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1.
PLoS One ; 17(8): e0272482, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35917376

RESUMO

DNA methylation regulates epigenetic gene expression in ischemic stroke. Decitabine attenuates ischemic stroke by inhibiting DNA methylation. However, the underlying mechanism of this effect is not known. A model of ischemic stroke in Sprague-Dawley rats was induced through middle cerebral artery occlusion followed by reperfusion step. The rats were randomly treated with decitabine or vehicle by a one-time intraperitoneal injection. Sham rats received similar treatments. Four days after treatment, the rats were perfused with saline or 4% paraformaldehyde after which the brain was excised. DNA methylation level and brain infarct volume were determined by dot blot and histochemistry, respectively. The cellular co-localization and quantitative analysis of DNA methylation were assessed by immunohistochemistry and expression levels of cdkn1b (p27) mRNA and protein were measured by qRT-PCR and immunohistochemistry, respectively. The proliferation of astrocytes and number of neurons were determined by immunohistochemistry. Rats treated with decitabine showed hypomethylation and reduced infarct volume in the cortex. DNA methylation was decreased in astrocytes. Decitabine upregulated p27 mRNA and protein expression levels and attenuated the proliferation of astrocytes in vivo and vitro. Decitabine promotes p27 gene expression possibly by inhibiting its DNA methylation, thereby decreases the proliferation of astrocytes, neuronal death and infarct volume after ischemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Astrócitos/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Proliferação de Células , Decitabina/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo
2.
Biomed Res Int ; 2022: 6066567, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35937396

RESUMO

Cancer-testis (CT) genes are typically expressed in the testes; however, they have been linked to aberrant expression in a variety of malignancies. MAGE-B family genes are an example of CT genes. Therefore, the overarching objective of this study was to examine the expressions of MAGE-B family genes in several patients with colon cancer (CC) to see if they might be employed as cancer biomarkers in the early phases of cancer detection and to improve treatment. In this investigation, RT-PCR was used to analyze MAGE-B family genes in neighboring normal colon (NC) tissue from 10 CC patients. In addition, the effect of DNA demethylation on the expression status of the MAGE-B1 gene was evaluated by RT-PCR in HCT116 and Caco-2 cells and by qRT-PCR for HCT116 only after treating both CC cell lines with varying concentrations of 5-aza-2'-deoxycytidine (1.0, 5.0, and 10.0 µM) for 48 or 72 hours. All MAGE-B family genes except for MAGE-B1 showed weak bands in several samples of NC tissues: MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, and MAGE-B6 genes were observed in 40%, 50%, 40%, 30%, and 60% of the NC samples, respectively. Nonetheless, they had strong bands in multiple samples of CC tissues, with 70%, 90%, 60%, 50%, and 90% of the CC samples, respectively. Interestingly, MAGE-B1 was detected in 60% of CC tissues but not in NC tissues, suggesting that it is a potential biomarker for early CC detection. MAGE-B1 expression was not observed in either untreated or DMSO-treated HCT116 cells after 48 or 72 hours of treatment. However, according to the RT-PCR and qRT-PCR results, the MAGE-B1 gene was overexpressed in the HCT116 cells treated with three different concentrations of 5-aza-2'-deoxycytidine. This shows that demethylation plays a crucial role in MAGE-B1 expression activation.


Assuntos
Antígenos de Neoplasias , Neoplasias do Colo , Antígenos de Neoplasias/genética , Células CACO-2 , Neoplasias do Colo/genética , Decitabina/farmacologia , Humanos , Masculino , Proteínas de Neoplasias/genética
3.
Clin Epigenetics ; 14(1): 89, 2022 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842682

