Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

<p>Abstract</p> <p>Background</p> <p>The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established.</p> <p>Methods</p> <p>Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the <it>in vitro </it>antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The <it>in vivo </it>antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an <it>in vitro </it>clonogenic assay.</p> <p>Results</p> <p>Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (<it>p57KIP2 </it>in HL-60 leukemic cells and <it>p16CDKN2A </it>in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 μM).</p> <p>Conclusion</p> <p>We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce <it>in vitro </it>clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer.</p

ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

Doxorubicin is an anthracycline drug that is one of the most effective and widely used anticancer agents for the treatment of both hematologic and solid tumors. The stress-activated protein kinases (SAPKs) are frequently activated by a number of cancer chemotherapeutics. When phosphorylated, the SAPKs initiate a cascade that leads to the production of proinflammatory cytokines. Some inhibitors of protein synthesis, known as ribotoxic stressors, coordinately activate SAPKs and lead to apoptotic cell death. We demonstrate that doxorubicin effectively inhibits protein synthesis, activates SAPKs, and causes apoptosis. Ribotoxic stressors share a common mechanism in that they require ZAK, an upstream MAP3K, to activate the pro-apoptotic and proinflammatory signaling pathways that lie downstream of SAPKs. By employing siRNA mediated knockdown of ZAK or administration of sorafenib and nilotinib, kinase inhibitors that have a high affinity for ZAK, we provide evidence that ZAK is required for doxorubicin-induced proinflammatory and apoptotic responses in HaCaT cells, a pseudo-normal keratinocyte cell line, but not in HeLa cells, a cancerous cell line. ZAK has two different isoforms, ZAK-α (91 kDa) and ZAK-β (51 kDa). HaCaT or HeLa cells treated with doxorubicin and immunoblotted for ZAK displayed a progressive decrease in the ZAK-α band and the appearance of ZAK-β bands of larger size. Abrogation of these changes after exposure of cells to sorafenib and nilotinib suggests that these alterations occur following stimulation of ZAK. We suggest that ZAK inhibitors such as sorafenib or nilotinib may be effective when combined with doxorubicin to treat cancer patients

Diet folate, DNA methylation and genetic polymorphisms of MTHFR C677T in association with the prognosis of esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Folic acid may affect the development of human cancers. However, few studies have evaluated the consumption of diet folate in the prognosis of patients with esophageal squamous cell carcinoma (ESCC).</p> <p>Methods</p> <p>One hundred and twenty five ESCC patients underwent esophagectomy between January 2005 and March 2006 in the Yangzhong People's Hospital were recruited and followed up. The effects of diet folate, aberrant DNA methylation of selected genes and methylenetetrahydrofolate reductase (<it>MTHFR</it>) C677T genetic polymorphisms on the prognosis of ESCC were evaluated by using Cox proportional hazard regression models.</p> <p>Results</p> <p>Our analysis showed an inverse association between diet folate intake and the risk of death after esophagectomy. The median survival time was 3.06 years for low or moderate folate consumption and over 4.59 years for high folate consumption. After adjusting for potential confounders, the hazard ratios (95% confidence interval) [HRs (95% CI)] were 0.72 (0.36-1.46) for moderate and 0.39 (0.20-0.78) for high folate intake, respectively (<it>P </it>for trend = 0.007). This preventive effect was more evident in patients carrying <it>MTHFR </it>677CC genotype. No significant relation was observed between aberrant DNA methylation of <it>P16</it>, <it>MGMT </it>and <it>hMLH1 </it>gene, as well as <it>MTHFR </it>C677T genetic polymorphisms and the prognosis of ESCC.</p> <p>Conclusions</p> <p>Our research indicated that diet folate intake may have benefits on the prognosis of ESCC after esophagectomy. From a practical viewpoint, the findings of our study help to establish practical intervention and surveillance strategies for managements of ESCC patients and can finally decrease the disease burden.</p

Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells

The USFDA approved "epigenetic drug", Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC

Suppression of Soft Tissue Sarcoma Growth by a Host Defense-Like Lytic Peptide

BACKGROUND: Soft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K3H3L9 on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum. METHODS: In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K3H3L9, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K3H3L9 was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed. RESULTS: The peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K3H3L9 in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group. CONCLUSION: These findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K3H3L9 in athymic and syngeneic mouse models

Promoter polymorphisms of DNMT3B and the risk of colorectal cancer in Chinese: a case-control study

<p>Abstract</p> <p>Background</p> <p>DNA-methyltransferase-3B (DNMT3B), which plays a role in DNA methylation, is usually aberrant expression involved in carcinogenesis. Polymorphisms of the DNMT3B gene may influence DNMT3B activity on DNA methylation in several cancers, thereby modulating the susceptibility to cancer.</p> <p>Methods</p> <p>DNMT3B -579G>T genotypes and -149C>T were determined by PCR-RFLP and sequencing in 137 colorectal cancer patients and 308 controls matched for age and sex, who did not receive radiotherapy or chemotherapy for newly diagnosed and histopathologically confirmed colorectal cancer. The association between two SNPs of the <it>DNMT3B </it>promoter and the risk of the development of colorectal cancer was analyzed in a population of Chinese.</p> <p>Results</p> <p>The allele frequency of -149C >T among patients and controls was 0.73% versus 0.65%, respectively. The allele frequency of -597G>T for patients and controls was 6.57% versus 11.53%, respectively. Individuals with at least one -149C>T allele were no at a significantly increase risk of colorectal cancer compared with those having a -149TT genotype. However, Individuals with at least one 579G>T allele were decreased risk of colorectal cancer compared with those having a -579TT genotype.</p> <p>Conclusion</p> <p>The relative distribution of -149C>T <it>DNMT3B </it>SNPs among a Chinese population can not be used as a stratification marker to predict an individual's susceptibility to colorectal cancer. However, the DNMT3B -579G>T polymorphism may contribute to the genetic susceptibility to colorectal cancer.</p

Azacytidine and Decitabine Induce Gene-Specific and Non-Random DNA Demethylation in Human Cancer Cell Lines

The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation

Mechanisms of Resistance to Decitabine in the Myelodysplastic Syndrome

Purpose: The DNA methylation inhibitor 5-aza-29-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS. Patients and Methods: We performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance. Results: Most genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P,.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P,.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P,.05).MCAM analysis showed decreased methylation of an average of 4.5 % (range 0.6%– 9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20 % of patients