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.