chain reaction. Protein was assayed using western blots. Cell death was assessed by propidium iodide staining, lactate dehydrogenase release, gasdermin-N domain abundance, and caspase-1 activation.ResultsThe human umbilical cord mesenchymal stem cell (hucMSC) exosomes were found to effectively improve the viability of NP cells and protect them from pyroptosis through targeting METTL14, with a methyltransferase catalyzing m6A modification. METTL14 was highly present in NP cells from IVDD patients, which stabilize NLRP3 mRNA in an IGFBP2-dependent manner. The elevated NLRP3 levels result in the increase of interleukin 1β (IL-1β) and IL-18 levels and trigger pyroptotic NP cell death. Such pathogenic axis could be blocked by hucMSC exosomes, which directly degrade METTL14 through exosomal miR-26a-5p.ConclusionsThe results of the current study revealed the beneficial effects of hucMSC exosomes on NP cells and determined a potential mechanism inducing IVDD.

Expanding the Phenotype of TRMT10A Mutations: Case Report and a Review of the Existing Cases

The tRNA methyltransferase 10 homologue A (TRMT10A) gene encodes tRNA methyl transferase, and biallelic loss of function mutations cause a recessive syndrome of intellectual disability, microcephaly, short stature and diabetes. A case with intellectual disability and distinctive features including microcephaly was admitted. She was diagnosed with epilepsy at 2.5 years old. At 3.6 years of age, severe short stature related to growth hormone (GH) deficiency was detected. She had an incidental diagnosis of diabetes at age 11.4 years which was negative for diabetes antibodies with persistent C-peptide level and she was treated with metformin. Spontaneous puberty did not begin until 15.7 years of age and she was found to have primary ovarian failure. A homozygous p.Arg127* mutation in TRMT10A was detected. In addition to the typical clinical features which characterize TRMT10A syndrome, we observed an unusual form of impaired glucose metabolism which presented in early childhood with hypoglycemia followed by diabetes in late childhood. GH deficiency and primary ovarian failure may also be additional findings of this syndrome. Patients with slow onset diabetes who are negative for auto-antibodies and have extra-pancreatic features should be tested for all known subtypes of monogenic diabetes.

Expression Pattern and Clinical Value of Key m6A RNA Modification Regulators in Abdominal Aortic Aneurysm.

BackgroundAberrant expression of N6-methyladenosine (m6A) RNA modification regulators plays a critical role in a variety of human diseases. However, their implication in abdominal aortic aneurysm (AAA) remains largely unknown. Herein, we sought to explore the general expression pattern and potential functions of m6A regulators in AAA.MethodsWe analyzed gene expression data of m6A regulators in human AAA and normal tissues from public GEO database. The R package and other tools such as m6A2Target database, Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, gene set variation analysis (GSVA), Search Tool for the Retrieval of Interacting Genes (STRING), starBase, miRDB and Cytoscape software were applied for bioinformatics analysis to investigate the downstream molecular mechanisms and upstream regulatory mechanisms for distinctly expressed regulators. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to validate the expression of key m6A regulators in our collected human AAA specimens.ResultsWe found that METTL14 and HNRNPC were the downregulated m6A regulators, and RBM15B was the upregulated methylation transferase in human AAA. The modified genes were primarily enriched in RNA catabolic process, regulation of translation, focal adhesion, transcription coregulator activity, ribosome, RNA transport, cell cycle, et al. METTL14, HNRNPC and RBM15B levels were correlated with the immune infiltration degree of Tcm, macrophages, mast cells, Tgd and NK CD56bright cells. A total of 154 and 76 target genes of three regulators were separately involved in body metabolism and autophagy in AAA disease, and their interactive relationships and hub genes were identified. The lncRNA-miRNA-mRNA interaction regulatory networks were also constructed for METTL14, HNRNPC and RBM15B. Based on our clinical tissue and serum samples, METTL14 exhibited lower expression levels in AAA and its rupture type, and low METTL14 expression was associated with high levels of WBC and CRP (all P < 0.05).ConclusionOur study presents an overview of the expression pattern and functional significance of m6A regulators in human AAA. Our findings will provide a valuable resource that may guide both mechanistic and therapeutic analyses about the role of key m6A regulators in AAA.

