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1.
Lancet Haematol ; 7(8): e566-e574, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32735836

RESUMO

BACKGROUND: The median overall survival of patients with high-risk myelodysplastic syndromes refractory to hypomethylating agents is less than 6 months. Currently, no standard therapy for such patients exists. Preclinical studies have shown that inhibition of the nuclear export protein exportin 1 (XPO1) causes nuclear accumulation of p53 and disruption of NF-κB signalling, both relevant targets for myelodysplastic syndromes. We therefore aimed to assess the safety and activity of selinexor in patients with myelodysplastic syndromes or oligoblastic acute myeloid leukaemia refractory to hypomethylating agents. METHODS: We did a single-centre, single-arm, phase 2 trial at the Memorial Sloan Kettering Cancer Center in the USA. We included patients 18 years or older with high-risk myelodysplastic syndromes or oligoblastic acute myeloid leukaemia (defined as blasts ≥20% but ≤30%) refractory to hypomethylating agents and with an Eastern Cooperative Oncology Group performance status score of 0-2. Eligible patients received 3-week long cycles of oral selinexor (60 mg twice per week for 2 weeks, followed by 1 week off). The primary outcome was overall response rate. Complete remission, partial remission, marrow complete remission, or haematological improvement were included in the response categories for assessing the primary endpoint. The activity analysis included all patients who completed at least one full-scheduled post-treatment disease assessment. All patients who were given selinexor were included in the safety analysis. This study is registered with ClinicalTrials.gov, NCT02228525. FINDINGS: Between Sept 23, 2014, and March 13, 2018, 25 patients were enrolled on this study. The median follow-up was 8·5 months (IQR 3·1-12·2). Two patients did not meet the full eligibility criteria after baseline assessment; therefore, 23 patients were evaluable for activity assessment. In the 23 evaluable patients, overall response rate was 26% (95% CI 10-48) in six patients with marrow complete remission, with an additional 12 patients (52%, 95% CI 31-73) achieving stable disease. The most common grade 3 or 4 adverse events were thrombocytopenia (eight [32%] of 25 patients) and hyponatraemia (five [20%]). There were no drug-related serious adverse events and no treatment-related deaths. INTERPRETATION: Selinexor showed responses in patients with myelodysplastic syndromes or oligoblastic acute myeloid leukaemia refractory to hypomethylating agents. Adverse events were manageable with supportive care implementation. Further studies are needed to compare selinexor with supportive care alone, and to identify patient subgroups that might benefit the most from selinexor treatment. FUNDING: Karyopharm Therapeutics.


Assuntos
Azacitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hidrazinas/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Triazóis/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Síndromes Mielodisplásicas/patologia , Segurança do Paciente , Prognóstico , Taxa de Sobrevida
2.
PLoS One ; 15(7): e0236192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692756

RESUMO

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of ß-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2'-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.


Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Via de Sinalização Wnt/genética , Azacitidina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Metilação de DNA/genética , Feminino , Humanos , Proteínas de Membrana/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Gene ; 758: 144958, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683073

RESUMO

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.


Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Azacitidina/farmacologia , Linhagem Celular , Citomegalovirus/genética , Fator de Iniciação 1 em Eucariotos/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Transdução Genética
4.
PLoS One ; 15(6): e0233784, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492024

RESUMO

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.


Assuntos
Clonagem de Organismos/veterinária , Citomegalovirus/genética , Técnicas de Transferência Nuclear/veterinária , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Animais , Animais Geneticamente Modificados , Azacitidina/farmacologia , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Cães , Transferência Embrionária/veterinária , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Gravidez , Transfecção , Transgenes
5.
Arterioscler Thromb Vasc Biol ; 40(8): 1854-1869, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32580634

RESUMO

OBJECTIVE: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. CONCLUSIONS: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , PTEN Fosfo-Hidrolase/fisiologia , Remodelação Vascular/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , PTEN Fosfo-Hidrolase/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Regiões Promotoras Genéticas
6.
Leukemia ; 34(11): 2951-2963, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32576961

RESUMO

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.


Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/farmacologia , Evolução Clonal , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Sinergismo Farmacológico , Feminino , GTP Fosfo-Hidrolases/genética , Humanos , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/mortalidade , Leucemia Mielomonocítica Crônica/patologia , Proteínas de Membrana/genética , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Cancer Res ; 80(14): 3046-3056, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32354737

RESUMO

Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations. SIGNIFICANCE: This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Azacitidina/análogos & derivados , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Azacitidina/farmacologia , Proliferação de Células , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nucleic Acids Res ; 48(9): 4797-4810, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32246716

RESUMO

Therapeutic targeting of epigenetic modulators offers a novel approach to the treatment of multiple diseases. The cellular consequences of chemical compounds that target epigenetic regulators (epi-drugs) are complex. Epi-drugs affect global cellular phenotypes and cause local changes to gene expression due to alteration of a gene chromatin environment. Despite increasing use in the clinic, the mechanisms responsible for cellular changes are unclear. Specifically, to what degree the effects are a result of cell-wide changes or disease related locus specific effects is unknown. Here we developed a platform to systematically and simultaneously investigate the sensitivity of epi-drugs at hundreds of genomic locations by combining DNA barcoding, unique split-pool encoding, and single cell expression measurements. Internal controls are used to isolate locus specific effects separately from any global consequences these drugs have. Using this platform we discovered wide-spread loci specific sensitivities to epi-drugs for three distinct epi-drugs that target histone deacetylase, DNA methylation and bromodomain proteins. By leveraging ENCODE data on chromatin modification, we identified features of chromatin environments that are most likely to be affected by epi-drugs. The measurements of loci specific epi-drugs sensitivities will pave the way to the development of targeted therapy for personalized medicine.


Assuntos
Epigênese Genética/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Azacitidina/farmacologia , Azepinas/farmacologia , Cromossomos Humanos , Metilação de DNA/efeitos dos fármacos , Genes Reporter , Loci Gênicos , Genômica/métodos , Histonas/metabolismo , Humanos , Células K562 , Análise de Sequência de DNA , Triazóis/farmacologia
9.
Cancer Res ; 80(12): 2441-2450, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32245794

RESUMO

The DNA methyltransferase inhibitors (DNMTi) 5-azacytidine and 5-aza-2-deoxycytidine have been approved for the treatment of different types of hematologic malignancies. However, only about 50% of patients respond to treatment. Therefore, a more comprehensive understanding of the molecular changes in patients treated with DNMTi is needed. Here, we examined gene expression profiles in a total of 150 RNA samples from two adult cohorts and one pediatric cohort with hematologic cancers taken before, during, and after treatment with 5-azacytidine (40 patients; 15 nonresponders, 25 responders). Using each patient as their own control, malignant cells showed preferential activation of a subset of evolutionarily young transposable elements (TE), including endogenous retroviral long terminal repeats (LTR), short and long interspersed nuclear elements (SINE and LINE), and the type I IFN pathway in responders, all independent of disease classification. Transfection of eight upregulated LTRs into recipient human cells in culture showed robust and heterogenous activation of six genes in the type I IFN pathway. These results, obtained in diverse hematologic disease entities, show that common targets (TE) activated by the same drug (5-azacytidine) elicit an immune response, which may be important for patient's responses to DNMTi. SIGNIFICANCE: Activation of specific classes of evolutionarily young transposable elements can lead to activation of the innate immune system.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Elementos de DNA Transponíveis/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Imunidade Inata/genética , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Estudos de Coortes , Elementos de DNA Transponíveis/genética , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Retrovirus Endógenos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/metabolismo , Masculino , Pessoa de Meia-Idade , Mimetismo Molecular/imunologia , RNA-Seq , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/efeitos dos fármacos
10.
Sci Rep ; 10(1): 6655, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313015

