Mechanism of 2′,3′-dimethoxyflavanone-induced apoptosis in breast cancer stem cells: Role of ubiquitination of caspase-8 and LC3

•Among the tested 42 flavonoids, 2′,3′-dimethoxyflavanone (DMF) exhibits the most potent toxicity towards breast cancer stem cells MCF-7-SCs.•DMF induces both intrinsic and extrinsic apoptosis through DNA damage and caspase cascade.•DMF induces accumulation and full activation of caspase-8, which results in apoptosis.•The autophagic protein LC3 plays an important role in apoptosis by activating caspase-8.

Accumulating evidence has displayed that targeting cancer stem cells (CSCs) is a very promising way for anti-cancer therapies. 2′,3′-Dimethoxyflavanone (2′,3′-DMF) showed the most potent toxicity of a group of 42 flavonoids tested in MCF-7-SC breast cancer stem cells. 2′,3′-DMF triggered intrinsic and extrinsic apoptosis by stimulating the cleavage of PARP and the activation of caspase-9, -8, and -3. Interestingly, 2′,3′-DMF induces a dramatic increase in the conversion of LC3, a well-known marker for autophagy. However, acidic vesicular organelles (AVOs), one of the autophagic flux markers were not detected. Co-treatment with chloroquine, the lysosomal inhibitor that blocks autophagic degradation did not show any change in the degree of LC3 conversion, implying that LC3 could play a role in the non-autophagic cell death of MCF-7-SC. We found that 2′,3′-DMF induces the ubiquitination of caspase-8, this resulted in an interaction between caspase-8 and LC3, which led to the aggregation and activation of caspase-8. Co-treating cells with 2′,3′-DMF and 3-methyladenine, an inhibitor of LC3 lipidation, reduced the activation of caspase-8. These findings provide novel insights into the anti-cancer effects of 2′,3′-DMF in breast cancer stem cells by revealing that it induced apoptosis in accompany with the activation of caspase-8 mediated by LC3 conversion.