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1.
Clin Epigenetics ; 14(1): 92, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35858918

RESUMO

BACKGROUND: SLC5A7 (solute carrier family 5 member 7), also known as choline transporter 1 (CHT1), is downregulated in colorectal cancer (CRC) and functions as a tumor suppressor. However, the mechanisms underlying the inactivation of SLC5A7 in CRC remain to be elucidated. RESULTS: In the present study, two broad-spectrum demethylation agents (azacitidine and decitabine) employed to treat CRC cells significantly upregulated SLC5A7 expression. Further results based on the CRC cohort and TCGA database indicated that SLC5A7 promoter methylation inversely correlated with SLC5A7 expression, and the status of SLC5A7 promotor methylation showed a promising prognostic value for patients with CRC. Next, the dCas9-multiGCN4/scFv-TET1CD-based precision demethylation system was constructed, which could significantly and specifically promote SLC5A7 expression in CRC cells through sgRNA targeting the SLC5A7 promoter. Both in vitro and in vivo experiments demonstrated that targeted demethylation of SLC5A7 by dCas9-multiGCN4/scFv-TET1CD-sgSLC5A7 inhibited tumor growth by stabilizing p53 and regulating downstream targets. CONCLUSIONS: Collectively, DNA promoter methylation caused inactivation of SLC5A7 in CRC, and targeted demethylation of SLC5A7 might be a therapeutic target for CRC and other cancers.


Assuntos
Neoplasias Colorretais , Simportadores , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Desmetilação , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Simportadores/genética , Simportadores/metabolismo , Simportadores/uso terapêutico
2.
BMC Med ; 20(1): 222, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35843958

RESUMO

BACKGROUND: At present, the extent and clinical relevance of epigenetic differences between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) remain largely unknown. Here, we conducted a study to describe the global DNA methylation landscape of UTUC and UCB and to address the prognostic value of DNA methylation subtype and responses to the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma (UC). METHODS: Using whole-genome bisulfite sequencing (n = 49 samples), we analyzed epigenomic features and profiles of UTUC (n = 36) and UCB (n = 9). Next, we characterized potential links between DNA methylation, gene expression (n = 9 samples), and clinical outcomes. Then, we integrated an independent UTUC cohort (Fujii et al., n = 86) and UCB cohort (TCGA, n = 411) to validate the prognostic significance. Furthermore, we performed an integrative analysis of genome-wide DNA methylation and gene expression in two UC cell lines following transient DNA methyltransferase inhibitor SGI-110 treatment to identify potential epigenetic driver events that contribute to drug efficacy. RESULTS: We showed that UTUC and UCB have very similar DNA methylation profiles. Unsupervised DNA methylation classification identified two epi-clusters, Methy-High and Methy-Low, associated with distinct muscle-invasive statuses and patient outcomes. Methy-High samples were hypermethylated, immune-infiltrated, and enriched for exhausted T cells, with poor clinical outcome. SGI-110 inhibited the migration and invasion of Methy-High UC cell lines (UMUC-3 and T24) by upregulating multiple antitumor immune pathways. CONCLUSIONS: DNA methylation subtypes pave the way for predicting patient prognosis in UC. Our results provide mechanistic rationale for evaluating SGI-110 in treating UC patients in the clinic.


Assuntos
Azacitidina , Carcinoma de Células de Transição , Metilação de DNA , Metilases de Modificação do DNA , Neoplasias da Bexiga Urinária , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Humanos , Prognóstico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo
3.
BMC Plant Biol ; 22(1): 278, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672704

