Kiziltepe, T. et al. 5-Azacytidine, a DNA methyltransferase inhibitor, induces ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity with doxorubicin and bortezomib against multiple myeloma cells. Mol. Cancer Ther. 6, 1718-1727

Harvard University, Cambridge, Massachusetts, United States
Molecular Cancer Therapeutics (Impact Factor: 5.68). 07/2007; 6(6):1718-27. DOI: 10.1158/1535-7163.MCT-07-0010
Source: PubMed


In this study, we investigated the cytotoxicity of 5-azacytidine, a DNA methyltransferase inhibitor, against multiple myeloma (MM) cells, and characterized DNA damage-related mechanisms of cell death. 5-Azacytidine showed significant cytotoxicity against both conventional therapy-sensitive and therapy-resistant MM cell lines, as well as multidrug-resistant patient-derived MM cells, with IC(50) of approximately 0.8-3 micromol/L. Conversely, 5-azacytidine was not cytotoxic to peripheral blood mononuclear cells or patient-derived bone marrow stromal cells (BMSC) at these doses. Importantly, 5-azacytidine overcame the survival and growth advantages conferred by exogenous interleukin-6 (IL-6), insulin-like growth factor-I (IGF-I), or by adherence of MM cells to BMSCs. 5-Azacytidine treatment induced DNA double-strand break (DSB) responses, as evidenced by H2AX, Chk2, and p53 phosphorylations, and apoptosis of MM cells. 5-Azacytidine-induced apoptosis was both caspase dependent and independent, with caspase 8 and caspase 9 cleavage; Mcl-1 cleavage; Bax, Puma, and Noxa up-regulation; as well as release of AIF and EndoG from the mitochondria. Finally, we show that 5-azacytidine-induced DNA DSB responses were mediated predominantly by ATR, and that doxorubicin, as well as bortezomib, synergistically enhanced 5-azacytidine-induced MM cell death. Taken together, these data provide the preclinical rationale for the clinical evaluation of 5-azacytidine, alone and in combination with doxorubicin and bortezomib, to improve patient outcome in MM.

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    • "In vitro studies have shown that azacytidine alone or in combination with other antitumor agents induces tumor cell apoptosis. It has been reported that azacytidine induced ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity in multiple myeloma cells [8]. It has also been demonstrated that a combination of azacytidine and arsenic trioxide generates a synergistic anti-tumor activity in myeloma [9]. "
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    ABSTRACT: Azacytidine is an inhibitor of DNA methyltransferase and is known to be an anti-leukemic agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with azacytidine at clinically relevant concentrations to induce necrosis through oxidative stress. Necrotic myeloma cells exhibit unique characteristics, including enrichment of the cell-bound albumin and overexpression of endoplasmic reticulum (ER)- and mitochondrial-specific chaperones, which were not observed in other necrotic cells, including HUH-7, A2780, A549, and Hoc1a. Proteomic analysis shows that HSP60 is the most abundant up-regulated mitochondrial specific chaperone, and azacytidine-induced overexpression of HSP60 is confirmed by western blot analysis. In contrast, expression levels of cytosolic chaperones such as HSP90 and HSP71 were down-regulated in azacytidine-treated myeloma cells, concomitant with an increase of these chaperones in the cell culture medium, suggesting that mitochondrial chaperones and cytosolic chaperones behave differently in necrotic myeloma cells; ER- and mitochondrial-chaperones being retained, and cytosolic chaperones being released into the cell culture medium through the ruptured cell membrane. Our data suggest that HSP60 is potentially a new target for multiple myeloma chemotherapy.
    Full-text · Article · Jun 2013 · Proteome Science
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    • "It has been previously shown that cytoxicity of 5-azadC to mammalian cells can be mediated through covalent DNMT-DNA adducts, which in turn cause DNA damage that activates ATR signaling (3,20). Here, we find that 5-azadC treatment produces γ-H2AX foci (Figure 1A and B), which has also been reported earlier (3). "
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    ABSTRACT: 5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.
    Full-text · Article · Apr 2013 · Nucleic Acids Research
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    • "A similar trend was seen in the ratio of cleaved:uncleaved PARP. 5azaC is also known to induce DNA double strand breaks in cells [48], which can be estimated by evaluating levels of phosphorylated histone variant γH2AX. A synergistic increase in the level of γH2AX was observed in response to 5azaC plus ING1b compared to 5azaC or ING1b alone. "
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    ABSTRACT: Inhibitor of Growth (ING) proteins are epigenetic "readers" that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin. Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo. These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.
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