Different bisphenols induce non-monotonous changes in miRNA expression and LINE-1 methylation in two cell lines

Abstract 4,4ʹ-Isopropylidenediphenol (bisphenol A, BPA), a chemical substance that is widely used mainly as a monomer in the production of polycarbonates, in epoxy resins, and in thermal papers, is suspected to cause epigenetic modifications with potentially toxic consequences. Due to its negative health effects, BPA is banned in several products and is replaced by other bisphenols such as bisphenol S and bisphenol F. The present study examined the effects of BPA, bisphenol S, bisphenol F, p,pʹ-oxybisphenol, and the BPA metabolite BPA β-d-glucuronide on the expression of a set of microRNAs (miRNAs) as well as long interspersed nuclear element-1 methylation in human lung fibroblast and Caco-2 cells. The results demonstrated a significant modulation of the expression of different miRNAs in both cell lines including miR-24, miR-155, miR-21, and miR-146a, known for their regulatory functions of cell cycle, metabolism, and inflammation. At concentrations between 0.001 and 10 µg/ml, especially the data of miR-155 and miR-24 displayed non-monotonous and often significant dose–response curves that were U- or bell-shaped for different substances. Additionally, BPA β-d-glucuronide also exerted significant changes in the miRNA expression. miRNA prediction analysis indicated effects on multiple molecular pathways with relevance for toxicity. Besides, long interspersed nuclear element-1 methylation, a marker for the global DNA methylation status, was significantly modulated by two concentrations of BPA and p,pʹ-oxybisphenol. This pilot study suggests that various bisphenols, including BPA β-d-glucuronide, affect epigenetic mechanisms, especially miRNAs. These results should stimulate extended toxicological studies of multiple bisphenols and a potential use of miRNAs as markers.

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Epigenetic Regulation of MicroRNA Expression In Hodgkin Lymphoma

Blood ◽

10.1182/blood.v116.21.3885.3885 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3885-3885

Author(s):

Alfons Navarro ◽

Tania Díaz ◽

Marina Díaz Beyá ◽

Antonio Martinez ◽

Gerardo Ferrer ◽

...

Keyword(s):

Dna Methylation ◽

B Cells ◽

Tumor Suppressor ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Mirna Expression ◽

Methylation Status ◽

Promoter Regions ◽

Tumor Suppressor Mirnas

Abstract Abstract 3885 Background: In recent years, microRNAs (miRNAs) have emerged as key regulators of carcinogenesis. miRNA expression is deregulated in multiple hematological malignancies. One of the mechanisms that can affect miRNA expression is methylation in the promoter regions of miRNA genes. The main objective of the present study was to identify tumor suppressor miRNAs that are silenced by alterations in gene methylation in Hodgkin Lymphoma (HL). In addition, since demethylating agents could affect miRNA expression, we evaluated the in vitro effectiveness of 5-Aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, in HL cell lines. Methods: To detect miRNAs regulated by methylation, we analyzed the expression of 670 mature miRNAs in two HL cell lines, L-428 and L-1236, before and after AZA treatment, by TaqMan Human MicroRNA Arrays V2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2-ΔΔCt method using RNU48 as endogenous control. To validate the methylation status, genomic DNA samples from four HL cell lines (L-428, L-1236, HDMYZ and L-540) and peripheral B-cells from healthy donors were modified by sodium bisulfite using the EZ DNA Methylation kit (Zymo Research). The DNA methylation status of these samples was analyzed by methylation specific PCR (MSP) after sodium bisulfite modification of DNA. To evaluate AZA in vitro effectiveness, we treated the four HL cell lines daily with 20 nM, 250 nM, 1 μM and 5 μM of AZA or DMSO (vehicle control), and we assayed proliferation with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) after 72 hours of treatment. Results: After AZA treatment in the L-428 and L-1236 HL cell lines, 32 miRNAs (of 670 analyzed) were expressed de novo, 15 of which were present in both cell lines. miR-34a, miR-203, miR-342, miR-105, miR-490, miR-375 and the miR-500 family had well-defined CpG islands in their promoter regions. The MSP analysis showed that miR-34a, miR-203, miR-490 and miR-375 were methylated in L-428 and L-1236 HL cell lines, but not in the normal B-cells. miR-342 was methylated in L-1236 HL cell line, but not in L-428 nor normal B-cells. To further validate this we analyzed the methylation pattern in HDMYZ and L-540 HL cell lines and found that only miR-203, miR-375 and miR-490 were methylated in all 4 HL cell lines analyzed. We observed an AZA dose-dependent reduction in proliferation in all four HL cell lines. After 72 hours of treatment with AZA at 5μM, proliferation significantly decreased by 29% in L-428, 36% in L-1236, 28% in HDMYZ and 22% in L-540. Moreover, the analysis of miRNA levels during the treatment showed a re-expression of methylated miRNAs after 48 hours. The methylation analysis of these miRNAs in laser captured microdisected HL cells from patients and functional analysis are ongoing and complete data will be reported. Conclusions: In summary, HL exhibits a characteristic epigenetic pattern which includes the methylation of key miRNAs during the carcinogenesis process, such as miR-34a and miR-203, which have previously been shown to act as tumor suppressors in lung, colorectal and other cancers. Moreover, we present evidence that demethylating agents allow the re-expression of these tumor suppressor miRNAs, suggesting that they could be a promising novel treatment for HL patients. Supported by FIS grant (PS09/00547). Disclosures: No relevant conflicts of interest to declare.

