Preclinical evaluation of the antineoplastic action of 5-aza-2'-deoxycytidine and different histone deacetylase inhibitors on human Ewing's sarcoma cells

<p>Abstract</p> <p>Background</p> <p>Most patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC).</p> <p>Results</p> <p>Here, we used a clonogenic assay to evaluate the <it>in vitro </it>antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line.</p> <p>Conclusion</p> <p>These results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.</p

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

A Perspective on the Comparative Antileukemic Activity of 5-Aza-2′-deoxycytidine (Decitabine) and 5-Azacytidine (Vidaza)

5-Aza-2′-deoxycytidine (5-AZA-CdR, decitabine, Dacogen®) and 5-azacytidine (5-AC, Vidaza®) are epigenetic agents that have been approved for the clinical treatment of the hematological malignancy myelodysplastic syndrome (MDS) and are currently under clinical evaluation for the treatment of acute myeloid leukemia (AML). Most investigators currently classify 5-AZA-CdR and 5-AC as inhibitors of DNA methylation, which can reactivate tumor suppressor genes silenced by this epigenetic event. Examination of the pharmacology of these analogues reveals important differences with respect to their molecular mechanism of action. The action of 5-AZA-CdR is due to its incorporation into DNA. 5-AC is a riboside analogue that is incorporated primarily into RNA. A small fraction of 5-AC is converted to its deoxyribose form by ribonucleotide reductase and subsequently incorporated into DNA. The incorporation of 5-AC into RNA can interfere with the biological function of RNA and result in an inhibition protein synthesis. Microarray analysis revealed that both these analogues target the expression of different cohorts of genes. Preclinical studies show that 5-AZA-CdR is a more effective antileukemic agent than 5-AC. One explanation for this observation is that 5-AC blocks the progression of some leukemic cells from G&lt;sub&gt;1&lt;/sub&gt; into S phase, and this protects these cells from the chemotherapeutic action of this riboside analogue related to its incorporation&lt;strong&gt; &lt;/strong&gt;into DNA. However, differences in chemotherapeutic efficacy of these related analogues have not been clearly demonstrated in clinical trials in patients with hematological malignancies. These observations should be taken into consideration in the design of new clinical trials using 5-AZA-CdR or 5-AC in patients with MDS and AML

Epigenetic therapy of non-small cell lung cancer using decitabine (5-aza-2’-deoxycytidine, DAC)

Epigenetic analysis shows that many genes that suppress malignancy are silenced by aberrant DNA methylation in lung cancer. Many of these genes are interesting targets for reactivation by the inhibitor of DNA methylation, decitabine (5-aza-2’-deoxycytidine, DAC). A pilot study on intense dose DAC showed promising results in patients with metastatic non-small cell lung cancer (NSCLC). However, subsequent clinical studies using low dose DAC were not very effective against NSCLC and interest in this therapy diminished. Recently, interesting responses were observed in a patient with NSCLC following treatment with a combination of the related inhibitor of DNA methylation, 5-azacytidine and an inhibitor of histone deacetylation. This finding has generated a renewed interest in the epigenetic therapy of lung cancer. Preclinical studies indicate that DAC has remarkable chemotherapeutic potential for tumor therapy. This epigenetic agent has a delayed and prolonged epigenetic action on tumor cells. This delayed action should be taken into consideration in the design and evaluation of clinical studies on DAC. Future research should be directed at finding the optimal dose-schedule of de DAC for the treatment of NSCLC

Enhancement of the Antileukemic Action of the Inhibitors of DNA and Histone Methylation: 5-Aza-2′-Deoxycytidine and 3-Deazaneplanocin-A by Vitamin C

Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2′-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule