FIGURE 6

GRP78 suppression by ADQ leads to a β-catenin destabilization and an autophagy inhibition in breast CSCs. (A) Western blotting analysis showed that ADQ treatment for 48 h could inhibit GRP78 protein expression levels in MDA-MB-231 cells and MCF-7 cells in the presence of taxol. All values represent the means ± SD (n = 3, *p < 0.05, **p < 0.01 vs. Control group; ^#p < 0.05, ^##p < 0.01 vs. Taxol group). (B) Mammospheres assay and immunofluorescence analysis suggested that ADQ administration for 48 h could attenuate self-renewal and autophagic activities in GRP78^high MDA-MB-231 CSCs. (C) Western blotting analysis demonstrated that the autophagy activity in GRP78-overexpressed breast CSCs was significantly limited by the combination of 3-MA and ADQ. (D) Immunofluorescence experiments showed that ADQ not only limited the transfer of β-catenin from the cytoplasm to the nucleus, but also decreased the expression of β-catenin in GRP78^high MDA-MB-231 CSCs. (E) ADQ treatment for 48 h could attenuate β-catenin and ABCG2 expressions in GRP78^high MDA-MB-231 CSCs. (F)–(G) Cell number and sphere formation assay using GRP78^high MDA-MB-231 CSCs transfected with or without siβ-catenin after the indicated treatment. All values represent the means ± SD (n = 3, *p < 0.05, **p < 0.01 vs. Control group; ^#p < 0.05, ^##p < 0.01 vs. Taxol group). (H) Western blotting assay showing the effects of ADQ on the β-catenin proteasome degradation pathway in GRP78^high MDA-MB-231 CSCs. (I) Co-immunoprecipitation and ubiquitination array revealed that GRP78 overexpression reduced poly-ubiquitination accumulation of β-catenin in breast CSCs, while ADQ administration notably promoted the ubiquitination of β-catenin in breast CSCs.