Effect of 6-TG on apoptosis and cell cycle in MCF-7 breast cancer cells and its mechanisms through the regulation of DNMT1
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ABSTRACT:
PROVIDER: PRJNA534122 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8943430_1.fastq.gz | Fastqsanger.gz | |||
| SRR8943430_2.fastq.gz | Fastqsanger.gz | |||
| SRR8943431_1.fastq.gz | Fastqsanger.gz | |||
| SRR8943431_2.fastq.gz | Fastqsanger.gz | |||
| SRR8943432_1.fastq.gz | Fastqsanger.gz |
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Project description:Thioguanine (6-TG) as conventional drug has anti-tumor activity in various cancer cell lines. However, the effect of 6-TG on luminal subtypes breast cancer (ER+, PR+) is elusive. In this study, effect of 6-TG on MCF-7 breast cancer cell line (ER+, PR+) and its mechanisms were investigated. MCF-7 cells were treated with 6-TG and IC50 value was measured by cell counting kit-8 assay. DEGs were confirmed by RNA-seq. Apoptosis and cell cycle consequences were checked by flow cytometry and western blot. Our results showed that colony formation decreased obviously after 6-TG treatment. Moreover, with the treatment of 6-TG cell apoptosis percentage increased. DNMT1 mRNA and protein expression decreased and FAS expression increased in both expression level. Meanwhile, 6-TG treatment also induced MCF-7 cells to occur G2/M phase cell cycle arrest. Overall, our results indicated that 6-TG suppressed cell proliferation, induced G2/M phase arrest through DNMT1 mediated p53-dependent p21 signaling, and accelerated cell apoptosis through activated extrinsic apoptosis pathway which was caused by DNMT1 mediated FAS signaling.
2020-03-02 | GSE130161 | GEO
Project description:The dynamic balance of DNA methylation and demethylation is required for erythropoiesis. Our previous transcriptomic analyses revealed that DNA methyltransferase 1 (DNMT1) is abundantly expressed in erythroid cells at all developmental stages. However, the role and molecular mechanisms of DNMT1 in human erythropoiesis remain unknown. Here we found that DNMT1 deficiency led to cell cycle arrest of erythroid progenitors which was partially rescued by treatment with a p21 inhibitor UC2288. Mechanically, this is due to decreased DNA methylation of p21 promoter, leading to upregulation of p21 expression. In contrast, DNMT1 deficiency led to increased apoptosis during terminal stage by inducing endoplasmic reticulum (ER) stress in a p21 independent manner. ER stress was attributed to the upregulation of RPL15 expression due to the decreased DNA methylation at RPL15 promoter. The upregulated RPL15 expression subsequently caused a significant upregulation of core ribosomal proteins (RPs) and thus ultimately activated all branches of unfolded protein response (UPR)leading to the excessive ER stress, suggesting a role of DNMT1 in maintaining protein homeostasis during terminal erythroid differentiation. Furthermore, the increased apoptosis was significantly rescued by the treatment of ER stress inhibitor TUDCA. Our findings demonstrate the stage-specific role of DNMT1 in regulating human erythropoiesis and provide new insights into regulation of human erythropoiesis.
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Project description:Background: Cellular apoptosis is a central mechanism the chemotherapy leveraged to treat human cancers. Cytosine methylations installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Methods: Cisplatin and doxorubicin were used to induce cell apoptosis in three differed types of osteosarcoma cell lines, respectively. Bisulfite-PCR, MS-PCR, and MeDIP experiments were employed to evaluate promoter cytosine methylation, and MeRIP were performed to investigate mRNA cytosine methylation. MeRIP-seq revealed the common target genes regulated by DNMT1 and NSun2. Particular chimeric reporter constructs were generated and dual luciferase assays dissected corresponding regulations via DNA and/or mRNA methylation. Cell viability assay, apoptosis analysis by flow cytometry, and tumor xenograft model were utilized to determine osteosarcoma drug resistance. Results: Promoter methylation by DNMT1 dialogues with mRNA methylation by NSun2 to coordinately regulate osteosarcoma cell apoptosis. DNMT1 was induced in osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was dampened. DNMT1 was found to represses NSun2 expression by methylating NSun2 promoter. Moreover, DNMT1 and NSun2 regulate anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced these anti-apoptotic gene expressions via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma to apoptosis. Conclusions: Our findings establish a crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and add additional layers to the control of gene expression at different epigenetic levels.