RESUMO

BACKGROUND: Several reports have provided crucial evidence in animal models that epigenetic modifications, such as DNA methylation, may be involved in psychostimulant-induced stable changes at the cellular level in the brain. Epigenetic editors DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs) coordinate expression of gene networks, which then manifest as long-term behavioural changes. However, the extent to which aberrant DNA methylation is involved in the mechanisms of substance use disorder in humans is unclear. We previously demonstrated that cocaine modifies gene transcription, via DNA methylation, throughout the brain and in peripheral blood cells in mice. RESULTS: We treated human peripheral blood mononuclear cells (PBMCs) from healthy male donors (n = 18) in vitro with psychostimulants (amphetamine, cocaine). After treatment, we assessed mRNA levels and enzymatic activities of TETs and DNMTs, conducted genome-wide DNA methylation assays and next-generation sequencing. We found that repeated exposure to psychostimulants decreased mRNA levels and enzymatic activity of TETs and 5-hydroxymethylation levels in PBMCs. These data were in line with observed hyper- and hypomethylation and mRNA expression of marker genes (IL-10, ATP2B4). Additionally, we evaluated whether the effects of cocaine on epigenetic editors (DNMTs and TETs) and cytokines interleukin-6 (IL-6) and IL-10 could be reversed by the DNMT inhibitor decitabine. Indeed, decitabine eliminated cocaine's effect on the activity of TETs and DNMTs and decreased cytokine levels, whereas cocaine increased IL-6 and decreased IL-10. CONCLUSIONS: Our data suggest that repeated psychostimulant exposure decreases TETs' enzymatic activity in PBMCs. Co-treatment with decitabine reversed TETs' levels and modulated immune response after repeated cocaine exposure. Further investigation is needed to clarify if TET could represent a putative biomarker of psychostimulant use and if DNMT inhibition could have therapeutic potential.


Assuntos
Cocaína , Metilação de DNA , Animais , Cocaína/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilases de Modificação do DNA/genética , Decitabina/farmacologia , Humanos , Interleucina-10/genética , Interleucina-6/genética , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , RNA Mensageiro/genética
4.
J Int Med Res ; 50(7): 3000605221112024, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35869624

RESUMO

OBJECTIVE: Combination therapy has become the hallmark of lung cancer treatment, as it reduces the dosage intensity of individual drugs while increasing their efficacy. In the current study, we analyzed the combinatorial effect of decitabine and aspirin on non-small cell lung cancer (NSCLC) cell growth. METHODS: In this study, we investigated the combinatorial effect of decitabine and aspirin by MTT, colony formation, and Transwell assays. We also explored the underlying molecular mechanism via a series of in vitro and in vivo experiments. RESULTS: The combination of decitabine and aspirin regulated cell viability and migration in vitro. Moreover, the combination therapy suppressed tumor cell growth by inhibiting the ß-catenin/STAT3 signaling pathway. Our study also found that the regimen increased the phosphorylation of ß-catenin and decreased the expression of STAT3 and ß-catenin. CONCLUSION: The combined administration of decitabine and aspirin significantly reduced tumor growth compared with single-agent treatment and the control in vivo. The study results indicated that decitabine and aspirin could suppress NSCLC cell growth and metastasis via the ß-catenin/STAT3 signaling pathway.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Aspirina/farmacologia , Aspirina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Decitabina/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
5.
Pathol Res Pract ; 236: 154007, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35810565