RESUMO

Retinopathy continues to progress even when diabetic patients try to control their blood sugar, but the molecular mechanism of this 'metabolic memory' phenomenon remains elusive. Retinal mitochondria remain damaged and vicious cycle of free radicals continues to self-propagate. DNA methylation suppresses gene expression, and diabetes activates DNA methylation machinery. Our aim was to investigate the role of DNA methylation in continued compromised mitochondrial dynamics and genomic stability in diabetic retinopathy. Using retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four days, and retinal microvessels from streptozotocin-induced diabetic rats in poor glycemia for four months, followed by normal glycemia for four additional months, DNA methylation of mitochondrial fusion and mismatch repair proteins, Mfn2 and Mlh1 respectively, was determined. Retinopathy was detected in trypsin-digested microvasculature. Re-institution of good glycemia had no beneficial effect on hypermethylation of Mfn2 and Mlh1 and retinal function (electroretinogram), and the  retinopathy continued to progress. However, intervention of good glycemia directly with DNA methylation inhibitors (Azacytidine or Dnmt1-siRNA), prevented Mfn2 and Mlh1 hypermethylation, and ameliorated retinal dysfunction and diabetic retinopathy. Thus, direct regulation of DNA methylation can prevent/reverse diabetic retinopathy by maintaining mitochondrial dynamics and DNA stability, and prevent retinal functional damage.


Assuntos
Azacitidina/farmacologia , DNA Mitocondrial/genética , Diabetes Mellitus Experimental/terapia , Retinopatia Diabética/terapia , Epigênese Genética , Hiperglicemia/terapia , Mitocôndrias/genética , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Progressão da Doença , Eletrorretinografia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Glucose/efeitos adversos , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/genética , Hiperglicemia/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Estreptozocina/administração & dosagem
11.
Int J Biochem Cell Biol ; 122: 105733, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32114121

RESUMO

Recent studies have shown that cardiac fibroblasts (CFs) can be transformed into induced cardiomyocytes (iCMs). This phenomenon represents a potential method for rescuing damaged myocardia after myocardial infarction. The mechanism underlying cardiac reprogramming regulation must be clarified to improve the induction efficiency of iCMs. In this study, we treated CFs with 5-aza for 24 h and added TGF-ß inhibitor A83-01 for 2 weeks in vitro to investigate the effect of inhibiting fibrosis on myocardial differentiation. Inhibition of TGF-ß1 activity with A83-01 significantly decreased the expressions of collagen III and α-SMA and increased the expression of myocardial specific marker cTnT and gap junction protein Cx43 in CFs, enhanced cardiac reprogramming as opposed to 2 weeks with 5-aza alone. Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis at the 14th day postinduction of A83-01 revealed that the expression of genes involved in cardiac development increased in the presence of 5-aza. These findings suggest that the addition of A83-01 remarkably inhibits profibrotic signalling and improved the efficiency of iCMs, provide new insights into the molecular mechanisms of cardiac reprogramming and promote the use of iCMs in clinical applications.


Assuntos
Azacitidina/uso terapêutico , Fibroblastos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Humanos , Ratos , Transdução de Sinais
12.
Int J Clin Oncol ; 25(6): 1105-1114, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32206938

