Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells
Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells
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DOI：
10.4161/auto.6083
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发表时间：
2008-07-01
期刊：
影响因子：
13.3
通讯作者：
Ozpolat, Bulent
中科院分区：
文献类型：
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作者：
Akar, Ugur;Chaves-Reyez, Arturo;Ozpolat, Bulent
细胞凋亡(程序性细胞死亡类型I)和自噬(类型II)是调节细胞死亡和动态平衡的重要机制。原癌基因Bcl2在50-70%的乳腺癌中过度表达，可能导致对化疗、放疗和激素治疗诱导的细胞凋亡产生抵抗。在这里，我们研究了Bcl2在乳腺癌细胞自噬中的作用。在MCF-7乳腺癌细胞中，通过siRNA沉默Bcl2，可下调Bcl2蛋白水平(85%)，抑制细胞生长(71%)，抑制克隆形成(79%)，并通过自噬而不是细胞凋亡导致细胞死亡(高达55%)。丫啶橙染色、电子显微镜和GFP-LC-3(GFP-ATG8)转基因MCF-7细胞自噬体膜上GFP-LC3-II的积聚证实了自噬的诱导。通过siRNA沉默Bcl2还可以诱导自噬的标志LC-3-II、ATG5和Beclin-1自噬促进蛋白的表达。ATG5基因敲除可显著抑制Bcl2siRNA诱导的LC3-II表达、GFP-LC3-II标记的自噬阳性细胞数和自噬细胞死亡(p<；0.05)。此外，高剂量阿霉素(IC95，1 mU M)诱导细胞凋亡，而低剂量(IC50，0.07 mU M)仅诱导自噬和Beclin-1表达。当与Bc l-2 siRNA结合时，阿霉素(IC50)增强了自噬，表现为GFP-LC3-II染色的自噬小体数量增加(点状阳性)。这些结果首次证明了靶向沉默Bcl2可诱导MCF-7乳腺癌细胞自噬细胞死亡，并且Bcl2siRNA可单独或与化疗联合用于过表达Bcl2的乳腺癌细胞的治疗。
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 mu M) induced apoptosis but at a low dose (IC50, 0.07 mu M) 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.