Molecular docking, drug likeliness and in silico ADMET study of bioactive compounds against DNA methyltransferase

There is no proper cure for cancer even after so many years and with every progressing year as the drugs are getting more resistant and toxic, chances for recovering from cancer reduces. This present work represents the potential role of active natural metabolites present in common spices as proposed anticancer molecule. We report molecular docking of ferulic acid, crocetin, cinamaldehyde, fernesol and alpha tumerone against cancer target enzyme, DNA methyl transferase to determine their strong binding affinity, studied various others in silico properties like the drug likeliness behavior and absorption, distribution, metabolism, elimination and toxicity (ADMET) behavior. Our preliminary study reveals that all the five natural metabolites have the potential to behave like drugs, follows Lipinski’s rule of 5 and are non-carcinogenic. These bioactive compounds shows better solubility, good absorption and permeability and are non-toxic to mammalian cell Molecular docking shows crocetin and alpha tumerone to be a good inhibitor for DNA methyl transferase with a high total binding energy of −44.84 K cal/mol and −41.44 Kcal/mol respectively. Asn 779, Gln 188, Glu 303, Tyr 681, His 677, Glu 593 and Arg 840 of the target enzyme are involved in the hydrogen bond formation with all the five compounds. Our initial finding concludes that all the five bioactive compounds have the potential for proposing them as anticancer drugs.

HDAC6 Substrate Discovery Using Proteomics-Based Substrate Trapping: HDAC6 Deacetylates PRMT5 to Influence Methyltransferase Activity

Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.

Effect of DNA Methylation on LPS-Induced Expression of Tumour Necrosis Factor Alpha (TNF-α) in Bovine Mammary Epithelial Cells

Background: Cow mastitis is a major disease that affects dairy industry worldwide. Although the inflammatory response induced by lipopolysaccharide (LPS) in bovine mammary epithelial cells (BMECs) is similar to the response to pathogenic bacteria, the underlying regulatory mechanisms remain unclear. This study aimed to clarify the effect of DNA methylation on LPS-induced expression of tumour necrosis factor alpha (TNF-α) in BMECs. Methods: The mammary epithelial cells were treated with LPS and DNA methylation inhibition 5-Aza-2’-deoxycytodine (5Aza). Expression of TNF-α, IL-6, BNBD-5, DNA methyltransferases (DNMT1, DNMT2, DNMT3A and DNMT3B) and DNA methylation at TNF-α regions, were analyzed using quantitative realtime PCR (qRT-PCR), Elisa and bisulfite sequencing PCR (BSP). Result: Our results showed that LPS significantly increased the expression of inflammatory factors, including interleukin-6 (IL-6), bovine neutrophil beta-defensins (BNBD-5) and TNF-α. Further, we observed that the DNA methylation inhibitor, 5-Aza-2'-deoxycytosine (5-Aza), enhanced LPS-induced TNF-α mRNA expression. In addition, we found that LPS treatment significantly decreased the methylation levels of specific CpG sites in the TNF-α promoter (at -245 and -323) and inhibited the expression of DNA methyl transferases (DNMT1, DNMT2, DNMT3A and DNMT3B). Our results indicate that LPS promotes the expression of TNF-α in BMECs by inhibiting DNA methylation in the gene promoter region.

Development of Maize Hybrids With Enhanced Vitamin-E, Vitamin-A, Lysine, and Tryptophan Through Molecular Breeding.

