title: Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.
abstract: Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Dong Eun
authors.initials: DE
authors.lastname: Kim
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Yunha
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea.
authors.forename: Dong-Hyung
authors.initials: DH
authors.lastname: Cho
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Seong-Yun
authors.initials: SY
authors.lastname: Jeong
authors.affiliation: Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Sung-Bae
authors.initials: SB
authors.lastname: Kim
authors.affiliation: Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Department of Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Nayoung
authors.initials: N
authors.lastname: Suh
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Jung Shin
authors.initials: JS
authors.lastname: Lee
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Eun Kyung
authors.initials: EK
authors.lastname: Choi
authors.affiliation: Neural Injury Research Center and Department of Neurology, Asan Medical Center, Seoul, Korea.
authors.forename: Jae-Young
authors.initials: JY
authors.lastname: Koh
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Jung Jin
authors.initials: JJ
authors.lastname: Hwang
authors.affiliation: Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea ; Department of Urology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
authors.forename: Choung-Soo
authors.initials: CS
authors.lastname: Kim
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D001943:Breast Neoplasms / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: C502778:ATG5 protein, human; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020849:Raloxifene Hydrochloride; C025946:3-methyladenine; D055372:AMP-Activated Protein Kinases; D020169:Caspases; D000225:Adenine
keywords: AMPK; ATP; autophagy; breast cancer; raloxifene