Email this page

Print this page

Research Paper

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 and Bulent Ozpolat

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.

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. In this study, 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 preautopghagosomal and 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 and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p<0.05). Furthermore, doxorubicin at a high dose (IC95, 1μM) 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 autophagosomes in untransfected and GFP-LC3-II-stained punctuated pattern positive cells upon GFP-LC-3 transfection. 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.

Authors

Ugur Akar

University of Texas MD Anderson Cancer Center

Arturo Chaves-Reyez

University of Texas MD Anderson Cancer Center

Magaly Barria

University of Texas MD Anderson Cancer Center

Ana Tari

University of Texas MD Anderson Cancer Center

Angela Sanguino

University of Texas MD Anderson Cancer Center

Yasuko Kondo

University of Texas MD Anderson Cancer Center

Seiji Kondo

University of Texas MD Anderson Cancer Center

Banu Arun

University of Texas MD Anderson Cancer Center

Gabriel Lopez-Berestein

University of Texas MD Anderson Cancer Center

Bulent Ozpolat

University of Texas MD Anderson Cancer Center

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.