Malnutrition is a widespread problem that affects human health, society, and the economy. Traditional maize that serves as an important source of human nutrition is deficient in vitamin-E, vitamin-A, lysine, and tryptophan. Here, favorable alleles of vte4 (α-tocopherol methyl transferase), crtRB1 (β-carotene hydroxylase), lcyE (lycopene ε-cyclase), and o2 (opaque2) genes were combined in parental lines of four popular hybrids using marker-assisted selection (MAS). BC1F1, BC2F1, and BC2F2 populations were genotyped using gene-based markers of vte4, crtRB1, lcyE, and o2. Background selection using 81–103 simple sequence repeats (SSRs) markers led to the recovery of recurrent parent genome (RPG) up to 95.45%. Alpha (α)-tocopherol was significantly enhanced among introgressed progenies (16.13 μg/g) as compared to original inbreds (7.90 μg/g). Provitamin-A (proA) (10.42 μg/g), lysine (0.352%), and tryptophan (0.086%) were also high in the introgressed progenies. The reconstituted hybrids showed a 2-fold enhancement in α-tocopherol (16.83 μg/g) over original hybrids (8.06 μg/g). Improved hybrids also possessed high proA (11.48 μg/g), lysine (0.367%), and tryptophan (0.084%) when compared with traditional hybrids. The reconstituted hybrids recorded the mean grain yield of 8,066 kg/ha, which was at par with original hybrids (mean: 7,846 kg/ha). The MAS-derived genotypes resembled their corresponding original hybrids for the majority of agronomic and yield-related traits, besides characteristics related to distinctness, uniformity, and stability (DUS). This is the first report for the development of maize with enhanced vitamin-E, vitamin-A, lysine, and tryptophan.

Survival Prediction Analysis in Glioblastoma With Diffusion Kurtosis Imaging.

Simple SummaryGlioblastoma (GBM) is the most common and aggressive primary brain tumor. Diffusion kurtosis imaging (DKI) has characterized non-Gaussian diffusion behaviors in brain normal tissue and gliomas, but there are very limited efforts in investigating treatment responses of kurtosis in GBM. This study aimed to investigate whether any parameter derived from the DKI is a significant predictor of overall survival (OS). We found that the large mean, 80 and 90 percentile kurtosis values in the contrast enhanced gross tumor volume (Gd-GTV) on post-Gd T1-weighted images pre-RT were significantly associated with reduced OS. In the multivariate Cox model, the mean kurtosis Gd-GTV pre-RT after considering effects of age, extent of surgery, and methylation were significant predictors of OS. In addition, the 80 and 90 percentile kurtosis values in Gd-GTV post RT were significantly associated with progression free survival (PFS). The DKI model demonstrates the potential to predict outcomes in the patients with GBM.PurposeNon-Gaussian diffusion behaviors in gliomas have been characterized by diffusion kurtosis imaging (DKI). But there are very limited efforts in investigating the kurtosis in glioblastoma (GBM) and its prognostic and predictive values. This study aimed to investigate whether any of the diffusion kurtosis parameters derived from DKI is a significant predictor of overall survival.Methods and MaterialsThirty-three patients with GBM had pre-radiation therapy (RT) and mid-RT diffusion weighted (DW) images. Kurtosis and diffusion coefficient (DC) values in the contrast enhanced gross tumor volume (Gd-GTV) on post-Gd T1 weighted images pre-RT and mid-RT were calculated. Univariate and multivariate Cox models were used to evaluate the DKI parameters and clinical factors for prediction of OS and PFS.ResultsThe large mean kurtosis values in the Gd-GTV pre-RT were significantly associated with reduced OS (p = 0.02), but the values at mid-RT were not (p > 0.8). In the multivariate Cox model, the mean kurtosis in the Gd-GTV pre-RT (p = 0.009) was still a significant predictor of OS after adjusting effects of age, O6-Methylguanine-DNA Methyl transferase (MGMT) methylation and extent of resection. In Gd-GTV post-RT, 80 and 90 percentile kurtosis values were significant predictors (p ≤ 0.05) for progression free survival (PFS).ConclusionThe DKI model demonstrates the potential to predict OS and PFS in the patients with GBM. Further development and histopathological validation of the DKI model will warrant its role in clinical management of GBM.