RESUMO

Colorectal cancer is known as the third most common cancer in both women and men. Genetic and epigenetic changes are major players contributing to colorectal carcinogenesis. Regulator of G-protein signaling 10 (RGS10) is a member of the RGS proteins, which negatively regulate several signaling pathways including cell survival and proliferation. We and others have previously shown that RGS10 expression is modulated by epigenetic modifications in ovarian cancer and suppression of RGS10 partially contributes to chemoresistance. Here, we further analyzed the roles and regulation of RGS10 in colon adenocarcinoma (COAD), using broad bioinformatics tools. We analyzed the expression profiles, promoter methylation state, prognostic value and effect of a hypomethylating agent on RGS10 expression. Results showed that RGS10 expression is higher in normal colon tissues than in tumor tissues. In addition, there is a negative correlation between DNA methylation and RGS10 transcript expression. We also observed that gene expression and promoter methylation of RGS10 in colorectal carcinoma patients were differently expressed depending on the tumor stage and microsatellite stability. DNA methylation was significantly increased in 18 probes of RGS10, which belongs to the high-risk group in COAD. In addition, pharmacological inhibition of DNA methyltransferase with decitabine reduced the six CpGsite-specific RGS10 hypermethylation in COAD. We also experimentally confirmed that RGS10 promoter activity was inhibited by treatment with decitabine in the HT-29 colorectal cell line. We further showed that decitabine treatment increases the RGS10 transcript expression in three different colorectal carcinoma cell lines. These results suggest that RGS10 expression is suppressed in the development of colorectal cancer and inhibition of DNA methylation may contribute to increasing overall survival rates of COAD patients.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Ovarianas , Proteínas RGS , Adenocarcinoma/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias Colorretais/patologia , Metilação de DNA/genética , Decitabina/metabolismo , Decitabina/farmacologia , Decitabina/uso terapêutico , Feminino , Proteínas de Ligação ao GTP/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Neoplasias Ovarianas/patologia , Proteínas RGS/genética , Proteínas RGS/metabolismo , Taxa de Sobrevida
6.
Med Oncol ; 39(10): 150, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35843988

RESUMO

BACKGROUND: Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. TNBC lacks targeted therapy receptors, rendering endocrine and HER2-targeted therapies ineffective. TNBC is typically treated with cytotoxic chemotherapy followed by surgery. Targeting epigenetic modifications could potentially be a new effective TNBC target therapy. The aim of this study is to examine the effects of epigenetic drugs, decitabine as DNA methyltransferase inhibitor (DNMTI) and vorinostat as histone deacetylase inhibitor (HDACI), and the ERß agonist DPN on ERα and ERß re-expressions in the MDA-MB-231 cells as a model of TNBC. METHODS: Using MTT assay, the IC50 of decitabine, vorinostat, and DPN on MDA-MB-231 cells were determined. The effects of all drugs alone or in combinations on MDA-MB-231 cells were evaluated. qRT-PCR was used to determine ERα & ERß gene expression. Caspase-3 activity and the protein expression levels of VEGF, Cyclin D1, and IGF-1 were assessed. RESULTS: Both ERα and ERß mRNA were re-expressed in different high levels in all treated groups, especially in the triple therapy group compared with control. Significantly, the triple drugs therapy showed the lowest levels of VEGF, Cyclin D1, and IGF-1 and the highest level of Caspase-3 activity, indicating a possible antitumor effect of ERß activation through decreasing proliferation and angiogenesis and increasing apoptosis in MDA-MB-231 cells. CONCLUSIONS: The antiproliferative effect of ERß could be retained when co-expressed with Erα using a powerful epigenetic combination of Decitabine and vorinostat with DPN.


Assuntos
Decitabina , Receptor beta de Estrogênio , Nitrilas , Propionatos , Neoplasias de Mama Triplo Negativas , Vorinostat , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Decitabina/farmacologia , Epigênese Genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , NAD/genética , NAD/metabolismo , NAD/farmacologia , Nitrilas/farmacologia , Propionatos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vorinostat/farmacologia
7.
Cell Rep ; 39(10): 110913, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35675776

RESUMO

An intronic (G4C2)n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G4C2)n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders.


Assuntos
Esclerose Amiotrófica Lateral , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA , Decitabina/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Neurônios/metabolismo , Nucleosídeos/metabolismo , RNA Antissenso/metabolismo
8.
BMC Cancer ; 22(1): 685, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729618

