Abstract
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor lapatinib could be overcome by the B cell CLL/lymphoma-2 (BCL-2) family antagonist obatoclax (GX15-070). Coadministration of lapatinib with obatoclax caused synergistic cell killing by eliciting autophagic cell death that was dependent upstream on mitochondrial reactive oxygen species generation and increased p62 levels and downstream on activation of p38 mitogen-activated protein kinase and inactivation of mammalian target of rapamycin. By immunohistochemical analysis, in drug combination-treated cells, microtubule-associated protein light chain 3 (LC3) associated with mitochondrial (cytochrome c oxidase), autophagosome (p62), and autolysosome (lysosomal associated membrane protein 2) proteins. Treatment of cells with 3-methyladenine or knockdown of beclin 1 was protective, whereas chloroquine treatment had no protective effect. Expression of myeloid cell leukemia-1 (MCL-1), compared with that of BCL-2 or BCL-2-related gene long isoform, protected against drug combination lethality. Lapatinib and obatoclax-initiated autophagy depended on NOXA-mediated displacement of the prosurvival BCL-2 family member, MCL-1, from beclin 1, which was essential for the initiation of autophagy. Taken together, our data argue that lapatinib and obatoclax-induced toxic autophagy is due to impaired autophagic degradation, and this disturbance of autophagic flux leads to an accumulation of toxic proteins and loss of mitochondrial function.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the Department of Defense [Breast Cancer Idea Award W81XWH-10-1-0009]; National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK52825]; National Institutes of Health National Cancer Institute [Grants CA141703, CA150218, F32-CA85159] (the last of these to Y.T. by way of the Massey Cancer Center).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- HER
- human epidermal growth factor receptor
- PI3K
- phosphatidylinositol 3-kinase
- MAPK
- mitogen-activated protein kinase
- BCL-2/Bcl-2
- B-cell CLL/lymphoma-2
- MCL-1
- myeloid cell leukemia-1
- BCL-XL
- BCL-2-related gene long isoform
- BAX
- BCL-2-associated x protein
- BAK
- BCL-2 antagonist killer
- mTOR
- mammalian target of rapamycin
- 3-MA
- 3-methyladenine
- FBS
- fetal bovine serum
- GFP
- green fluorescent protein
- LC3
- microtubule-associated protein light chain 3
- ATG
- autophagy related
- ATM
- ataxia telangiectasia mutated
- shRNA
- short hairpin RNA
- BAD
- BCL-2 antagonist of cell death
- BIM
- BCL-2-interacting mediator of cell death
- BID
- BCL-2-interacting domain death agonist
- LAMP-2
- lysosomal associated membrane protein 2
- NAC
- N-acetylcysteine
- ROS
- reactive oxygen species
- DCFDA
- dichlorofluorescein diacetate
- si
- small interfering
- PBS
- phosphate-buffered saline
- PI
- propidium iodide
- DMSO
- dimethyl sulfoxide
- COX IV
- cytochrome c oxidase
- AT-101
- (R-(−)-gossypol acetic acid
- ABT-263
- 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide
- Δψ
- mitochondrial membrane potential
- AVOs
- acidic vesicular organelles
- MOMP
- mitochondrial outer membrane permeabilization
- ER
- Estrogen receptor
- GX
- obatoclax (GX15–070).
- Received November 17, 2011.
- Accepted January 4, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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