Selectively targeting cancer stem cells: Current and novel therapeutic strategies and approaches in the effective eradication of cancer.

Cancer stem cells (CSCs) are a subgroup of cells in malignant cancers, which possess self-renewal capacity, tumor-initiating capability, and pluripotency, as well as being responsible for tumor maintenance, metastasis, relapse, and chemoresistance. The treatment modalities previously established for cancer included surgery, chemotherapy, and radiotherapy. The majority of tumor cells of non-CSCs could be eradicated using conventional chemotherapy and radiotherapy. Therefore, novel and promising therapeutic strategies that selectively target CSCs are of great importance. In this review, we described different therapeutic strategies such as immunotherapy, metabolism-based therapeutic strategies, and additional potential therapeutic approaches (targeting microRNAs [miRNAs], histone deacetylase, and DNA methyl transferase) against CSCs. Taken together, due to the inefficiency of anticancer single therapies, targeting CSCs through their metabolism and using immunotherapy and miRNAs besides classical chemo- and radiotherapy may exert better therapeutic effects.

Identification of Novel Variants in Cleft Palate-Associated Genes in Brazilian Patients With Non-syndromic Cleft Palate Only

The identification of genetic risk factors for non-syndromic oral clefts is of great importance for better understanding the biological processes related to this heterogeneous and complex group of diseases. Herein we applied whole-exome sequencing to identify potential variants related to non-syndromic cleft palate only (NSCPO) in the multiethnic Brazilian population. Thirty NSCPO samples and 30 sex- and genetic ancestry-matched healthy controls were pooled (3 pools with 10 samples for each group) and subjected to whole-exome sequencing. After filtering, the functional affects, individually and through interactions, of the selected variants and genes were assessed by bioinformatic analyses. As a group, 399 variants in 216 genes related to palatogenesis/cleft palate, corresponding to 6.43%, were exclusively identified in the NSCPO pools. Among those genes are 99 associated with syndromes displaying cleft palate in their clinical spectrum and 92 previously related to cleft lip palate. The most significantly biological processes and pathways overrepresented in the NSCPO-identified genes were associated with the folic acid metabolism, highlighting the interaction between LDL receptor-related protein 6 (LRP6) and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) that interconnect two large networks. This study yields novel data on characterization of specific variants and complex processes and pathways related to NSCPO, including many variants in genes of the folate/homocysteine pathway, and confirms that variants in genes related to syndromic cleft palate and cleft lip-palate may cause NSCPO.

Effects of Selenium-enriched probiotics on ochratoxin A-induced kidney injury and DNMTs expressions in piglets

Effects of Selenium-enriched probiotics (SP) on ochratoxin A-induced kidney injury, growth performance, antioxidant injury, selenoprotein and DNA methylation transferases (DNMTs) expression of piglets were investigated in the article. A total of 48 piglets were randomly divided into 4 groups and fed with basal diet (Con, 0.15 mg Se/kg and OTA at 0.00 mg/kg), basal diets added with OTA (OTA, 0.40 mg OTA/kg), SP and OTA (SP1, 0.15 mg Se/kg and 0.40 mg OTA/kg), SP and OTA (SP2, 0.30 mg Se/kg and 0.40 mg OTA/kg) respectively for 42 days. From each group, six piglets were randomly selected for blood collection on Days 0 and 42 and three piglets were selected for tissue collection on Day 42.The results showed that OTA at 0.40 mg /kg significantly decreased growth performance of pigs, induced the histopathological lesions of kidney and increased urea and creatine levels of serum, decreased GPx and SOD activities, and increased MDA levels. OTA decreased GPx1, GPx4 and SelS expressions, and increased TR1, DNMT 1, DNMT3a and SOCS3 expressions. Both SP1 and SP2 improved OTA-induced poor growth performance, kidney injury, poor antioxidant statues, GPx1, SelS, TR1, SOCS3, DNMT1 and DNMT3a expressions in kidney of pigs. The effects of SP2 on the above parameters changes were better than that of SP1. SP increased GPx and SOD activities and decreased MDA levels changes induced by OTA treatment. These results suggest that SP may serve as a better feed additive for piglets under mycotoxin contamination environments.

