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Nanomolar treatment with epigenetic drug combination induces genome-wide methylation and expression alterations in neuro-ectodermal cell lines [DNA methylation]

ABSTRACT: Epigenetic alterations are a fundamental aspect of cancer cells, and epigenetic drugs are currently used in clinical practice for hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and show epigenetic defects of apoptotic pathways, which points to sensitivity towards epigenetic drugs in this patient group. The young age of these patients is accompanied by However, ongoing developmental processes regulated by epigenetic mechanisms may be deregulated by epigenetic drugs in this patient group that is characterized by young age. This prompted us to study molecular effects and side-effects of low dosage epigenetic drugs in neuro-ectodermal tumor cell lines of pediatric origin. Short term combination treatment of 5-aza-2`-deoxicytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages reduced proliferation, induced wide-spread demethylating effects in 17 NBL and 5 PNET cell lines, and was accompanied by large effects on gene-expression profiles. Approximately half of the genes that were significantly upregulated upon treatment demonstrated significant demethylating effects in their promoter regions. In NBL cell lines, almost every cellular pathway (193/200) investigated demonstrated altered expression upon treatment, and resulted in upregulation of known epigenetically regulated genes such as X-chromosomal, tissue-specific, and a limited number of imprinted genes, but also known tumor suppressor genes and oncogenes. In conclusion, genome-wide methylation and gene expression changes are induced DAC and TSA treatment at nanomolar dosages. This treatment affected more than 97% of cellular pathways investigated and further studies towards the effectiveness and side-effects of epigenetic drugs are desirable in pediatric tumors. Epigenetic alterations are a fundamental aspect of cancer cells, and epigenetic drugs are currently used in clinical practice for hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and show epigenetic defects of apoptotic pathways, which points to sensitivity towards epigenetic drugs in this patient group. The young age of these patients is accompanied by However, ongoing developmental processes regulated by epigenetic mechanisms may be deregulated by epigenetic drugs in this patient group that is characterized by young age. This prompted us to study molecular effects and side-effects of low dosage epigenetic drugs in neuro-ectodermal tumor cell lines of pediatric origin. Short term combination treatment of 5-aza-2`-deoxicytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages reduced proliferation, induced wide-spread demethylating effects in 17 NBL and 5 PNET cell lines, and was accompanied by large effects on gene-expression profiles. Approximately half of the genes that were significantly upregulated upon treatment demonstrated significant demethylating effects in their promoter regions. In NBL cell lines, almost every cellular pathway (193/200) investigated demonstrated altered expression upon treatment, and resulted in upregulation of known epigenetically regulated genes such as X-chromosomal, tissue-specific, and a limited number of imprinted genes, but also known tumor suppressor genes and oncogenes. In conclusion, genome-wide methylation and gene expression changes are induced DAC and TSA treatment at nanomolar dosages. This treatment affected more than 97% of cellular pathways investigated and further studies towards the effectiveness and side-effects of epigenetic drugs are desirable in pediatric tumors.

ORGANISM(S): Homo sapiens

PROVIDER: GSE35506 | GEO | 2012/07/01

SECONDARY ACCESSION(S): PRJNA156103

REPOSITORIES: GEO

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Project description:Reversal of gene promoter DNA hypermethylation and associated abnormal gene silencing is an attractive approach to cancer therapy. The DNA methylation inhibitor, decitabine (5-aza-2'-deoxycitidine), is proving efficacious for hematological neoplasms especially at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, but these may not explain the prolonged time to response seen in patients. Transient exposure of leukemic and solid tumor cells to clinically-relevant nanomolar doses, without causing immediate cytotoxicity or apoptosis, produces sustained reduced tumorigenicity, and for leukemia cells, diminished long-term self-renewal. These effects appear triggered by cellular reprogramming and include sustained decreases in promoter DNA methylation with associated gene re-expression, and anti-tumor changes in multiple key cellular regulatory pathways, most of which are high priority targets for pharmacologic anti-cancer strategies. Thus, low dose decitabine regimens appear to have broad applicability for cancer management. [Gene expression profiling] Leukemia cell lines Kasumi-1 and KG1A are treated with 10nM DAC during 72 hours and gene expression was assayed at day 3, 7 and 14 after the start of the treatment. Appropriate mock treated samples were used as control in each case. In addition, Kasumi-1 cells were also treated with a higher dose of DAC (500nM), 100nM ARA-C and 300 nM TSA, again controlled against mock treated Kasumi-1 cells, to separate dose and agent dependent effects. MCF7 was studied as an example of a solid tumor cell line. Therefore MCF7 cells were treated with 100nM DAC and results were assayed at day 1, day 3 and day 10. [Methylation profiling] The effects of the demethylating agent DAC were studied in the leukemia cell line Kasumi-1 over a 28 day time course. Intermediate time points were studied at days 3, 7, 14 and 21. These results were verfied in KG1A and KG1 leukemia cell lines, at one selected time point. The effects on one primary sample were also studied. Four normal leukemia samples (PL1, 2, 4 and 5) were used as general controls. The effect of DAC was compared to ARA-C, TSA. Both mock treated and day 3 DAC treated Kasumi-1 cells were repeated. These results were verified at one selected time point for the DAC treated MCF7 breast cancer cell line.

Project description:Purpose: We aimed to investigate the effect of several anti-leukemia drugs in combination with decitabine (DAC) on the proliferation of myeloid leukemia cells in vitro and in vivo, to select the most efficient combination group and explore associated mechanisms of these combination therapies. Experimental Design: After comparing with five anti-leukemia drugs in several different kinds of cell lines, the combination effect of idarubicin (IDA) with DAC was best. In vivo, by using microPET, TUNEL, and transmission electron microscopy, the inhibitory effects obtained by sequentially combining DAC with IDA, evidenced by evaluating tumor cell proliferation and cell apoptosis. Molecular studies were conducted using gene chip, which was used to explore associated pathways, and real-time quantitative reverse transcription-PCR, western blot and immunohistochemistry (IHC), used to assess regulation of Wnt/β-catenin pathway. Results: The sequential combination of DAC and IDA showed synergistic induction of cell death in U937, HEL, SKM-1 and cells isolated from AML patients. Importantly, the inhibition of tumor growth in the sequential combination group was found to be significantly higher than that of single drug group or control group in vivo. Moreover, sequential treatment with DAC and IDA induced apoptosis and depression of the Wnt/β-catenin pathway in both culture and animal studies. Conclusions: Our findings showed that sequentially combining decitabine with idarubicin had a synergistic anti-leukemia effect. These findings were attributed to demethylation of Wnt pathway inhibitors and downregulation of Wnt pathway nuclear targets observed in vitro and in vivo. After comparing with five anti-leukemia drugs in several different kinds of cell lines, the combination effect of idarubicin (IDA) with DAC was best. In vivo, by using microPET, TUNEL, and transmission electron microscopy, the inhibitory effects obtained by sequentially combining DAC with IDA, evidenced by evaluating tumor cell proliferation and cell apoptosis. Molecular studies were conducted using gene chip, which was used to explore associated pathways, and real-time quantitative reverse transcription-PCR, western blot and immunohistochemistry (IHC), used to assess regulation of Wnt/β-catenin pathway.

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Nanomolar treatment with epigenetic drug combination induces genome-wide methylation and expression alterations in neuro-ectodermal cell lines [DNA methylation]

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