RESUMO

BACKGROUND: Strawberry ripening involves a number of irreversible biochemical reactions that cause sensory changes through accumulation of sugars, acids and other compounds responsible for fruit color and flavor. The process, which is strongly dependent on methylation marks in other fruits such as tomatoes and oranges, is highly controlled and coordinated in strawberry. RESULTS: Repeated injections of the hypomethylating compound 5-azacytidine (AZA) into green and unripe Fragaria × ananassa receptacles fully arrested the ripening of the fruit. The process, however, was reversible since treated fruit parts reached full maturity within a few days after AZA treatment was stopped. Transcriptomic analyses showed that key genes responsible for the biosynthesis of anthocyanins, phenylpropanoids, and hormones such as abscisic acid (ABA) were affected by the AZA treatment. In fact, AZA downregulated genes associated with ABA biosynthetic genes but upregulated genes associated with its degradation. AZA treatment additionally downregulated a number of essential transcription factors associated with the regulation and control of ripening. Metabolic analyses revealed a marked imbalance in hormone levels, with treated parts accumulating auxins, gibberellins and ABA degradation products, as well as metabolites associated with unripe fruits. CONCLUSIONS: AZA completely halted strawberry ripening by altering the hormone balance, and the expression of genes involves in hormone biosynthesis and degradation processes. These results contradict those previously obtained in other climacteric and fleshly fruits, where AZA led to premature ripening. In any case, our results suggests that the strawberry ripening process is governed by methylation marks.


Assuntos
Fragaria , Ácido Abscísico/metabolismo , Antocianinas/metabolismo , Azacitidina/farmacologia , Fragaria/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Hormônios/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
4.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 30(3): 790-796, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-35680807

RESUMO

OBJECTIVE: To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells. METHODS: The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein. RESULTS: Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05),while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05). CONCLUSION: MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.


Assuntos
MicroRNAs , Mieloma Múltiplo , Apoptose , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/farmacologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Mieloma Múltiplo/genética , RNA Mensageiro/metabolismo
5.
Sci Rep ; 12(1): 9349, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35672334

RESUMO

Recent studies have indicated strong connections between epigenetic modulation and secondary metabolites in plants. It is vital to understand the roles of epigenetics in the production of secondary metabolites. In this study, the inhibitor of DNA methylation 5-azacytidine (5-Az) was used on the hairy roots of the medicinal plant Salvia miltiorrhiza to investigate its effect on secondary metabolite production, gene expression, methylation levels in genomic DNA and promoter regions. Our results showed that the contents of tanshinones in S. miltiorrhiza hairy roots increased by 1.5-5 times, and some genes in the biosynthesis pathway showed an upward trend. According to our NGS analysis, the methylation pattern in the promotor of the gene encoding copalyl diphosphate synthase (CPS) was altered, and 51 out of 145 cytosines were demethylated during 5-Az treatment. A total of 36 putative transcription factors (TFs) binding cites were identified in these demethylation sites. Among these TFs binding cites, cis-regulatory elements for the binding of NF-Y and MYB were frequently found in our results. This is the first report to demonstrate a possible mechanism of DNA methylation participating in tanshinone biosynthesis in S. miltiorrhiza hairy roots by modulating the CPS promoter and TFs binding sites.


Assuntos
Salvia miltiorrhiza , Abietanos , Azacitidina/metabolismo , Azacitidina/farmacologia , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Salvia miltiorrhiza/metabolismo
6.
Blood Cancer Discov ; 3(4): 346-367, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35532363

RESUMO

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. SIGNIFICANCE: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265.


Assuntos
Dioxigenases , Síndromes Mielodisplásicas , Pré-Leucemia , Azacitidina/farmacologia , Cromatina/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Hematopoese/genética , Humanos , Proteínas Proto-Oncogênicas/genética
7.
Invest New Drugs ; 40(4): 738-746, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35576022