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Design, Synthesis and Cytotoxicity Evaluation of New 3, 5-Disubstituted-2-Thioxoimidazolidinones

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666171129213838 ◽

2018 ◽

Vol 18 (4) ◽

pp. 573-582 ◽

Cited By ~ 1

Author(s):

Khaled R.A. Abdellatif ◽

Mostafa M. Elbadawi ◽

Mohammed T. Elsaady ◽

Amer A. Abd El-Hafeez ◽

Takashi Fujimura ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Topoisomerase I ◽

Cancer Cell Line ◽

Cytotoxic Agents ◽

Dependent Manner ◽

Human Lung Fibroblast ◽

Mcf 7

Background: Some 2-thioxoimidazolidinones have been reported as anti-prostate and anti-breast cancer agents through their inhibitory activity on topoisomerase I that is considered as a potential chemotherapeutic target. Objective: A new series of 3,5-disubstituted-2-thioxoimidazolidinone derivatives 10a-f and their S-methyl analogs 11a-f were designed, synthesized and evaluated for cytotoxicity against human prostate cancer cell line (PC-3), human breast cancer cell line (MCF-7) and non-cancerous human lung fibroblast cell line (WI-38). </P><P> Results and Method: While compounds 10a-f showed a broad range of activities against PC-3 and MCF-7 cell lines (IC50 = 34.0 – 186.9 and 24.6 – 147.5 µM respectively), the S-methyl analogs 11a-f showed (IC50 = 22.7 – 198.5 and 16.9 – 188.2 µM respectively) in comparison with 5-fluorouracil (IC50 = 60.7 and 40.7 µM respectively). 11c (IC50 = 22.7 and 29.2 µM) and 11f (IC50 = 28.7 and 16.9 µM) were the most potent among all compounds against both PC-3 and MCF-7 respectively with no cytotoxicity against WI-38. Conclusion: The newly synthesized compounds showed good activity against PC-3 and MCF-7 cell lines in comparison with 5-fluorouracil. Compounds 11c and 11f bound with human topoisomerase I similar to its known inhibitors and significantly inhibited its DNA relaxation activity in a dose dependent manner which may rationalize their molecular mechanism as cytotoxic agents.

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SUV39H1-Mediated DNMT1 is Participated in the Epigenetic Regulation of Smad3 in Cervical Cancer

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721110016 ◽

2020 ◽

Vol 20 ◽

Author(s):

Li Zhang ◽

Sijuan Tian ◽

Minyi Zhao ◽

Ting Yang ◽

Shimin Quan ◽

...

Keyword(s):

Cervical Cancer ◽

Cell Lines ◽

Epigenetic Regulation ◽

Promoter Region ◽

Methylation Status ◽

Regulation Mechanism ◽

Methylation Specific Pcr ◽

Specific Pcr ◽

Immune Factor ◽

Cancer Tissues

Background: Smad3 is a pivotal intracellular mediator for participating in the activation of multiple immune signal pathway. Objective: The epigenetic regulation mechanism of the positive immune factor Smad3 in cervical cancer remains unknown. Therefore, the epigenetic regulation on Smad3 is investigated in this study. Methods: The methylation status of SMAD3 was detected by Methylation-specific PCR (MS-PCR) and Quantitative Methylation-specific PCR (MS-qPCR) in cervical cancer tissues and cell lines. The underlying molecular mechanisms of SUV39H1-DNMT1-Smad3 regulation was elucidated using cervical cancer cell lines containing siRNA or/and overexpression system. Confirmation of the regulation of DNMT1 by SUV39H1 used Chromatin immunoprecipitation-qPCR (ChIP-qPCR). The statistical methods used for comparing samples between groups were paired t tests and one-way ANOVAs. Results: H3K9me3 protein which regulated by SUV39H1 directly interacts with the DNMT1 promoter region to regulate its expression in cervical cancer cells, resulting in the reduce expression of the downstream target gene DNMT1. In addition, DNMT1 mediates the epigenetic modulation of the SMAD3 gene by directly binding to its promoter region. The depletion of DNMT1 effectively restores the expression of Smad3 in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1-DNMT1 was found to correlate with Smad3 expression in accordance with the expression at the cellular level. Notably, the promoter region of SMAD3 was hypermethylated in cervical cancer tissues, and this hypermethylation inhibits the subsequent gene expression. Conclusion: These results indicate that SUV39H1-DNMT1 is a crucial Smad3 regulatory axis in cervical cancer. SUV39H1-DNMT1 axis may provide a potential therapeutic target for the treatment of cervical cancer.