RESUMO

BACKGROUND: Gastric cancer (GC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related mortality. In recent years, SAMD14 has been studied in various malignant cancers; however, little is known about the exact mechanisms of SAMD14 involvement in carcinogenesis and malignant progression. METHODS: 60 paired GC-normal gastric tissues were evaluated for their SAMD14 mRNA expression in relation to SAMD14 gene promoter methylation. GC patient survival was assessed by Kaplan-Meier analyses and a Cox's proportional hazard model was employed for multivariate analyses. RESULTS: SAMD14 expression was significantly inversely correlated with the Borrmann type (P = 0.017), lymph node metastasis (P = 0.006) and tumor-node-metastasis (TNM) stage (P = 0.033). Methylation-specific PCR (MSP) revealed hyper-methylation of the SAMD14 promoter in 56.7% (34/60) of the primary GC tissues tested and in 10% (6/60) of matched non-malignant tissues. The SAMD14 promoter methylation status was also related to pathological differentiation, Borrmann type, TNM stage and lymph node metastasis. The results showed SAMD14 expression was significantly downregulated in Borrmann type, lymph node metastasis and TNM stage, which showed significantly higher methylation. SAMD14 promoter hyper-methylation was significantly associated with a poor prognosis and could serve as an independent marker for survival using multivariate Cox regression analysis. CONCLUSIONS: Our results indicated that promoter methylation was a key mechanism contributing to the downregulation of SAMD14 in GC. SAMD14 may be an epigenetically silenced tumor suppressor gene, and hyper-methylation of the SAMD14 promoter may serve as a biomarker to predict the clinical outcome of GC.


Assuntos
Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Repressoras/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia
13.
Oncol Rep ; 43(3): 827-838, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32020216

RESUMO

Restoration of normal DNA promoter methylation and expression states of cancer­related genes may be an option for the prevention as well as the treatment of several types of cancer. Constitutional promoter methylation of BRCA1 DNA repair associated (BRCA1) gene is linked with a high risk of developing breast and ovarian cancer. Furthermore, hypomethylation of the proto­oncogene Î³ synuclein (SNCG) is associated with the metastasis of breast and ovarian cancer and reduced disease­free survival (DFS). In the present study, we evaluated the potential of curcumin to re­express hypermethylated BRCA1 and to suppress hypomethylated SNCG in triple­negative breast cancer (TNBC) cell line HCC­38, the estrogen receptor­negative/progesterone receptor­negative (ER­/PR­) cell line UACC­3199, and the ER+/PR+ cell line T47D. The cells were treated with 5 and 10 µM curcumin for 6 days and with 5­aza­2'­deoxycytidine (5'­aza­CdR) for 48 h. Methylation­specific PCR and bisulfite pyrosequencing assays were used to assess DNA promoter methylation while gene expression levels were analyzed using quantitative real­time PCR and immunoblotting. We found that curcumin treatment restored BRCA1 gene expression by reducing the DNA promoter methylation level in HCC­38 and UACC­3199 cells and that it suppressed the expression of SNCG by inducing DNA promoter methylation in T47D cells. Notably, 5'­aza­CdR restored BRCA1 gene expression only in UACC­3199, and not in HCC­38 cells. Curcumin­induced hypomethylation of the BRCA1 promoter appears to be realized through the upregulation of the ten­eleven translocation 1 (TET1) gene, whereas curcumin­induced hypermethylation of SNCG may be realized through the upregulation of the DNA methyltransferase 3 (DNMT3) and the downregulation of TET1. Notably, miR­29b was found to be reversely expressed compared to TET1 in curcumin­ and 5'­aza­CdR­treated cells, suggesting its involvement in the regulation of TET1. Overall, our results indicate that curcumin has an intrinsic dual function on DNA promoter methylation. We believe that curcumin may be considered a promising therapeutic option for treating TNBC patients in addition to preventing breast and ovarian cancer, particularly in cancer­free females harboring methylated BRCA1.


Assuntos
Proteína BRCA1/genética , Curcumina/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , gama-Sinucleína/genética , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
14.
Immunology ; 160(1): 38-51, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32027025

RESUMO

First discovered on the natural killer (NK) cell, the cell surface inhibitory receptor sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is known for regulating many important biological activities. However, the detail regulatory mechanism for Siglec-7 expression in NK cells currently remains unclear. In this study, we aimed to investigate how cell surface Siglec-7 expression is regulated and found that, in both NK cell lines and peripheral NK cells, transcription was the main regulatory step. Furthermore, when NK-92MI and peripheral NK cells were treated with DNA methyltransferase (DNMT) inhibitor, the CpG island, with 9 CpG sites, in 5' Siglec-7 promoter became noticeably hypomethylated, and Siglec-7 expression increased in both RNA transcript and surface protein. Within this CpG island, we identified both CpG 8 and CpG 9 as two key regulators responsible for Siglec-7 expression. Additionally, by using histone deacetylases (HDAC) inhibitor, butyric acid, we showed that Siglec-7 expression was also subjected to the histone modification. And a combined treatment with both 5-azacytidine and butyric acid showed an additive effect on Siglec-7 transcript expression in peripheral NK cells.