Abstract 990: Differential immunoregulatory effects of decitabine on lipid metabolism in doxurobicin versus tamoxifen treated breast cancer cells

Abstract Background: The treatment of hormonal positive breast cancer is still based on hormonal tamoxifen (TAM) based regimens in early stages, while based on chemotherapeutic doxorubicin (DOX) based regimens in late stages. DNA methyl transferase inhibitor (decitabine: DEC) is an epigenetic modulator with synergistic anticancer activity when combined with either TAM or DOX. Aims: The cytotoxic synergistic combinations of DEC with either DOX or TAM were examined for their effects on immunomodulation and lipid metabolism in hormonal positive breast cancer cells (MCF7). Material and Methods: Sulphorhodamine-B cytotoxic assay, flow cytometric analysis of cell cycle and apoptosis along with the florescent uptake of Rhodamine123 were adopted for testing the sensitivity of MCF7 cells to the drug combinations. Real time PCR was utilized to measure the genetic expression of immunoregulatory markers (FOXP3, TNFα, IFNγ and IL-6), lipid metabolism regulatory markers (FASN, CPT1, MLYCD, ACCα and AMPK) and apoptosis markers (BAX, BCL2, Caspase-8, AKT, mTOR). Western Blotting was used for the determination of EGFR, CK7, LC3B proteins expressions. Results: The combination with 500nM DEC caused synergistic 50% growth inhibition of MCF7 cells, in which the concentration required for DOX was reduced from 0.501 µM into 0.264µM, while in TAM, the concentration was reduced from 18µM into 12.4µM. The concentration of 500nM DEC induced the uptake of Rhodamine 123 and the expression of Caspase-8, IFNγ, IL-6 and AMPK in the combination with 20µM TAM or 0.25µM DOX compared with their respective single treatment concentrations. Also, both DEC combinations reduced the expressions of BAX/BCl2, EGFR, CK7, aromatase, LC3B and ACCα, but caused insignificant effect on FASN expression. The combination of 500nM DEC with 20µM TAM demonstrated significant accumulation of cells in the G0/G1 phase of the cell cycle (4.22%), and in the early apoptosis (8.97%), along with the increase of mTOR but decrease of CPT1A, MLYCD, TNFα genes expressions. On the other hand, the combination of 500nM DEC with 0.25µM DOX caused accumulation of 94.73% of cells in the late apoptosis, inhibition of the AKT and FOXP3 expressions, but induction of CPT1A. Conclusions: Epigenetic modifier (DEC) showed different synergistic immunomodulatory effects on lipid metabolism regulation markers when combined with chemotherapy (DOX) rather than hormonal therapy (TAM) in hormonal positive breast cancer cells. DEC induces extrinsic late apoptosis through the induction of CPT1A- mediated lipid metabolism along with FOXP3 and AKT inhibition in DOX treated cells, while it induces extrinsic early apoptosis through the inhibition of MLYCD, CPT1A and TNFα in TAM treated cells. Citation Format: Mariam Fouad, Mohammed Sayed-Ahmed. Differential immunoregulatory effects of decitabine on lipid metabolism in doxurobicin versus tamoxifen treated breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 990.