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Polyphenol-Enriched Blueberry Preparation Controls Breast Cancer Stem Cells by Targeting FOXO1 and miR-145

Molecules ◽

10.3390/molecules26144330 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4330

Author(s):

Jean-François Mallet ◽

Roghayeh Shahbazi ◽

Nawal Alsadi ◽

Chantal Matar

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Mirna Expression ◽

Epigenetic Mechanism ◽

Breast Cancer Cell Lines ◽

Relative Expression ◽

Target Proteins ◽

Pathological Characteristics

Scientific evidence supports the early deregulation of epigenetic profiles during breast carcinogenesis. Research shows that cellular transformation, carcinogenesis, and stemness maintenance are regulated by epigenetic-specific changes that involve microRNAs (miRNAs). Dietary bioactive compounds such as blueberry polyphenols may modulate susceptibility to breast cancer by the modulation of CSC survival and self-renewal pathways through the epigenetic mechanism, including the regulation of miRNA expression. Therefore, the current study aimed to assay the effect of polyphenol enriched blueberry preparation (PEBP) or non-fermented blueberry juice (NBJ) on the modulation of miRNA signature and the target proteins associated with different clinical-pathological characteristics of breast cancer such as stemness, invasion, and chemoresistance using breast cancer cell lines. To this end, 4T1 and MB-MDM-231 cell lines were exposed to NBJ or PEBP for 24 h. miRNA profiling was performed in breast cancer cell cultures, and RT-qPCR was undertaken to assay the expression of target miRNA. The expression of target proteins was examined by Western blotting. Profiling of miRNA revealed that several miRNAs associated with different clinical-pathological characteristics were differentially expressed in cells treated with PEBP. The validation study showed significant downregulation of oncogenic miR-210 expression in both 4T1 and MDA-MB-231 cells exposed to PEBP. In addition, expression of tumor suppressor miR-145 was significantly increased in both cell lines treated with PEBP. Western blot analysis showed a significant increase in the relative expression of FOXO1 in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Furthermore, a significant decrease was observed in the relative expression of N-RAS in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Our data indicate a potential chemoprevention role of PEBP through the modulation of miRNA expression, particularly miR-210 and miR-145, and protection against breast cancer development and progression. Thus, PEBP may represent a source for novel chemopreventative agents against breast cancer.

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DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2922-2927.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2922-2927

Author(s):

I L Andrulis ◽

M T Barrett

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary Cells ◽

Methylation Status ◽

Chinese Hamster ◽

Resistant Cell ◽

Methylation Pattern ◽

Asparagine Synthetase ◽

Synthetase Activity ◽

Dna Hypomethylation ◽

Ovary Cells

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

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A comparison between SOLiD 5500XLand Ion Torrent PGM-derived miRNA expression profiles in two breast cell lines

Genetics and Molecular Biology ◽

10.1590/1678-4685-gmb-2018-0351 ◽

2020 ◽

Vol 43 (2) ◽

Cited By ~ 1

Author(s):

Gabriela Pereira Branco ◽

Renan Valieris ◽

Lucas Venezian Povoa ◽

Luiza Ferreira de Araújo ◽

Gustavo Ribeiro Fernandes ◽

...

Keyword(s):

Cell Lines ◽

Mirna Expression ◽

Expression Profiles ◽

Ion Torrent ◽

Ion Torrent Pgm ◽

Breast Cell Lines ◽

Mirna Expression Profiles

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Bisphenol S and bisphenol F are less disruptive to cardiac electrophysiology and potentially safer for use in medical products, as compared to bisphenol A

10.1101/2021.02.16.431039 ◽

2021 ◽

Author(s):

Tomas Prudencio ◽

Luther Swift ◽

Devon Guerrelli ◽

Blake Cooper ◽

Marissa Reilly ◽

...

