title: BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production.
abstract: We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.
authors: Institute for Innovative Cancer Research; University of Ulsan College of Medicine; Asan Medical Center, Seoul, Korea.
authors: Yunha
authors: Y
authors: Kim
authors: Yong-Sook
authors: YS
authors: Kim
authors: Dong Eun
authors: DE
authors: Kim
authors: Jee Suk
authors: JS
authors: Lee
authors: Ji Hoon
authors: JH
authors: Song
authors: Ha-Gyeong
authors: HG
authors: Kim
authors: Dong-Hyung
authors: DH
authors: Cho
authors: Seong-Yun
authors: SY
authors: Jeong
authors: Dong-Hoon
authors: DH
authors: Jin
authors: Se Jin
authors: SJ
authors: Jang
authors: Hyang-Sook
authors: HS
authors: Seol
authors: Young-Ah
authors: YA
authors: Suh
authors: Seung Jin
authors: SJ
authors: Lee
authors: Choung-Soo
authors: CS
authors: Kim
authors: Jae-Young
authors: JY
authors: Koh
authors: Jung Jin
authors: JJ
authors: Hwang
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D001381:Azepines / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D045325:HCT116 Cells; D006649:Histocompatibility Antigens / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D061251:Primary Cell Culture; D011799:Quinazolines / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001381:Azepines; C518299:BIX 01294; D004791:Enzyme Inhibitors; D006649:Histocompatibility Antigens; D011799:Quinazolines; D017382:Reactive Oxygen Species; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase
keywords: BIX-01294; EHMT2/G9a; ROS; autophagy; histone methyltransferase