Abstract 1431: Role of emt biomarkers in mediating osimertinib resistance in non-small cell lung cancer

Abstract Osimeritinib (OR), a Tyrosine Kinase Inhibitor is a first-line therapy in EGFR-mutant NSCLC patients. Resistance to OR treatment may occur due to acquired C797S mutations, MET amplifications and ALK rearrangements and other mechanisms not clearly defined. EMT may be one of the reasons for TKI resistance. EMT is a process by which epithelial cells acquire mesenchymal characteristics that increase invasiveness. We hypothesize that loss of E-cadherin during EMT, results in abnormal localization of p120 catenin, an E-cadherin regulating protein, and Kaiso factor, a repressor of Wnt signaling, promoting oncogenesis. Protein arginine methyl transferase 1 (PRMT1) an EMT regulator catalyzes methylation of Twist1, which represses E-cadherin. However, their role in OR resistance is still unknown. We have made parental (P) NSCLC cells, H2170, H358, H1975 and H3255 resistant to OR. MTT assays were performed on the parental cells and OR resistant cells which showed that the H2170OR, H3255OR, H358OR, H1975OR cells had IC50 for OR, 2.8-14 fold higher in comparison to parental cells, respectively. We further performed qPCR to study the modulation of EMT biomarkers in OR cells as compared to parental cells and found upregulation of PRMT1, p120 catenin and Kaiso factor by 1.5-3.67 fold respectively in H1975 cells and 1.5-5.73 fold in H3255 cells. Immunofluorescence studies in H1975OR cells for expression of PRMT1 showed that there was 3.18 fold increase as compared to 2.21 fold increase in Erlotinib resistant H1975 (H1975ER) and 4.77 fold increase in H3255OR as compared to 2.27 fold increase in H3255ER. After transfection with p120 siRNA we found that OR efficacy was increased by 33% in comparison to mock siRNA and OR treated cells. The percentage wound closure after 24 hours was found to be 26.2% in the p120-catenin siRNA transfected cells as compared to 7.4%.in mock siRNA transfected cells (p&amp;lt;0.05) indicating its role in EMT and cell migration. Furthermore, our results showed increased expression of p120 catenin in smokers with 50% of smokers showing high expression compared to 0% and 5.8% of quit smokers and nonsmokers indicating that high p-120 expression was associated with smoking (P=0.002). In conclusion, biomarkers like PRMT1 may mediate the process of EMT by methylation of Twist1 and upregulation of p120 that prevents transcriptional repression of Kaiso factor target genes, promoting EMT in OR and ER resistant cells. p-120 expression was found to be higher in smokers compared to quit/nonsmokers indicating that it may have a potential role in lung cancer. Citation Format: Kavya Sri Racherla, Katrina Dovalovsky, Neelu Puri. Role of emt biomarkers in mediating osimertinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1431.

Cost-effectiveness analysis of genotype screening and therapeutic drug monitoring in patients with inflammatory bowel disease treated with azathioprine therapy: a Chinese healthcare perspective using real-world data.

BackgroundThis study aimed to analyze the cost-effectiveness of combining screening for thiopurine methyl transferase (TPMT) and nucleotide triphosphate diphosphatase (NUDT15) defective alleles with therapeutic drug monitoring (TDM) in Chinese patients with inflammatory bowel disease (IBD) treated with azathioprine (AZA).MethodsWe evaluated the cost-effectiveness of combining screening for NUDT15 and TPMT deficiency with TDM in patients receiving AZA treatment over a 1-year horizon by developing a decision tree model. Real-world data and published literature were used to derive model inputs. The model’s primary outcomes included quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs). One-way and probabilistic sensitivity analyses were used to address uncertainty.ResultsCompared to NUDT15 genotyping, the combined TPMT/NUDT15 genotyping strategy cost an additional $13.83, yielding an ICER of $3,929.54/QALY, which was under the willingness-to-pay level of $30,425 per QALY in China. Compared to strategies with singular TPMT genotyping or no genotyping, the combined TPMT/NUDT15 genotyping strategy gained 0.00406 and 0.00782 QALYs and reduced the cost by $25.15 and $99.06, respectively. Additionally, incorporating TDM of AZA was more effective and less expensive than strategies without TDM. One-way sensitivity analysis revealed the expense attached to severe myelotoxicity to be the factor with the greatest influence in the present research. The application of the combined genotype screening strategy with TDM of AZA treatment was found to have a 91.7% chance of being cost-effective.ConclusionsFor Chinese patients with IBD who receive an AZA regimen, a strategy involving combined NUDT15/TPMT genotype screening prior to treatment initiation and incorporating TDM for treatment management is cost-effective compared to strategies involving genotyping of NUDT15 or TPMT alone or genotyping without TDM.