RESUMO

BACKGROUND: Glutathione-S transferases (GSTs) comprise a series of critical enzymes involved in detoxification of endogenous or xenobiotic compounds. Among several GSTs, Glutathione S-transferases mu (GSTM) has been implicated in a number of cancer types. However, the prognostic value and potential functions of the GSTM family genes have not been investigated in lung adenocarcinoma (LUAD). METHODS: We examined the expression of GSTM5 in LUAD and identified associations among GSTM5 expression, clinicopathological features, survival data from the Cancer Genome Atlas (TCGA). The correlation between GSTM5 DNA methylation and its expression was analyzed using the MEXPRESS tool and UCSC Xena browser. The methylation status of GSTM5 in the promoter region in lung cancer cells was measured by methylation-specific PCR (MSP). After 5-aza-2'-deoxycytidine treatment of lung cancer cells, expression of GSTM5, cell proliferation and migration were assessed by RT-PCR, CCK-8 and transwell assays, respectively. RESULTS: The results showed that GSTM5 was abnormally down-regulated in LUAD patients' tissues, and patients with low GSTM5 expression level had significantly shorter OS. Cox regression analyses revealed that GSTM5 was associated with overall survival (OS) of LUAD patients, which expression was an independent prognostic indicator in terms of OS (hazard ratio: 0.848; 95% CI: 0.762-0.945; P = 0.003). In addition, we found the promoter region of GSTM5 was hypermethylated in the tumor tissue compared with adjacent normal tissues, and the average methylation level of GSTM5 were moderately correlated with its expression. Moreover, methylation-specific PCR also showed that the GSTM5 gene promoter was hypermethylated in lung cancer cells, and treatment with 5-Aza-CdR can restore the gene expression and inhibit cell proliferation and migration. Finally, Gene Set Enrichment Analysis (GSEA) revealed that low GSTM5 expression was significantly related to DNA repair pathways. CONCLUSION: Our data demonstrate that low GSTM5 expression and its high DNA methylation status may act as a novel putative molecular target gene for LUAD.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA/metabolismo , Metilação de DNA , Decitabina , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Glutationa Transferase , Humanos , Neoplasias Pulmonares/patologia , Prognóstico , Transferases/genética , Transferases/metabolismo
10.
Blood Adv ; 6(14): 4107-4121, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35561310

RESUMO

Exploring the repertoire of peptides presented on major histocompatibility complexes (MHCs) helps identify targets for immunotherapy in many hematologic malignancies. However, there is a paucity of such data for diffuse large B-cell lymphomas (DLBCLs), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the enhancer of zeste homolog 2 (EZH2)-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon-γ (IFN-γ), restored MHC expression in DLBCL. In DLBCL, peptides presented on MHCs were identified via mass spectrometry after treatment with tazemetostat or decitabine alone or in combination with IFN-γ. Such treatment synergistically increased the expression of MHC class I surface proteins up to 50-fold and the expression of class II surface proteins up to threefold. Peptides presented on MHCs increased to a similar extent for both class I and class II MHCs. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells for 2 of 3 cell lines and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from the regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands that are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFN-γ can expand the repertoire of MHC ligands presented on DLBCLs by revealing suppressed epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets.


Assuntos
Linfoma Difuso de Grandes Células B , Proteínas RGS , Decitabina/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama , Ligantes , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Peptídeos/metabolismo
11.
Methods Mol Biol ; 2451: 559-567, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35505032

RESUMO

Photofrin-based photodynamic therapy (PDT) is approved for clinical use by the US Food and Drug Administration and the European Medicines Agency and is among the most widely used photosensitizer for the treatment of cancer. It was broadly reported that both the innate and the adaptive arms of immune response can be activated by PDT and play a critical role in the anticancer outcome of this treatment. PDT leads to the induction of acute local inflammation that includes leukocyte infiltration as well as increased activation and production of pro-inflammatory factors and cytokines. These events can lead to the development of systemic and specific antitumor immune response. Combining Photofrin-PDT with the epigenetic agent 5-aza-2'-deoxycytidine results in potentiated antitumor effects in vivo. Understanding the molecular mechanisms underlying this phenomenon would be invaluable for clinical development of this therapeutic approach. This chapter describes a detailed protocol allowing evaluation of specific antitumor immune response induced by PDT.