RESUMO

Myelodysplastic syndromes (MDS) are clonal malignancies of multipotent hematopoietic stem cells, characterized by ineffective hematopoiesis leading to cytopenia. Hypomethylating agents, including azacitidine, have been used for treating MDS with some success; however, the overall survival rate remains poor and, therefore, finding new therapies is necessary. Selinexor, which exerts anticancer effects against some hematologic tumors, is a nuclear export protein inhibitor that blocks cell proliferation and induces apoptosis in various cancer cell lines. We investigated the effects of combined selinexor and azacitidine administration on two MDS cell lines, namely SKM-1 and MUTZ-1. Cells were subjected to a proliferation assay, and the effects of each drug alone, and in combination, were compared. Changes in apoptosis and the cell cycle between groups were also analyzed. Western blotting was conducted to identify the underlying mechanism of action of combined selinexor and azacitidine therapy. The results revealed that the combination of selinexor and azacitidine synergistically inhibited MDS cell proliferation and arrested the cell cycle at the G2/M phase. This combination also promoted MDS cell apoptosis and enhanced p53 accumulation in the nucleus, thereby allowing p53 to be activated and to function as a tumor suppressor. Overall, our results indicate that the combination of selinexor and azacitidine may be a promising approach for treating MDS.


Assuntos
Síndromes Mielodisplásicas , Neoplasias , Azacitidina/farmacologia , Humanos , Hidrazinas/farmacologia , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Neoplasias/tratamento farmacológico , Triazóis , Proteína Supressora de Tumor p53
8.
Dokl Biochem Biophys ; 503(1): 76-79, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35538282

RESUMO

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.


Assuntos
Azacitidina , Linfoma de Burkitt , Leucemia , Fator 1 de Transcrição de Octâmero , Animais , Azacitidina/farmacologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Técnicas de Cultura de Células , Camundongos , Fator 1 de Transcrição de Octâmero/antagonistas & inibidores , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
9.
Oncol Rep ; 48(1)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35593315

RESUMO

The in­depth study of melanoma pathogenesis has revealed that epigenetic modifications, particularly DNA methylation, is a universal inherent feature of the development and progression of melanoma. In the present study, the analysis of the tumor suppressor gene growth arrest­specific transcript 5 (GAS5) demonstrated that its expression was downregulated in melanoma, and its expression level had a certain negative association with its methylation modification level. The promoter of GAS5 presented with detectable CpG islands, and methylation­specific polymerase chain reaction analysis demonstrated that GAS5 was actually modified by methylation in melanoma tissues and cells; however, no methylation modification of GAS5 was detected in normal tissues. Following the treatment of melanoma cells with 5­Aza­2'­deoxycytidine (5­Aza­dC), GAS5 methylation was significantly reversed. The analysis of melanoma cell proliferation revealed that 5­Aza­dC inhibited A375 and SK­MEL­110 cell proliferation in a time­dependent manner. Further analysis of apoptosis demonstrated that 5­Aza­dC significantly increased the apoptosis level of the two cell lines. Moreover, migration analysis of melanoma cells revealed that 5­Aza­dC significantly reduced cell migration. Furthermore, 5­Aza­dC significantly decreased the invasive ability of the two cell lines. However, when the expression of GAS5 was silenced, the effects of 5­Aza­dC on cell proliferation, apoptosis, invasion and migration were not significant. Furthermore, the subcutaneous injection of A375 cells in nude mice successfully resulted in xenograft tumor formation. However, following an intraperitoneal injection of 5­Aza­dC, the volume and weight of xenograft tumors and Ki­67 expression were significantly reduced, and caspase­3 activity and GAS5 expression were enhanced; following the silencing of GAS5, the antitumor effect of 5­Aza­dC was significantly blocked. On the whole, the present study demonstrates that 5­Aza­dC inhibits the growth of melanoma, and its function may be related to the methylation modification of GAS5.


Assuntos
Melanoma , RNA Longo não Codificante , Animais , Apoptose , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Camundongos , Camundongos Nus , RNA Longo não Codificante/genética
10.
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
11.
Fungal Biol ; 126(5): 385-394, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35501034

