ORIGINAL PAPER siRNA-mediated knock-down of DFF45 amplifies doxorubicin therapeutic effects in breast cancer cells Fatemeh Bagheri & Shahrokh Safarian & Mohamadreza Baghaban Eslaminejad & Nader Sheibani Accepted: 21 October 2013 /Published online: 26 November 2013 # International Society for Cellular Oncology 2013 Abstract Purpose RNA interference (RNAi) has become a promising tool for cancer therapy. Small interfering RNAs (siRNAs) can synergistically enhance the cell killing effects of drugs used in cancer treatment. Here we examined the effects of siRNA- mediated DNA fragmentation factor 45 (DFF45) gene silenc- ing on breast cancer cell viability, cell cycle arrest, and apo- ptosis in the presence and absence of doxorubicin. Methods We designed three siRNAs, which target different regions of the DFF45 mRNA. Gene silencing was confirmed by real time RT-PCR and Western blot analyses. The impact of DFF45 siRNA, doxorubicin, and their combination on the viability, cell cycle and apoptosis of T-47D and MDA-MB- 231 breast cancer cells were determined by MTT, PI staining, annexin V binding, caspase-3 activity, DNA laddering, and chromatin condensation assays. Results Based on flow cytometric analyses, we found that silencing of DFF45 alone had little effect on apoptosis, espe- cially in T-47D cells. However, when used in combination with doxorubicin (0.33 μM) a significant increase (P <0.05) in apoptosis was observed in T-47D and MDA-MB-231 cells, i.e., ~2.5- and 3-fold, respectively. Caspase-3 activity, chromatin condensation, as well as DNA laddering supported increased apoptosis in the combinatorial treatment. Cell cycle arrest in both cell lines occurred at lower levels after siRNA + doxorubicin treatment compared to doxorubicin only. Conclusions Our data indicate that DFF45 gene silencing, when applied in combination with doxorubicin, may offer a novel therapeutic strategy for the treatment of breast cancer. Keywords DFF45 siRNA . Apoptosis . Breast cancer . Doxorubicin . Cancer therapy 1 Introduction Numerous physiological growth-control mechanisms that di- rect cell proliferation and tissue homeostasis are linked to apoptosis. Tumor cells are often resistant to apoptosis and this resistance can be an important cause for cancer development [1–3]. Since effective chemotherapy depends on the induction of apoptosis, cancers with defects in cell death signaling path- ways are particularly difficult to treat [4]. Tumor cells can acquire resistance to apoptosis by an increase in expression of anti-apoptotic proteins or by a down-regulation or mutation of pro-apoptotic proteins. Thus, interference with apoptotic path- ways could be an effective means for cancer therapy [5]. One important event during late stages of apoptosis is DNA laddering. The main enzyme in this process is the DNA fragmentation factor 40 (DFF40), which is also called caspase activated DNase (CAD) [6]. DFF40 normally exists as a heterodimer with a 45 kDa protein (termed DFF45) through a conserved domain of 80 amino acids in its N-terminus [7]. The DFF45 protein has an inhibitory effect on DFF40, and acts as a chaperone affecting the appropriate folding of DFF40 and, thereby, the activation of its nuclease activity [8]. Upon the induction of apoptosis, DFF45 is cleaved by caspase-3 and released from DFF40. The hence activated DFF40 cleaves F. Bagheri : S. Safarian (*) Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran e-mail: safarian@khayam.ut.ac.ir F. Bagheri : M. B. Eslaminejad (*) Department of Stem Cells and Developmental Biology at Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran e-mail: eslami@royaninstitute.org N. Sheibani Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA Cell Oncol. (2013) 36:515–526 DOI 10.1007/s13402-013-0157-1