Assuntos
Éter de Diematoporfirina , Fotoquimioterapia , Decitabina/farmacologia , Decitabina/uso terapêutico , Éter de Diematoporfirina/farmacologia , Éter de Diematoporfirina/uso terapêutico , Epigênese Genética , Imunidade , Triazenos , Estados Unidos
12.
Support Care Cancer ; 30(8): 7031-7038, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35585204

RESUMO

PURPOSE: The purpose of this study was to identify the incidence, sites and main pathogens, and risk factors for infectious complications occurring in patients with adult acute myeloid leukemia (AML) during the first course of venetoclax combined with decitabine or azacitidine. METHODS: A retrospective cohort analysis was performed of 81 patients with AML older than 14 years who received the first cycle of venetoclax combined with a hypomethylating agent (HMA) between March 2018 and March 2021 at our institution. Infectious complications, if any, were documented. RESULTS: Among a total of 81 cases of AML, 59 (72.8%) patients occurred infections, including fever without an identifiable source (28.8%), clinically documented infections (40.7%), and microbiologically documented infections (30.5%). The most commonly isolated organism in culture was Candida albicans, followed by Klebsiella pneumonia, and Pseudomonas aeruginosa. The 4-week and 8-week mortality rates were 3.7% and 7.4%, respectively. In multivariate analysis, a high proportion of blasts in bone marrow, decreased hemoglobin level, and fever with or without a documented infection at baseline were significant independent risk factors for infectious complications. CONCLUSION: Compared with conventional chemotherapy, the incidence of infectious complications of venetoclax combined with decitabine or azacitidine significantly decreased. Pretreatment high leukemia burden and fever were independent risk factors for infections.


Assuntos
Azacitidina , Leucemia Mieloide Aguda , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Azacitidina/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes , Decitabina/efeitos adversos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Estudos Retrospectivos , Sulfonamidas , Resultado do Tratamento
13.
Thromb Res ; 215: 5-13, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35580466

RESUMO

BACKGROUND: DNA methylation regulates gene expression by inhibiting transcription factor binding to promoter and regulatory regions. Acute hypoxia during altitude exposure is associated with decreased natural anticoagulants and morbid thrombotic events. Thrombomodulin (TM) is a high affinity thrombin binding receptor protein, vital for vascular homeostasis. The purpose of this study is to determine gene expression regulation via methylation of TM gene in high altitude hypoxia induced deep vein thrombosis (DVT) patients. MATERIALS AND RESULTS: Percent 5-methyl cytosine analysis showed increased methylation in high altitude DVT patients (HAP) as compared to high altitude control (HAC) and seal level control (Control) subjects, while TM protein and mRNA levels were decreased in high altitude DVT patients as compared to other two groups. Bisulfite sequencing analysis indicated increased methylation in TM promoter in high altitude DVT patients compared to high altitude controls. Flow cytometry analysis showed decreased TM expression in hypoxia induced primary human umbilical vein endothelial cells (HUVECs). Treatment with specific DNA methyltransferase (DNMT) inhibitor-decitabine during hypoxia, restored TM expression. in vitro global methylation assay showed increased methylation in hypoxia group. Specific concentration of decitabine in hypoxia decreased global methylation showing a direct correlation between DNMTs and methylation. Selective dose of decitabine restored TM levels in HUVECs. DNMT1 and DNMT3B proteins showed to mediate the overall expression of TM. CONCLUSION: TM emerged as a potential candidate for methylation in high altitude DVT patients, regulated by hypoxia-induced epigenetic mechanism. Hypoxia culminates in methylation of DNA sequences in the promoter region of TM gene and increased the expression of DNMT1 and DNMT3B per se in primary HUVECs. Critical DNA methylation events were found to be compromised in high altitude DVT patients.