Keyword(s):

Bisphenol A ◽

Safety Profile ◽

Intracellular Signaling ◽

Cardiac Electrophysiology ◽

Field Potential ◽

Production Volume ◽

Bisphenol S ◽

Bisphenol F ◽

Bisphenol Analogues ◽

Whole Heart

ABSTRACTBackgroundBisphenol A (BPA) is a high-production volume chemical that is commonly used to manufacture consumer and medical-grade plastic products. Due to its ubiquity, the general population can incur daily environmental exposure to BPA, while heightened BPA exposure has been reported in intensive care patients and industrial workers. Due to health concerns, structural analogues are being explored as replacements for BPA.ObjectiveThis study aimed to examine the direct nongenomic effects of BPA on cardiac electrophysiology and compare its safety profile to recently developed alternatives, including BPS (bisphenol S) and BPF (bisphenol F).MethodsWhole-cell voltage-clamp recordings were performed on cell lines transfected with Nav1.5, hERG, or Cav1.2. Results of single channel experiments were validated by conducting electrophysiology studies on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and intact, whole heart preparations.ResultsOf the chemicals tested, BPA was the most potent inhibitor of both fast (INa-P) and late (INa-L) sodium channel (IC50 = 55.3 and 23.6 μM, respectively), L-type calcium channel (IC50 = 30.8 μM) and hERG channel current (IC50 = 127 μM). The inhibitory effects of BPA and BPF on L-type calcium channels were supported by microelectrode array recordings, which revealed shortening of the extracellular field potential (akin to QT interval). Further, BPA and BPF exposure impaired atrioventricular conduction in intact, whole heart experiments. BPS did not alter any of the cardiac electrophysiology parameters tested.DiscussionResults of this study demonstrate that BPA and BPF exert an immediate inhibitory effect on cardiac ion channels, and that BPS may be a safer alternative. Intracellular signaling or genomic effects of bisphenol analogues were not investigated; therefore, additional mechanistic studies are necessary to fully elucidate the safety profile of bisphenol analogues on the heart.

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Baicalein, a Natural Anti-Cancer Compound, Alters MicroRNA Expression Profiles in Bel-7402 Human Hepatocellular Carcinoma Cells

Cellular Physiology and Biochemistry ◽

10.1159/000470815 ◽

2017 ◽

Vol 41 (4) ◽

pp. 1519-1531 ◽

Cited By ~ 22

Author(s):

Beibei Bie ◽

Jin Sun ◽

Jun Li ◽

Ying Guo ◽

Wei Jiang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Lines ◽

Mirna Expression ◽

Expression Profiles ◽

Akt Pathway ◽

Qrt Pcr ◽

Cycle Arrest ◽

Mirna Expression Profiles

Background/Aims: Baicalein has been shown to possess significant anti-hepatoma activity by inhibiting cell proliferation. Whether the anti-proliferative effect of baicalein is related to its modulation of miRNA expression in hepatocellular carcinoma (HCC) is still unknown. Methods: The anti-proliferative effects of baicalein on HCC cell line Bel-7402 was assessed by detecting the proliferation activity, cell cycle distribution, expression changes of p21/CDKN1A, P27/CDKN1B, total Akt and phosphoryted AKT. Microarray analysis was conducted to determine the miRNA expression profiles in baicalein-treated or untreated Bel-7402 cells and then validated by qRT-PCR in two HCC cell lines (Bel-7402 and Hep3B). The gain-of-function of miR-3127-5p was performed by detecting anti-proliferative effects after transfecting miRNA mimics in cells. Finally, the expression level of miR-3127-5p in different HCC cell lines was determined by qRT-PCR. Results: Baicalein was able to inhibit the proliferation of Bel-7402 cells by inducing cell cycle arrest at the S and G2/M phase via up-regulating the expression of p21/CDKN1A and P27/CDKN1B and suppressing the PI3K/Akt pathway. Baicalein could alter the miRNA expression profiles in Bel-7402 cells. Putative target genes for differentially expressed miRNAs could be enriched in terms of cell proliferation regulation, cell cycle arrest and were mainly involved in MAPK, PI3K-Akt, Wnt, Hippo and mTOR signaling pathways. MiR- 3127-5p, one of up-regulated miRNAs, exhibits low expression level in several HCC cell lines and its overexpression could inhibit cell growth of Bel-7402 and Hep3B cell lines by inducing S phase arrest by up-regulating the expression of p21and P27 and repressing the PI3K/Akt pathway. Conclusions: Modulation of miRNA expression may be an important mechanism underlying the anti-hepatoma effects of baicalein.

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