title: siRNA-mediated knock-down of DFF45 amplifies doxorubicin therapeutic effects in breast cancer cells.
abstract: OBJECTIVE
  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 silencing on breast cancer cell viability, cell cycle arrest, and apoptosis 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, especially 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.
authors.affiliation: Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Bagheri
authors.forename: Shahrokh
authors.initials: S
authors.lastname: Safarian
authors.forename: Mohamadreza Baghaban
authors.initials: MB
authors.lastname: Eslaminejad
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C109664:caspase-activated DNase inhibitor; D004317:Doxorubicin; D053148:Caspase 3