Assuntos
Metilação de DNA , Trombomodulina/genética , Trombose Venosa , Altitude , Decitabina/administração & dosagem , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/metabolismo , Regiões Promotoras Genéticas , Trombose Venosa/genética
14.
BMC Pediatr ; 22(1): 312, 2022 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-35624441

RESUMO

BACKGROUND: Myelodysplastic syndrome (MDS) is a rare disease in children and the treatment option before the allogeneic hematopoietic stem cell transplantation (allo-HSCT) is rarely reported. Our main objective was to report our single-center experience with the DNA-hypomethylating agent, decitabine-combined minimally myelosuppressive regimen (DAC + MMR) bridged allo-HSCT in children with MDS. METHODS: Twenty-eight children with de novo MDS who underwent allo-HSCT between 2011 and 2020 were enrolled. Patients were divided into subgroups (refractory cytopenia of childhood [RCC] and advanced MDS [aMDS]) and treated by HSCT alone or pre-transplant combination treatment based on risk stratification. The patients' clinical characteristics, treatment strategies and outcomes were retrospectively evaluated. RESULTS: Twenty patients with aMDS had received pre-transplant treatment (three were treated with decitabine alone, thirteen with DAC + MMR, and four with acute myeloid leukemia type [AML-type] induction therapy). DAC + MMR was well tolerated and the most common adverse events were myelosuppression and gastrointestinal reaction. DAC + MMR had shown an improved marrow complete remission (mCR) compared with AML-type chemotherapy (13/13, 100% versus 2/4, 50%, P = 0.044). The median follow-up for total cohort was 53.0 months (range, 2.3-127.0 months) and the 4-year overall survival (OS) was 71.4 ± 8.5%. In the subgroup of aMDS, pretreatment of DAC + MMR resulted in a much better survival rate than AML-type chemotherapy (84.6 ± 10.0% versus 0.0 ± 0.0%, P < 0.001). CONCLUSIONS: The DAC + MMR bridged allo-HSCT may be recommended as a novel and effective approach.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Síndromes Mielodisplásicas , Criança , Decitabina/uso terapêutico , Humanos , Síndromes Mielodisplásicas/terapia , Doenças Raras , Estudos Retrospectivos
15.
Int J Mol Sci ; 23(10)2022 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-35628480

RESUMO

In myelodysplastic syndrome (MDS), resistance to hypomethylating agents (HMA) portends a poor prognosis, underscoring the importance of understanding the molecular mechanisms leading to HMA-resistance. In this study, P39 and Kasumi-1 cells and their azacitidine-resistant and decitabine-resistant sublines were evaluated comparatively with transcriptomic and methylomic analyses. Expression profiling and genome-wide methylation microarray showed downregulation of PTEN associated with DNA hypermethylation in P39 cell lines resistant to azacitidine and decitabine. This pattern of PTEN dysregulation was also confirmed in a cohort of patients failing treatment with HMA. DNA hypomethylation of MDM2 was detected with downregulation of MDM2 in HMA resistant cell lines. Long-read sequencing revealed significant RNA hypomethylation of MDM2 resulting in alternative splicing and production of a truncated MDM2 transcript in azacitidine-resistant P39 cells. The expression of this MDM2 truncated transcript was also significantly increased in HMA-resistant patients compared with HMA-responsive patients. In conclusion, epigenetic and epi-transcriptomic dysregulation of PTEN and MDM2 were associated with resistance to hypomethylating agents.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Segunda Neoplasia Primária , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas c-mdm2 , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Metilação de DNA , Decitabina/farmacologia , Epigênese Genética , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Segunda Neoplasia Primária/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-mdm2/genética
16.
Sci Rep ; 12(1): 5776, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35388081

RESUMO

Global changes in DNA methylation are observed in development and disease, and single-cell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of average DNA methylation levels. By targeting high-copy SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each scTEM-seq library. Parallel transcriptome analysis is also performed to link global DNA methylation estimates with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Our method reveals global DNA methylation heterogeneity induced by decitabine treatment of KG1a cells associated with altered expression of immune process genes. We also compare global DNA methylation estimates to expression of transposable elements and find a predominance of negative correlations. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond.