XSP10 and SlSAMT, Fusarium wilt disease responsive genes of tomato (Solanum lycopersicum L.) express tissue specifically and interact with each other at cytoplasm in vivo.

The online version contains supplementary material available at 10.1007/s12298-021-01025-y.

Application of methyltransferases in microbial synthesis of natural products

Methyltransferases (MTs) constitute a large group of enzymes that catalyze the transfer of a methyl moiety, most frequently from S-adenosyl-L-methionine, to their substrates. It plays an essential role in regulation of gene expression and synthesis of many natural compounds. Owing to its broad substrate spectrum, MTs make important contributions to diversify the spectrum of products through methylation modifications. Recently, great progress has been made in application of MTs for the biosynthesis of various natural products including phenylpropane compounds, fragrances, hormones and antibiotics, which is summarized in this review. Moreover, we highlighted the strategies of using MTs for efficient production and for expanding the diversity of these methylated natural products, and discussed the current challenges and future prospects in this area.

Development and Characterization of the Novel Orally Bioavailable EZH1/2 Dual Inhibitor DS-3201

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27) and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 function as catalytic subunits of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Many reports suggest that inhibiting PRC2 activity through EZH2 knockdown gives rise to anti-cancer effect against hematological and solid cancers.To date, some EZH2 selective inhibitors have demonstrated anti-cancer potential in preclinical and clinical studies. In spite of this promise, there is concern that the efficacy anticipated with EZH2 selective inhibitors may be compromised due to compensation by EZH1 activity.Herein, we describe the novel, orally bioavailable EZH1/2 dual inhibitor DS-3201. This compound strongly and selectively inhibited the methyltransferase activity of both EZH1 and EZH2 with an IC50 value of 10 nM and 6.0 nM, respectively in cell-free enzymatic assays. This compound also suppressed trimethylation of H3K27 in cells (IC50: 0.55 nM) more potently than EZH2 selective inhibitors.In order to identify the anti-cancer spectrum of DS-3201, we tested its in vitro growth inhibition activity against a panel of cancer cell lines including a variety of hematological cancers. Among the panel, hematological cell lines showed particular sensitivity to DS-3201, with GI50 values less than 100 nM, including 1) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines harboring fusion genes, such as MLL-AF9, MLL-AF4, or AML1-ETO, 2) diffuse large B-cell lymphoma (DLBCL) cell lines having EZH2 gain-of-function (GOF) mutations, 3) most of the peripheral T-cell lymphoma and multiple myeloma cell lines tested. Our data thus shows this compound has broader anti-cancer activity than EZH2 selective inhibitors, especially in hematology cancers. We also evaluated the in vivo anti-tumor efficacy of DS-3201 against KARPAS-422, a DLBCL tumor xenograft with an EZH2 GOF mutation. A 100 mg/kg once daily oral administration of DS-3201 caused nearly complete tumor regression without any body weight loss and 25 mg/kg administration also showed clear tumor growth delay.Based on these results, we initiated clinical studies of DS-3201 for patients with lymphoma (Clinical trial information: NCT02732275) and for those with AML and ALL (NCT03110354). DisclosuresHonma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Kanno:Asubio Pharma Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Hirasawa:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Takizawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Yasumatsu:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Horiuchi:Daiichi Sankyo Co., Ltd.: Employment. Nakao:Daiichi Sankyo Co., Ltd.: Employment. Suzuki:Daiichi Sankyo Co., Ltd.: Employment. Yamasaki:Daiichi Sankyo Co., Ltd.: Employment. Nakajima:Daiichi Sankyo Co., Ltd.: Employment. Hayakawa:Daiichi Sankyo Co., Ltd.: Employment. Yamazaki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo Co., Ltd.: Employment.