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

• Published in: Molecular cancer therapeutics 2007-06, Vol.6 (6), p.1718-1727
• Main Authors: Kiziltepe, Tanyel, Hideshima, Teru, Catley, Laurence, Raje, Noopur, Yasui, Hiroshi, Shiraishi, Norihiko, Okawa, Yutaka, Ikeda, Hiroshi, Vallet, Sonia, Pozzi, Samantha, Ishitsuka, Kenji, Ocio, Enrique M, Chauhan, Dharminder, Anderson, Kenneth C
• Format: Article
• Language: English
• Subjects: 5-Azacytidine; Antineoplastic Agents - pharmacology; apoptosis; Apoptosis - drug effects; Azacitidine - pharmacology; Boronic Acids - pharmacology; Bortezomib; Cell Division - drug effects; Cell Line, Tumor; Cell Survival - drug effects; DNA Damage; DNA, Neoplasm - drug effects; Doxorubicin - pharmacology; Drug Synergism; Enzyme Inhibitors - pharmacology; Humans; methyltransferase inhibitor; Multiple Myeloma - pathology; myeloma; Pyrazines - pharmacology
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-07-0010

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 ∼0.8–3 μmol/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. [Mol Cancer Ther 2007;6(6):1718–27]