RESUMO

Endophytic fungi are capable of producing a great diversity of bioactive metabolites. However, the presence of silent and lowly expressed genes represents a main challenge for the discovery of novel secondary metabolites with different potential uses. Epigenetic modifiers have shown to perturb the production of fungal metabolites through the induction of silent biosynthetic pathways leading to an enhanced chemical diversity. Moreover, the addition of bioprecursors to the culture medium has been described as a useful strategy to induce specific biosynthetic pathways. The aim of this study was to assess the effects of different chemical modulators on the metabolic profiles of an endophytic fungal strain of Cophinforma mamane (Botryosphaeriaceae), known to produce 3 thiodiketopiperazine (TDKP) alkaloids (botryosulfuranols A-C), previously isolated and characterized by our team. Four epigenetic modifiers, 5-azacytidine (AZA), sodium butyrate (SB), nicotinamide (NIC), homoserine lactone (HSL) as well as 2 amino acids, l-phenylalanine and l-tryptophan, as bioprecursors of TDKPs, were used. The metabolic profiles were analysed by UHPLC-HRMS/MS under an untargeted metabolomics approach. Our results show that the addition of the two amino acids in C. mamane culture and the treatment with AZA significantly reduced the production of the TDKPs botryosulfuranols A, B and C. Interestingly, the treatment with HSL significantly induced the production of different classes of diketopiperazines (DKPs). The treatment with AZA resulted as the most effective epigenetic modifier for the alteration of the secondary metabolite profile of C. mamane by promoting the expression of cryptic genes.


Assuntos
Aminoácidos , Ascomicetos , Aminoácidos/metabolismo , Ascomicetos/metabolismo , Azacitidina/metabolismo , Azacitidina/farmacologia , Epigênese Genética
12.
Biochim Biophys Acta Bioenerg ; 1863(6): 148566, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35489443

RESUMO

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.


Assuntos
Traumatismo por Reperfusão Miocárdica , Trifosfato de Adenosina , Animais , Azacitidina/farmacologia , Citocromos b/genética , DNA Mitocondrial/genética , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos
13.
Biochem Biophys Res Commun ; 609: 75-83, 2022 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-35421632

RESUMO

The present study was to identify abnormal methylation genes implicated in esophageal squamous cell carcinoma (ESCC). Genomic methylation alterations in ESCC tissues were analyzed using laser-microdissection and whole-genome bisulfite sequencing. CXCL14 promoter was frequently hypermethylated in ESCC tissues. The correlation of CXCL14 hypermethylation status and the mRNA and protein expression levels were validated using nested methylation-specific PCR (nMS-PCR), RNAscope in situ hybridization (RISH) and Western blot. RISH results showed completely negative CXCL14 expression in 34.3% (34/99) ESCC, compared with those in the basal layer cells of normal epithelia. Low expression of CXCL14 was more present in patients with lower differentiation. The anticancer role of CXCL14 has been commonly associated with immune regulation in the literature. Here, we observed by functional analysis that CXCL14 can also act as a tumor suppressor in ESCC cells. 5-Aza-dC treatment suppressed CXCL14 methylation and up-regulated the expression of CXCL14. Ectopic expression of CXCL14 suppressed the proliferation, invasion, tumor growth, and lung metastasis of ESCC cells. Both ectopic expression and induction of CXCL14 with 5-Aza-dC inhibited the activity of SRC, MEK1/2 and STAT3 in ESCC cells, while activated EGFR. Importantly, a combination of CXCL14 expression and SRC or EGFR inhibitor dramatically repressed the proliferation of ESCC cells and the growth of xenografts. Our findings revealed a direct tumor suppressor role of CXCL14, but not through the immune system. The data suggest that for ESCC patients with low level CXCL14, increasing CXCL14 expression combined with inhibition of SRC or EGFR might be a promising therapeutic strategy.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Metilação de DNA , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fenótipo
14.
Cells ; 11(7)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35406777