Assuntos
Antígenos de Diferenciação Mielomonocítica/genética , Epigênese Genética/imunologia , Células Matadoras Naturais/imunologia , Lectinas/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Azacitidina/farmacologia , Ácido Butírico/farmacologia , Linhagem Celular , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/imunologia , Epigênese Genética/efeitos dos fármacos , Código das Histonas/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Lectinas/metabolismo , Regiões Promotoras Genéticas/genética , RNA-Seq , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/imunologia
15.
Ann Hematol ; 99(4): 693-701, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32025842

RESUMO

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Chemotherapy with cytotoxic agents is the standard of care, but is associated with a high rate of adverse events. Elderly patients are frequently intolerant to such treatment, presenting a very poor prognosis. The hypomethylating agents (HMA) azacitidine or decitabine represent one of the main therapeutic alternatives for these patients. Isocitrate dehydrogenase inhibitors (IDH) constitute another therapeutic class with DNA methylation effects in AML. In this article, we review the use of first- and second-generation HMA and IDH inhibitors in AML. The data collected demonstrated that HMA are generally considered effective and safe for AML patients who are not eligible for standard chemotherapy. The combination of azacitidine or decitabine with venetoclax was recently approved by the US Food and Drug Administration (FDA) for older AML patients and those unfit for intense chemotherapy. IDH inhibitors also showed encouraging results for relapsed/refractory AML patients harboring an IDH mutation and received FDA approval. Therefore, recent studies have led to the emergence of new therapeutic options using HMA and IDH inhibitors for specific groups of AML patients, representing an important step in the treatment of this aggressive malignancy. New options should emerge from the ongoing studies in the coming years.


Assuntos
Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Idoso , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/administração & dosagem , Azacitidina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Ensaios Clínicos como Assunto , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Decitabina/administração & dosagem , Decitabina/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Metanálise como Assunto , Estudos Multicêntricos como Assunto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Sulfonamidas/administração & dosagem , Resultado do Tratamento
16.
J Oncol Pharm Pract ; 26(5): 1285-1288, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32054413

RESUMO

INTRODUCTION: Polycythemia vera is a myeloproliferative neoplasm (MPN) characterized by increased red blood cell mass. The natural evolution of this MPN is to progress to an anemic/cytopenic phase also known as "spent" phase prior to transformation into an accelerated and/or an overt leukemic phase. CASE REPORT: Herein, we describe a case of a patient with polycythemia vera transitioning though a "spent" phase to an MPN in accelerated phase (MPN-AP). The patient had anemia, thrombocytopenia, neutrophilia and increased blasts in the bone marrow. Management and outcome: Upon treatment with four cycles of 5-azacitidine, the patient's polycythemia vera reversed back to the proliferative phase. Serial phlebotomies were again required. DISCUSSION: Reversal of a "spent" phase by 5-azacitidine back to a proliferative polycythemia vera phase requiring phlebotomies has not been previously reported in the scientific literature. We might witness similar cases in the literature in the future years, which could lead to yet another therapeutic indication of this important pharmacologic agent.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Flebotomia/métodos , Policitemia Vera/tratamento farmacológico , Idoso de 80 Anos ou mais , Anemia/sangue , Anemia/tratamento farmacológico , Anemia/terapia , Azacitidina/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Feminino , Humanos , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/terapia , Policitemia Vera/sangue , Policitemia Vera/terapia
17.
J Antibiot (Tokyo) ; 73(6): 410-413, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32060485

