Project description:Our study had shown that DAC treatment enhanced immunogenecity of EL4 cells. To explore the mechanisms of DAC-induced tumor immunity, we carried out cDNA microarray analyses to compare the differential expression of genes between DAC and PBS treated EL4 cells. Two-condition experiment, PBS treated EL4 vs. Decitabine treated EL4 cells. Biological replicates: 3 PBS treated, 3 Decitabine treated, independently treatment and harvested. One replicate per array.
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:Genome-wide DNA methylation profiling of primary AML samples treated with 100nM decitabine (DAC), cytarabine (AraC), or DMSO. Eight distinct AML samples were grown using an in vitro stromal co-culture system for 4 days and then treated with either DAC, Ara-C or DMSO for 3 days. DNA was prepared for genome-wide methylation analysis with the Illumina Infinium 450k Human DNA methylation BeadChip. DNA from each sample/treatment was analyzed on duplicate arrays.
Project description:Genome-wide DNA methylation profiling of primary AML samples treated with 100nM decitabine (DAC), cytarabine (AraC), or DMSO. Eight distinct AML samples were grown using an in vitro stromal co-culture system for 4 days and then treated with either DAC, Ara-C or DMSO for 3 days. DNA was prepared for genome-wide methylation analysis with the Illumina Infinium 450k Human DNA methylation BeadChip. DNA from each sample/treatment was analyzed on duplicate arrays. Bisulfite-converted DNA from 24 samples was hybridised to the Illumina Infinium 450k Human Methylation Beadchip in duplicate (replicates are indicated by array plate number).
Project description:The combination of the demethylating agent Decitabine (DAC) and the retinoic acid ATRA has shown potent antileukemic action both in vitro and in vivo. The likely synergistic mechanism of action remains unknown. Both DAC and retinoid acids are known to affect methylation. DAC inhibits DNA-methyltransferases by covalent bonding, thereby causing demethylation after several replication cycles (Stresemann and Lyko, 2008). Depending on gene and entity, both demethylation (Das et al., 2010; Di Croce et al., 2002) as well as hypermethylation (Das et al., 2010) have been induced by treatment with retinoids. Therefore, a synergistic mechanism of action regarding the methylome to explain the combinatorial strong antileukemic effect of DAC+ATRA was investigated.
Project description:In order to further study the mechanism of decitabine (DAC) on MDS cell lines, 1uM DAC was used to treat SKM-1 and MDS-L cell lines, and the molecular pathway changes regulated by DAC were explored. Genes whose expression changes more than 2.0 times after drug treatment are defined as regulated genes. Taking the intersection change of the two cell lines, it was found that compared with the control, 541 genes were up-regulated in the intersection of gene changes treated with DAC, and 762 genes were down-regulated. Gene expression profile analysis shows that DAC can significantly affect various cell biological processes, including antiviral response, immune response, inflammatory response and cell apoptosis.
Project description:A novel hypomethylating agent NTX-301 is a promising therapeutic agent for AML patients. To examine its mechanisms of action, we produced methylome data upon treatment with NTX-301 or decitabine (DAC) in MV4-11 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.