Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Ugur Akar, Arturo Chaves-Reyez, Magaly Barria, Ana Tari, Angela Sanguino, Yasuko Kondo, Seiji Kondo, Banu Arun, Gabriel Lopez-Berestein, Bulent Ozpolat

Research output: Contribution to journalArticlepeer-review

201 Scopus citations

Abstract

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC95, 1 μM) induced apoptosis but at a low dose (IC50, 0.07 μM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC50) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.

Original languageEnglish (US)
Pages (from-to)669-679
Number of pages11
JournalAutophagy
Volume4
Issue number5
DOIs
StatePublished - Jul 1 2008

Keywords

  • Apoptosis
  • Autophagy
  • Bcl-2
  • Beclin-1
  • Breast cancer
  • siRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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