RESUMO

5-Azacytidine (5-azaC), a methyltransferase inhibitor and anticancer drug, can promote several cellular stress responses such as apoptosis, autophagy, and senescence. The action of 5-azaC is complex and can be modulated by dose, time of treatment, and co-administration with oxidants. Insulinoma is a rare pancreatic neuroendocrine tumor with limited chemotherapeutic options. In the present study, two cellular models of insulinoma were considered, namely NIT-1 and ß-TC-6 mouse cells, to evaluate the effects of 5-azaC post-treatment during hydrogen peroxide-induced oxidative stress. 5-azaC attenuated the development of oxidant-induced senescent phenotype in both cell lines. No pro-apoptotic action of 5-azaC was observed in cells treated with the oxidant. On the contrary, 5-azaC stimulated an autophagic response, as demonstrated by the increase in phosphorylated eIF2α and elevated pools of autophagic marker LC3B in oxidant-treated ß-TC-6 cells. Notably, autophagy resulted in increased necrotic cell death in ß-TC-6 cells with higher levels of nitric oxide compared to less affected NIT-1 cells. In addition, 5-azaC increased levels of RNA methyltransferase Trdmt1, but lowered 5-mC and m6A levels, suggesting Trdmt1 inhibition. We postulate that the 5-azaC anticancer action may be potentiated during oxidative stress conditions that can be used to sensitize cancer cells, at least insulinoma cells, with limited drug responsiveness.


Assuntos
Antineoplásicos , Insulinoma , Neoplasias Pancreáticas , Animais , Autofagia , Azacitidina/farmacologia , Metiltransferases , Camundongos , Oxidantes , Estresse Oxidativo , Neoplasias Pancreáticas/tratamento farmacológico
15.
Chemistry ; 28(26): e202200640, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35285586

RESUMO

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.


Assuntos
Azacitidina , Inibidores Enzimáticos , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Metilação de DNA , Decitabina/farmacologia , Decitabina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Humanos
16.
Drug Resist Updat ; 61: 100805, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35227933

RESUMO

Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Idoso , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Decitabina/farmacologia , Decitabina/uso terapêutico , Resistência a Medicamentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética
17.
Genes (Basel) ; 13(3)2022 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-35328043

RESUMO

The ionotropic glutamate receptor 6 (GluR6 or GRIK2) gene is transcribed by two cell-type-specific promoters in neuronal and non-neuronal cells, which results in five different transcript variants. The purpose of this study was to explore cell-type-specific silencing of these promoters by epigenetic mechanisms. The neuronal and non-neuronal promoter sequences were cloned upstream of the luciferase gene in the pGL3 luciferase reporter vector. Promoter susceptibility to methylation was confirmed by 5-azacytidine and trichostatin treatment, and the status of CpG dinucleotides was determined by bisulfite sequencing of the promoter was determined by bisulfite sequences. GluR6A transcript variant was expressed in the brain, and GluR6B was most abundant in tumor cell lines. The neuronal promoter was methylated in non-neuronal cell lines. The treatment with 5-azacytidine and trichostatin upregulated transcription of the GluR6 gene, and methylation of the GluR6 promoter sequence in the luciferase reporter system led to downregulation of the luciferase gene transcription. Bisulfite sequencing revealed methylation of 3 and 41 CpG sites in non-neuronal and neuronal promoters, respectively. The differential activation/silencing of GluR6 promoters suggests that the transcript variants of GluR6 are involved in tissue-specific biological processes and their aberrant regulation in tumor cells may contribute to distinct properties of tumor cells.


Assuntos
Carcinoma , Metilação de DNA , Azacitidina/farmacologia , Carcinoma/genética , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA/genética , Humanos , Luciferases/genética
18.
Int J Mol Sci ; 23(3)2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35163334

RESUMO

One important role of epigenetic regulation is controlling gene expression in development and homeostasis. However, little is known about epigenetics' role in regulating opsin expression. Cell cultures (HEK 293, Y79, and WERI) producing different levels of opsins were treated with 5-aza-2'-deoxycytidine (5-Aza-dc) and/or sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA) for 72 h. Global DNA methylation, site-specific methylation, and expressions of opsins were measured by LUMA assay, bisulfite pyrosequencing, and qPCR, respectively. Mouse retinal explants from wild-type P0/P1 pups were ex vivo cultured with/without 5-Aza-dc or SAHA for 6 days. The morphology of explants, DNA methylation, and expressions of opsins was examined. The drugs induced global DNA hypomethylation or increased histone acetylation in cells, including DNA hypomethylation of rhodopsin (RHO) and L-opsin (OPN1LW) and a concomitant increase in their expression. Further upregulation of RHO and/or OPN1LW in HEK 293 or WERI cells was observed with 5-Aza-dc and either SB or SAHA combination treatment. Mouse retinal explants developed normally but had drug-dependent differential DNA methylation and expression patterns of opsins. DNA methylation and histone acetylation directly regulate opsin expression both in vitro and ex vivo. The ability to manipulate opsin expression using epigenetic modifiers enables further study into the role of epigenetics in eye development and disease.