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2020-07-31 | GSE155467 | GEO
Project description:Dramatic change in DNA methylation patterns and levels both globally and/or locus-specifically is associated with the process of cell linage specification and somatic reprogramming. We found the expression of DNA methyltransferase 1 (Dnmt1) is tightly regulated by the features of cell cycle, which we referred as cell cycle-directed Dnmt1 expression adjustment. To further investigate how DNA methylation is affected by this mechanism, we applied reduced representation bisulphate sequencing (RRBS) to map genome-wide DNA methylation on mouse embryonic fibroblasts (MEFs). To characterize how this mechanism affects somatic reprogramming, samples of MEFs induced by Yamanaka factors (Sox2, Klf4, Oct4, cMyc) was included. The cell cycle was accelerated by shRNA targeted to p53, and the expression of Dnmt1 was manipulated genetically. We find 5mC level remains constant in regardless of cell proliferation rate, a result from the adjusted expression of Dnmt1. However, repressed expression of Dnmt1 in fast-proliferating cells results in global DNA demethylation. The patterns of DNA methylation and demethylation affected by this mechanism is sensitive to CpG densities. Generally, the results demonstrate cell cycle-directed Dnmt1 expression adjustment as an mechanism for the insurance of the stability of genomic 5mC inheritance.
2017-10-13 | GSE93058 | GEO
Project description:Cancer’s global burden highlights the urgent need for more effective therapies. Metalbased drugs, particularly copper complexes, offer promising alternatives due to copper’s diverse biological functions. This study investigates the antitumor potential of two novel dinuclear Cu²⁺ complexes, [Cu2(µ-CH3COO)(L)(OH2)]·2H2O (R9) and [Cu2(µ-OH)(HL)(OH2)]ClO4·2H2O (R10), in MCF-7 breast cancer cells. Both compounds exhibited greater cytotoxicity than cisplatin, with IC₅₀ values of 1.01 ± 0.09 μM (R9) and 1.27 ± 0.14 μM (R10), while showing selectivity toward cancer cells, as indicated by higher IC₅₀ values in healthy MCF10A cells. Treated MCF-7 cells showed increased granularity, mitochondrial membrane depolarization, and elevated reactive oxygen species. At IC₅₀ concentrations, cell cycle analysis revealed Sub-G1 accumulation and DNA fragmentation (TUNEL assay), indicating apoptosis via intrinsic pathways, supported by caspase 9 activation. Label-free proteomics revealed distinct mechanisms for R10 compared to cisplatin. In R10-treated cells, key downregulated pathways included actin cytoskeleton regulation, viral carcinogenesis, and PI3K-Akt signaling; upregulated pathways involved ribosome biogenesis, necroptosis, and metabolism. Apoptosis-related proteins such as lamin B1, NRAS, and MAPKs were downregulated, while AIFM1 was upregulated. These findings support the potential of dinuclear Cu²⁺ complexes as effective antitumor agents with mechanisms distinct from cisplatin, possibly offering superior efficacy through apoptosis induction.
2025-11-24 | PXD064464 | Pride
Project description:To generate association of DNA methylation level and gene expression profile, we applied RNA-sequencing on mouse embryonic fibroblasts (MEFs) after the modulation of cell cycle and Dnmt1 expression level. To characterize how these factors affect somatic reprogramming, samples of MEFs induced by Yamanaka factors (Sox2, Klf4, Oct4, cMyc) was also included. The cell cycle was accelerated by shRNA targeted to p53, and the expression of Dnmt1 was manipulated by either over expression or knock down.
2017-10-13 | GSE93416 | GEO
Project description:The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on DNMT1. Functional studies further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that is preferentially at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.
2023-07-26 | PXD043031 | Pride
Project description:BACKGROUND: Benzo[a]pyrene (BaP) is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR) induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA) changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP) 1A1 and CYP1B1, which are involved in BaP metabolism. RESULTS: Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE), the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. CONCLUSIONS: This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell cycle. Our results suggest that growth kinetics within a target-cell population may be important determinants of susceptibility and response to a genotoxic agent.
2012-02-02 | GSE26917 | GEO
Project description:Targeting cell cycle and apoptosis to overcome chemoresistance in AML
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