RESUMO

The effects of epigenetic modulation on secondary metabolite biosynthesis were investigated with five Aspergillus species cultured in the presence of either the DNA methyltransferase inhibitor 5-azacitidine or the histone deacetylase inhibitor vorinostat. With Aspergillus calidoustus and Aspergillus westerdijkiae, fermentation in the presence of vorinostat (100 µM) induced significant changes in secondary metabolite profile with examples of both induction and repression. We identified putative biosynthetic gene clusters for emericellamide in A. calidoustus and ochratoxin in A. westerdijkiae. A substantial induction in production levels was observed for two secondary metabolites: the diketopiperazine alkaloid phenylahistin in A. calidoustus and the polyketide penicillic acid in A. westerdijkiae, indicating the potential of epigenetic regulation for the activation of silent fungal biosynthetic pathways.


Assuntos
Aspergillus/metabolismo , Azacitidina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Vorinostat/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Família Multigênica , Metabolismo Secundário/genética
18.
Nature ; 579(7798): 284-290, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103175

RESUMO

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.


Assuntos
Epigênese Genética , Terapia Genética , Células Supressoras Mieloides/fisiologia , Neoplasias/terapia , Microambiente Tumoral , Animais , Azacitidina/farmacologia , Benzamidas/farmacologia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Camundongos , Células Supressoras Mieloides/citologia , Metástase Neoplásica/terapia , Neoplasias/cirurgia , Piridinas/farmacologia , Receptores CCR2/genética , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efeitos dos fármacos
19.
BMC Genet ; 21(1): 3, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941450

RESUMO

BACKGROUND: DNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC). RESULTS: Our data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression. CONCLUSION: These findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.


Assuntos
Azacitidina/farmacologia , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/efeitos dos fármacos , Epigenômica , Epigênese Genética/efeitos dos fármacos , Epigenômica/métodos , Éxons , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons , Regiões Promotoras Genéticas , Ligação Proteica , Sítio de Iniciação de Transcrição
20.
Plant Physiol Biochem ; 147: 91-100, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855819

RESUMO

Release of bud dormancy is a prerequisite for the growth resumption and production in perennial plants such as tree peony. DNA methylation plays a pivotal role in regulating gene expression. In this study, combination of morphologic observation and DNA methylation analysis indicated that 5-azacytidine (5-azaC) application for 7 d declined 5 mC quantities and promoted dormancy release. After 5-azaC treatment, total 174,341 unigenes and 1818 differentially expression genes (DEGs) were obtained by RNA-seq, of which there were 1194 DEGs after 1 d 5-azaC treatment (AD1 vs CD1), and 624 DEGs after 7 d (AD7 vs CD7), respectively. The KEGG pathway analysis identified that totally 10 DEGs annotated in DNA replication pathway were enriched when AD7 compared with CD7. Furthermore, the expression patterns of several DEGs by real-time quantitative RT-PCR were consistent with that of RNA-seq data. 5-azaC application significantly decreased the expression levels of DNA methyltransferase genes, PsCMT3, PsMET1 and PsDRM2, and increased the transcript of demethylase gene PsROS1. Simultaneously, total methyltransferases activity decreased, and demethylase activity was induced by 5-azaC. In summary, application of 5-azaC inhibited the expression of the genes related to growth and development in short-term, indicating a possible toxic effect to plant, and its long-term effect was to induce hypomethylation by increasing demethylase genes transcripts and decreasing the expressions of methyltransferase genes, and then activate cell cycle, DNA replication and glycol-metabolism processes, which subsequently accelerated dormancy release. All these would provide a new strategy to further understand the molecular mechanism of dormancy release in tree peony.


Assuntos
Azacitidina , Metilação de DNA , Flores , Paeonia , Dormência de Plantas , Azacitidina/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Paeonia/efeitos dos fármacos , Dormência de Plantas/efeitos dos fármacos , Dormência de Plantas/genética
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