Assuntos
Elementos de DNA Transponíveis , Leucemia Mieloide Aguda , Metilação de DNA , Elementos de DNA Transponíveis/genética , Decitabina/farmacologia , Epigênese Genética , Humanos , Leucemia Mieloide Aguda/genética , Análise de Célula Única
18.
EBioMedicine ; 79: 103985, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35429693

RESUMO

BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Repressão Epigenética , Infecções por HIV , HIV-1 , Ubiquitina-Proteína Ligases , Latência Viral , Síndrome de Imunodeficiência Adquirida , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Decitabina/metabolismo , Infecções por HIV/genética , HIV-1/fisiologia , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/genética
19.
Leukemia ; 36(6): 1654-1665, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35459873

RESUMO

Peripheral T-cell lymphoma (PTCL) is a rare, heterogenous malignancy with dismal outcomes at relapse. Hypomethylating agents (HMA) have an emerging role in PTCL, supported by shared mutations with myelodysplasia (MDS). Response rates to azacitidine in PTCL of follicular helper cell origin are promising. Guadecitabine is a decitabine analogue with efficacy in MDS. In this phase II, single-arm trial, PTCL patients received guadecitabine on days 1-5 of 28-day cycles. Primary end points were overall response rate (ORR) and safety. Translational sub-studies included cell free plasma DNA sequencing and functional genomic screening using an epigenetically-targeted CRISPR/Cas9 library to identify response predictors. Among 20 predominantly relapsed/refractory patients, the ORR was 40% (10% complete responses). Most frequent grade 3-4 adverse events were neutropenia and thrombocytopenia. At 10 months median follow-up, median progression free survival (PFS) and overall survival (OS) were 2.9 and 10.4 months respectively. RHOAG17V mutations associated with improved PFS (median 5.47 vs. 1.35 months; Wilcoxon p = 0.02, Log-Rank p = 0.06). 4/7 patients with TP53 variants responded. Deletion of the histone methyltransferase SETD2 sensitised to HMA but TET2 deletion did not. Guadecitabine conveyed an acceptable ORR and toxicity profile; decitabine analogues may provide a backbone for future combinatorial regimens co-targeting histone methyltransferases.


Assuntos
Azacitidina , Linfoma de Células T Periférico , Azacitidina/efeitos adversos , Azacitidina/análogos & derivados , Decitabina/uso terapêutico , Genômica , Humanos , Linfoma de Células T Periférico/tratamento farmacológico , Linfoma de Células T Periférico/genética , Síndromes Mielodisplásicas/patologia , Recidiva Local de Neoplasia/induzido quimicamente , Neutropenia/induzido quimicamente , Resultado do Tratamento
20.
Bone Marrow Transplant ; 57(7): 1063-1071, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35459877

RESUMO

The optimal conditioning regimen for high-risk myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains elusive. This study aimed to explore the anti-leukemic efficacy and toxicity of Decitabine (Dec, 20 mg/m2/day, day -11 to -7) intensified BUCY2 vs. traditional regimen in high-risk MDS population. We retrospectively evaluated 93 consecutive high-risk MDS patients undergoing allo-HSCT in our institution, comparing discrepancies in clinical characteristics and outcomes between cases using Dec-intensified BUCY2 (n = 52) and traditional BUCY2 regimen (n = 41). Three-year cumulative incidence of relapse after Dec-intensified BUCY2 conditioning was remarkably lower than that of patients using BUCY2 regimen (20.2% vs. 39.0%, p = 0.034). Overall survival and disease-free survival at 3 years for Dec-intensified BUCY2 group were 70.2% and 64.9%, respectively, which were significantly improved when compared with BUCY2 group (51.1% and 43.9%, p = 0.031 and p = 0.027). Furthermore, overall survival and disease-free survival for MDS cases receiving cytoreduction therapy were dramatically better than patients in non-cytoreduction group (p = 0.041, p = 0.047). In summary, the Dec-intensified conditioning regimen could be effective and feasible, providing prominent recurrence control with moderate toxicity for high-risk MDS patients. These patients might also benefit from pre-transplant cytoreductive therapeutic schedules. Larger randomized controlled trials are still needed to further confirm these conclusions.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Bussulfano , Decitabina/uso terapêutico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Estudos Retrospectivos , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo/efeitos adversos
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