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
ABSTRACT 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.
- SourceAvailable from: Masoud Manjili[Show abstract] [Hide abstract]
ABSTRACT: Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza-Rev and cryopreserved. Subsequent stem cell mobilization was followed by high-dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up-regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)-specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.British Journal of Haematology 07/2012; 158(6):700-11. DOI:10.1111/j.1365-2141.2012.09225.x · 4.96 Impact Factor
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ABSTRACT: In our model, we aimed to explore the cytotoxicity of 5-Aza-2'-deoxycytidine (5-Aza-CdR) against the colorectal cell line, Lovo, and further characterize the possible mechanisms. After Lovo cells were treated with 5-Aza-CdR at different concentrations for different periods of time, the cell viability was examined using an MTT assay and apoptosis was examined using both flow cytometry and DNA laddering. To examine the mechanisms by which Lovo cells respond to 5-Aza-CdR, we measured both caspase 3 activity as well as DNA damage. Western blotting and RT-PCR assays were used to assess the changes in the expression levels of P53, P21(Waf1/Cip1), runt-related transcription factor 3 (RUNX 3), DNA methyltransferases (DNMTs) and matrix metalloproteinases (MMPs). Additionally, we performed gelatin zymography to examine the effects of 5-Aza-CdR on metastasis. We observed that the growth and survival advantages of Lovo cells were overcome with 5-Aza-CdR treatment at limited concentrations. Mechanistic exploration demonstrated that 5-Aza-CdR was incorporated into the DNA to induce DNA damage in Lovo cells, which was evidenced by activation of P53, P21(Waf1/Cip1) and a caspase-independent cell apoptosis pathway. Also, further experiments preliminarily suggested that 5-Aza-CdR results in the deletion of DNMT 3a and DNMT 3b, but not DNMT 1, which reactivates the expression of RUNX 3. Finally, our data revealed that 5-Aza-CdR potentially reduces the activity and expression of MMP 2. These data greatly enhance our understanding of how human cancer cells respond to 5-Aza-CdR and also reveal a new role for 5-Aza-CdR in improving patient outcome in human colorectal cancer.Life Sciences 02/2009; 84(9-10):311-20. DOI:10.1016/j.lfs.2008.12.015 · 2.30 Impact Factor