Assuntos
Metilação de DNA , Histonas , Acetilação , Animais , Azacitidina/farmacologia , Decitabina/farmacologia , Epigênese Genética , Expressão Gênica , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Opsinas/genética , Opsinas/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo
19.
Cancer Genomics Proteomics ; 19(2): 205-228, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35181589

RESUMO

BACKGROUND/AIM: Prediction of response to azacitidine (AZA) treatment is an important challenge in hematooncology. In addition to protein coding genes (PCGs), AZA efficiency is influenced by various noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs), and transposable elements (TEs). MATERIALS AND METHODS: RNA sequencing was performed in patients with myelodysplastic syndromes or acute myeloid leukemia before AZA treatment to assess contribution of ncRNAs to AZA mechanisms and propose novel disease prediction biomarkers. RESULTS: Our analyses showed that lncRNAs had the strongest predictive potential. The combined set of the best predictors included 14 lncRNAs, and only four PCGs, one circRNA, and no TEs. Epigenetic regulation and recombinational repair were suggested as crucial for AZA response, and network modeling defined three deregulated lncRNAs (CTC-482H14.5, RP11-419K12.2, and RP11-736I24.4) associated with these processes. CONCLUSION: The expression of various ncRNAs can influence the effect of AZA and new ncRNA-based predictive biomarkers can be defined.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , RNA Longo não Codificante , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Epigênese Genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , RNA Longo não Codificante/genética
20.
Mol Med Rep ; 25(4)2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35169860

RESUMO

In recent years, researchers have found that epigenetics plays an important role in the occurrence and development of hepatocellular carcinoma (HCC). DNA methylation is involved in the proliferation and metastasis of HCC. However, the junctophilin 3 (JPH3) level and the potential regulatory mechanism of its DNA methylation in HCC remain uncertain. In the present study, 73 HCC samples were enrolled to analyze the expression of JPH3. Reverse­transcription quantitative PCR, western blotting and immunohistochemistry were used to detect the expression of JPH3 in HCC. Kaplan­Meier method and Cox regression analysis were applied to evaluate the prognostic impact of JPH3 on HCC patients. DNA methylation­specific PCR and bisulfite Sanger sequencing were used to detect the degree of DNA methylation of JPH3 in HCC. The demethylation drug 5­Aza­2'­deoxycytidine (5­Aza) was used to reduce the DNA methylation of JPH3. The role of JPH3 in the malignant biological behavior of HCC by promoting epithelial­mesenchymal transition (EMT) was confirmed by functional cell experiments. The results showed that JPH3 exhibited low levels in HCC tissues and cell lines. HCC patients with low expression of JPH3 had poor survival outcomes. JPH3 had higher DNA methylation levels in HCC tissues and cell lines. When the demethylation drug 5­Aza was used to reduce the degree of methylation of JPH3, its protein expression level was significantly increased and it significantly inhibited the malignant biological behavior of HCC cells. Additionally, effective increase in the expression of JPH3 through gene regulation technology also inhibited the proliferation, invasion and migration of HCC cells. After altering the DNA methylation level of JPH3, the EMT of HCC cells was also affected. Therefore, our study demonstrated the inactivation of JPH3 by promoter methylation and its function as a tumor suppressor in HCC. JPH3 may serve as a biomarker for early diagnosis and as a potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Adulto , Idoso , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas
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